Stephen Quake

All Publications

• Identification of a colonial chordate histocompatibility gene. Science Voskoboynik, A., Newman, A. M., Corey, D. M., Sahoo, D., Pushkarev, D., Neff, N. F., Passarelli, B., Koh, W., Ishizuka, K. J., Palmeri, K. J., Dimov, I. K., Keasar, C., Fan, H. C., Mantalas, G. L., Sinha, R., Penland, L., Quake, S. R., Weissman, I. L. 2013; 341 (6144): 384-387

  Abstract

  Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

  View details for DOI 10.1126/science.1238036

  View details for PubMedID 23888037

• Lineage structure of the human antibody repertoire in response to influenza vaccination. Science translational medicine Jiang, N., He, J., Weinstein, J. A., Penland, L., Sasaki, S., He, X., Dekker, C. L., Zheng, N., Huang, M., Sullivan, M., Wilson, P. C., Greenberg, H. B., Davis, M. M., Fisher, D. S., Quake, S. R. 2013; 5 (171): 171ra19-?

  Abstract

  The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

  View details for DOI 10.1126/scitranslmed.3004794

  View details for PubMedID 23390249

• Migration of Cells in a Social Context 57th Annual Meeting of the Biophysical-Society Vedel, S., Tay, S., Johnston, D. M., Bruus, H., Quake, S. R. CELL PRESS. 2013: 147A–147A

  View details for Web of Science ID 000316074301249

• Proteome-wide protein interaction measurements of bacterial proteins of unknown function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Meier, M., Sit, R. V., Quake, S. R. 2013; 110 (2): 477-482

  Abstract

  Despite the enormous proliferation of bacterial genome data, surprisingly persistent collections of bacterial proteins have resisted functional annotation. In a typical genome, roughly 30% of genes have no assigned function. Many of these proteins are conserved across a large number of bacterial genomes. To assign a putative function to these conserved proteins of unknown function, we created a physical interaction map by measuring biophysical interaction of these proteins. Binary protein--protein interactions in the model organism Streptococcus pneumoniae (TIGR4) are measured with a microfluidic high-throughput assay technology. In some cases, informatic analysis was used to restrict the space of potential binding partners. In other cases, we performed in vitro proteome-wide interaction screens. We were able to assign putative functions to 50 conserved proteins of unknown function that we studied with this approach.

  View details for DOI 10.1073/pnas.1210634110

  View details for Web of Science ID 000313906600027

  View details for PubMedID 23267104

• Migration of cells in a social context PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vedel, S., Tay, S., Johnston, D. M., Bruus, H., Quake, S. R. 2013; 110 (1): 129-134

  Abstract

  In multicellular organisms and complex ecosystems, cells migrate in a social context. Whereas this is essential for the basic processes of life, the influence of neighboring cells on the individual remains poorly understood. Previous work on isolated cells has observed a stereotypical migratory behavior characterized by short-time directional persistence with long-time random movement. We discovered a much richer dynamic in the social context, with significant variations in directionality, displacement, and speed, which are all modulated by local cell density. We developed a mathematical model based on the experimentally identified "cellular traffic rules" and basic physics that revealed that these emergent behaviors are caused by the interplay of single-cell properties and intercellular interactions, the latter being dominated by a pseudopod formation bias mediated by secreted chemicals and pseudopod collapse following collisions. The model demonstrates how aspects of complex biology can be explained by simple rules of physics and constitutes a rapid test bed for future studies of collective migration of individual cells.

  View details for DOI 10.1073/pnas.1204291110

  View details for Web of Science ID 000313630300038

  View details for PubMedID 23251032

  View details for PubMedCentralID PMC3538227

• Correlation of gene expression and genome mutation in single B-cells. PloS one Weinstein, J. A., Zeng, X., Chien, Y., Quake, S. R. 2013; 8 (6)

  Abstract

  High-throughput measurement of gene-expression and immune receptor repertoires have recently become powerful tools in the study of adaptive immune response. However, despite their now-widespread use, both tend to discard cell identity by treating cell populations in bulk, and therefore lose the correlation between genetic variability and gene-expression at the single cell level. In order to recover this information, we developed a method to simultaneously measure gene expression profiles and genome mutations in single cells. We applied this method by quantifying the relationships between gene expression and antibody mutation in ensembles of individual B-cells from immunized mice. The results reveal correlations reflecting the manner in which information propagates between a B-cell's antigen receptors, its gene expression, and its mutagenic machinery, and demonstrate the power of this approach to illuminate both heterogeneity and physiology in cell populations.

  View details for DOI 10.1371/journal.pone.0067624

  View details for PubMedID 23840752

• Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage. Nat Commun. Dodsworth, J. A., Blainey, P. C., Murugapiran, S. K., Swingley, W. D., Ross, C. A., Tringe, S. G., Quake, S. 2013; 4: 1854

  View details for DOI 10.1038/ncomms2884.

• The genome sequence of the colonial chordate Botryllus schlosseri. Elife. Voskoboynik, A., Neff, N. F., Sahoo, D., Newman, A. M., Pushkarev, D., Koh, W., Quake, S. 2013; 2: e00569

  View details for DOI 10.7554/eLife. 00569.

• Genetic measurement of memory B-cell recall using antibody repertoire sequencing. Vollmers, C., Sit, R. V., Weinstein, J. A., Dekker, C. L., Quake, S, R. 2013

  View details for DOI 10.1073/pnas. 1312146110

• The genome sequence of the colonial chordate, Botryllus schlosseri. eLife Voskoboynik, A., Neff, N. F., Sahoo, D., Newman, A. M., Pushkarev, D., Koh, W., Passarelli, B., Fan, H. C., Mantalas, G. L., Palmeri, K. J., Ishizuka, K. J., Gissi, C., Griggio, F., Ben-Shlomo, R., Corey, D. M., Penland, L., White, R. A., Weissman, I. L., Quake, S. R. 2013; 2

  Abstract

  Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.

  View details for DOI 10.7554/eLife.00569

  View details for PubMedID 23840927

• Microfluidic serial digital to analog pressure converter for arbitrary pressure generation and contamination-free flow control LAB ON A CHIP Yu, F., Horowitz, M. A., Quake, S. R. 2013; 13 (10): 1911-1918

  Abstract

  Multilayer microfluidics based on PDMS (polydimethylsiloxane) soft lithography have offered parallelism and integration for biological and chemical sciences, where reduction in reaction volume and consistency of controlled variables across experiments translate into reduced cost, increased quantity and quality of data. One issue with push up or push down microfluidic control concept is the inability to provide multiple control pressures without adding more complex and expensive external pressure controls. We present here a microfluidic serial DAC (Digital to Analog Converter) that can be integrated with any PDMS device to expand the device's functionality by effectively adding an on-chip pressure regulator. The microfluidic serial DAC can be used with any incompressible fluids and operates in a similar fashion compared to an electronic serial DAC. It can be easily incorporated into any existing multilayer microfluidic devices, and the output pressure that the device generates could be held for extensive times. We explore in this paper various factors that affect resolution, speed, and linearity of the DAC output. As an application, we demonstrate microfluidic DAC's ability for on-chip manipulation of flow resistance when integrated with a simple flow network. In addition, we illustrate an added advantage of using the microfluidic serial DAC in preventing back flow and possible contamination.

  View details for DOI 10.1039/c3lc41394b

  View details for Web of Science ID 000317937300011

  View details for PubMedID 23529280

• Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics. PloS one Streets, A. M., Sourigues, Y., Kopito, R. R., Melki, R., Quake, S. R. 2013; 8 (1)

  Abstract

  An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

  View details for DOI 10.1371/journal.pone.0054541

  View details for PubMedID 23349924

• Systematic reconstruction of RNA functional motifs with high-throughput microfluidics NATURE METHODS Martin, L., Meier, M., Lyons, S. M., Sit, R. V., Marzluff, W. F., Quake, S. R., Chang, H. Y. 2012; 9 (12): 1192-U85

  Abstract

  We present RNA-mechanically induced trapping of molecular interactions (RNA-MITOMI), a microfluidic platform that allows integrated synthesis and functional assays for programmable RNA libraries. The interaction of a comprehensive library of RNA mutants with stem-loop-binding protein precisely defined the RNA structural and sequence features that govern affinity. The functional motif reconstructed in a single experiment on our platform uncovers new binding specificities and enriches interpretation of phylogenetic data.

  View details for DOI 10.1038/NMETH.2225

  View details for Web of Science ID 000312093500022

  View details for PubMedID 23142872

• A Single-Cell Genome for Thiovulum sp. APPLIED AND ENVIRONMENTAL MICROBIOLOGY Marshall, I. P., Blainey, P. C., Spormann, A. M., Quake, S. R. 2012; 78 (24): 8555-8563

  Abstract

  We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

  View details for DOI 10.1128/AEM.02314-12

  View details for Web of Science ID 000311213200007

  View details for PubMedID 23023751

• High-Performance Binary Protein Interaction Screening in a Microfluidic Format ANALYTICAL CHEMISTRY Meier, M., Sit, R., Pan, W., Quake, S. R. 2012; 84 (21): 9572-9578

  Abstract

  The standard procedure to increase microfluidic chip performance is to grow the number of parallel test systems on the chip. This process is accompanied by miniaturizing biochemical workflows and micromechanical elements, which is often a major challenge for both engineering fields. In this work, we show that it is possible to substantially increase the runtime performance of a microfluidic affinity assay for protein interactions by simultaneously engineering fluid logics and assay chemistry. For this, synergistic effects between the micro- and chemical architecture of the chip are exploited. The presented strategy of reducing the runtime rather than size and volume of the mechanical elements and biological reagent compartments will, in general, be of importance for future analytical test systems on microfluidic chips to overcome performance barriers.

  View details for DOI 10.1021/ac302436y

  View details for Web of Science ID 000310664600097

  View details for PubMedID 23051662

• Detecting Prenatal Microdeletions Non-Invasively: Case of a 22q11.2 Deletion Annual Meeting of the Association-for-Molecular-Pathology Gu, W., Fan, H., Blumenfeld, Y., El-Sayed, Y., Quake, S. ELSEVIER SCIENCE INC. 2012: 638–38

  View details for Web of Science ID 000310178600021

• Quantitative Analysis of the Human Airway Microbial Ecology Reveals a Pervasive Signature for Cystic Fibrosis SCIENCE TRANSLATIONAL MEDICINE Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. R. 2012; 4 (153)

  Abstract

  Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator. Disruption of electrolyte homeostasis at mucosal surfaces leads to severe lung, pancreatic, intestinal, hepatic, and reproductive abnormalities. Loss of lung function as a result of chronic lung disease is the primary cause of death from CF. Using high-throughput sequencing to survey microbes in the sputum of 16 CF patients and 9 control individuals, we identified diverse microbial communities in the healthy samples, contravening conventional wisdom that healthy airways are not significantly colonized. Comparing these communities with those from the CF patients revealed significant differences in microbial ecology, including differential representation of uncultivated phylotypes. Despite patient-specific differences, our analysis revealed a focal microbial profile characteristic of CF. The profile differentiated case and control groups even when classically recognized CF pathogens were excluded. As a control, lung explant tissues were also processed from a group of patients with pulmonary disease. The findings in lung tissue corroborated the presence of taxa identified in the sputum samples. Comparing the sequencing results with clinical data indicated that diminished microbial diversity is associated with severity of pulmonary inflammation within our adult CF cohort.

  View details for DOI 10.1126/scitranslmed.3004458

  View details for Web of Science ID 000309525600003

  View details for PubMedID 23019655

• Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia SCIENCE TRANSLATIONAL MEDICINE Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Vyas, P., Weissman, I. L., Quake, S. R., Majeti, R. 2012; 4 (149)

  Abstract

  Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.

  View details for DOI 10.1126/scitranslmed.3004315

  View details for Web of Science ID 000308491600005

  View details for PubMedID 22932223

• Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm CELL Wang, J., Fan, H. C., Behr, B., Quake, S. R. 2012; 150 (2): 402-412

  Abstract

  Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

  View details for DOI 10.1016/j.cell.2012.06.030

  View details for Web of Science ID 000306595700018

  View details for PubMedID 22817899

• Non-invasive prenatal measurement of the fetal genome NATURE Fan, H. C., Gu, W., Wang, J., Blumenfeld, Y. J., El-Sayed, Y. Y., Quake, S. R. 2012; 487 (7407): 320-?

  Abstract

  The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.

  View details for DOI 10.1038/nature11251

  View details for Web of Science ID 000306506500033

  View details for PubMedID 22763444

• Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB) GENOME RESEARCH Pamp, S. J., Harrington, E. D., Quake, S. R., Relman, D. A., Blainey, P. C. 2012; 22 (6): 1107-1119

  Abstract

  Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.

  View details for DOI 10.1101/gr.131482.111

  View details for Web of Science ID 000304728100012

  View details for PubMedID 22434425

  View details for PubMedCentralID PMC3371716

• Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines PLOS ONE Powell, A. A., Talasaz, A. H., Zhang, H., Coram, M. A., Reddy, A., Deng, G., Telli, M. L., Advani, R. H., Carlson, R. W., Mollick, J. A., Sheth, S., Kurian, A. W., Ford, J. M., Stockdale, F. E., Quake, S. R., Pease, R. F., Mindrinos, M. N., Bhanot, G., Dairkee, S. H., Davis, R. W., Jeffrey, S. S. 2012; 7 (5)

  Abstract

  To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.

  View details for DOI 10.1371/journal.pone.0033788

  View details for Web of Science ID 000305335000005

  View details for PubMedID 22586443

  View details for PubMedCentralID PMC3346739

• High Throughput Sequencing of the Human Antibody Repertoire in Response to Influenza Vaccination 99th Annual Meeting of the American-Association-of-Immunologists Jiang, N., He, J., Weinstein, J., Penland, L., Saaki, S., He, X., Dekker, C., Wilson, P., Greenberg, H., Davis, M., Fisher, D., Quake, S. AMER ASSOC IMMUNOLOGISTS. 2012

  View details for Web of Science ID 000304659700423

• Identification of a cKit(+) Colonic Crypt Base Secretory Cell That Supports Lgr5(+) Stem Cells in Mice GASTROENTEROLOGY Rothenberg, M. E., Nusse, Y., Kalisky, T., Lee, J. J., Dalerba, P., Scheeren, F., Lobo, N., Kulkarni, S., Sim, S., Qian, D., Beachy, P. A., Pasricha, P. J., Quake, S. R., Clarke, M. F. 2012; 142 (5): 1195-?

  Abstract

  Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

  View details for DOI 10.1053/j.gastro.2012.02.006

  View details for Web of Science ID 000303113600038

  View details for PubMedID 22333952

  View details for PubMedCentralID PMC3911891

• Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns NATURE PROTOCOLS Sanchez-Freire, V., Ebert, A. D., Kalisky, T., Quake, S. R., Wu, J. C. 2012; 7 (5): 829-838

  Abstract

  Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.

  View details for DOI 10.1038/nprot.2012.021

  View details for Web of Science ID 000303359300002

  View details for PubMedID 22481529

  View details for PubMedCentralID PMC3657501

• Sizing Up Cell-Free DNA CLINICAL CHEMISTRY Quake, S. 2012; 58 (3): 489-490

  View details for DOI 10.1373/clinchem.2011.174250

  View details for Web of Science ID 000300934900003

  View details for PubMedID 22235102

• DNA Sequencing Clinical Applications of New DNA Sequencing Technologies CIRCULATION Dewey, F. E., Pan, S., Wheeler, M. T., Quake, S. R., Ashley, E. A. 2012; 125 (7): 931-944

  View details for DOI 10.1161/CIRCULATIONAHA.110.972828

  View details for Web of Science ID 000300949800019

  View details for PubMedID 22354974

• Profiling maternal plasma cell-free RNA by RNA-sequencing: a comprehensive approach 32nd Annual Pregnancy Meeting of the Society-for-Maternal-Fetal-Medicine (SMFM) Koh, W., Fan, C., Blumenfeld, Y., Wong, R., El-Sayed, Y., Kogut, E., Quake, S. MOSBY-ELSEVIER. 2012: S324–S324

  View details for Web of Science ID 000298889900725

• Microfluidic very lage scale integration (mVLSI) with integrated micromechnical valves Lab on a Chip Araci, I. E., Quake, Stephen, R. 2012; 12 (16): 2803-2806

  View details for DOI 10.1029/c21c40258k

• Microfluidic very large scale integration (mVLSI) with integrated micromechanical valves LAB ON A CHIP Araci, I. E., Quake, S. R. 2012; 12 (16): 2803-2806

  Abstract

  Microfluidic chips with a high density of control elements are required to improve device performance parameters, such as throughput, sensitivity and dynamic range. In order to realize robust and accessible high-density microfluidic chips, we have fabricated a monolithic PDMS valve architecture with three layers, replacing the commonly used two-layer design. The design is realized through multi-layer soft lithography techniques, making it low cost and easy to fabricate. By carefully determining the process conditions of PDMS, we have demonstrated that 8 × 8 and 6 × 6 μm(2) valve sizes can be operated at around 180 and 280 kPa differential pressure, respectively. We have shown that these valves can be fabricated at densities approaching 1 million valves per cm(2), substantially exceeding the current state of the art of microfluidic large-scale integration (mLSI) (thousands of valves per cm(2)). Because the density increase is greater than two orders of magnitude, we describe this technology as microfluidic very large scale integration (mVLSI), analogous to its electronic counterpart. We have captured and tracked fluorescent beads, and changed the electrical resistance of a fluidic channel by using these miniaturized valves in two different experiments, demonstrating that the valves are leakproof. We have also demonstrated that these valves can be addressed through multiplexing.

  View details for DOI 10.1039/c2lc40258k

  View details for Web of Science ID 000306523800007

  View details for PubMedID 22714259

• High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation LAB ON A CHIP Wu, A. R., Kawahara, T. L., Rapicavoli, N. A., van Riggelen, J., Shroff, E. H., Xu, L., Felsher, D. W., Chang, H. Y., Quake, S. R. 2012; 12 (12): 2190-2198

  Abstract

  Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.

  View details for DOI 10.1039/c2lc21290k

  View details for Web of Science ID 000304448700012

  View details for PubMedID 22566096

• Digital PCR provides absolute quantitation of viral load for an occult RNA virus JOURNAL OF VIROLOGICAL METHODS White, R. A., Quake, S. R., Curr, K. 2012; 179 (1): 45-50

  Abstract

  Using a multiplexed LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a prefabricated microfluidic device that monitored absolute viral load in native and immortalized cell lines, overall precision of detection, and the absolute detection limit of an occult RNA virus GB Virus Type C (GBV-C). RT-dPCR had on average a 10% lower overall coefficient of variation (CV, a measurement of precision) for viral load testing than RT-qPCR and had a higher overall detection limit, able to quantify as low as three 5'-UTR molecules of GBV-C genome. Two commercial high-yield in vitro transcription kits (T7 Ribomax Express by Promega and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA genome. THP-1 cells (a model of monocytic derived cells) were transfected with GBV-C, yielding infectious virions that replicated over a 120h time course and could be infected directly. This study provides the first evidence of GBV-C replication in monocytic derived clonal cells. Thus far, it is the only study using a microfluidic device that measures directly viral load of mammalian RNA virus in a digital format without need for a standard curve.

  View details for DOI 10.1016/j.jviromet.2011.09.017

  View details for Web of Science ID 000300207700008

  View details for PubMedID 21983150

• Single-cell dissection of transcriptional heterogeneity in human colon tumors NATURE BIOTECHNOLOGY Dalerba, P., Kalisky, T., Sahoo, D., Rajendran, P. S., Rothenberg, M. E., Leyrat, A. A., Sim, S., Okamoto, J., Johnston, D. M., Qian, D., Zabala, M., Bueno, J., Neff, N. F., Wang, J., Shelton, A. A., Visser, B., Hisamori, S., Shimono, Y., Van De Wetering, M., Clevers, H., Clarke, M. F., Quake, S. R. 2011; 29 (12): 1120-U11

  Abstract

  Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.

  View details for DOI 10.1038/nbt.2038

  View details for Web of Science ID 000298038700023

  View details for PubMedID 22081019

  View details for PubMedCentralID PMC3237928

• The RootChip: An Integrated Microfluidic Chip for Plant Science PLANT CELL Grossmann, G., Guo, W., Ehrhardt, D. W., Frommer, W. B., Sit, R. V., Quake, S. R., Meier, M. 2011; 23 (12): 4234-4240

  Abstract

  Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.

  View details for DOI 10.1105/tpc.111.092577

  View details for Web of Science ID 000299677700010

  View details for PubMedID 22186371

• Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Weissman, I. L., Quake, S. R., Majeti, R. AMER SOC HEMATOLOGY. 2011: 4–4

  View details for Web of Science ID 000299597100005

• Partial Genome Assembly for a Candidate Division OP11 Single Cell from an Anoxic Spring (Zodletone Spring, Oklahoma) APPLIED AND ENVIRONMENTAL MICROBIOLOGY Youssef, N. H., Blainey, P. C., Quake, S. R., Elshahed, M. S. 2011; 77 (21): 7804-7814

  Abstract

  Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.

  View details for DOI 10.1128/AEM.06059-11

  View details for Web of Science ID 000296568200043

  View details for PubMedID 21908640

• Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding NATURE BIOTECHNOLOGY Lu, R., Neff, N. F., Quake, S. R., Weissman, I. L. 2011; 29 (10): 928-U229

  Abstract

  Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.

  View details for DOI 10.1038/nbt.1977

  View details for Web of Science ID 000296273000020

  View details for PubMedID 21964413

• The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line NATURE BIOTECHNOLOGY Xu, X., Nagarajan, H., Lewis, N. E., Pan, S., Cai, Z., Liu, X., Chen, W., Xie, M., Wang, W., Hammond, S., Andersen, M. R., Neff, N., Passarelli, B., Koh, W., Fan, H. C., Wang, J., Gui, Y., Lee, K. H., Betenbaugh, M. J., Quake, S. R., Famili, I., Palsson, B. O., Wang, J. 2011; 29 (8): 735-U131

  Abstract

  Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.

  View details for DOI 10.1038/nbt.1932

  View details for Web of Science ID 000293696500026

  View details for PubMedID 21804562

  View details for PubMedCentralID PMC3164356

• An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells PLOS ONE Glotzbach, J. P., Januszyk, M., Vial, I. N., Wong, V. W., Gelbard, A., Kalisky, T., Thangarajah, H., Longaker, M. T., Quake, S. R., Chu, G., Gurtner, G. C. 2011; 6 (6)

  Abstract

  An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

  View details for DOI 10.1371/journal.pone.0021211

  View details for Web of Science ID 000292033700046

  View details for PubMedID 21731674

  View details for PubMedCentralID PMC3120839

• Dynamic Chromatin Localization of Sirt6 Shapes Stress- and Aging-Related Transcriptional Networks PLOS GENETICS Kawahara, T. L., Rapicavoli, N. A., Wu, A. R., Qu, K., Quake, S. R., Chang, H. Y. 2011; 7 (6)

  Abstract

  The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-κB and regulate some NF-κB target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-α. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-κB subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-κB signaling in stress response and aging.

  View details for DOI 10.1371/journal.pgen.1002153

  View details for Web of Science ID 000292386300062

  View details for PubMedID 21738489

• Universal noninvasive detection of solid organ transplant rejection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Snyder, T. M., Khush, K. K., Valantine, H. A., Quake, S. R. 2011; 108 (15): 6229-6234

  Abstract

  It is challenging to monitor the health of transplanted organs, particularly with respect to rejection by the host immune system. Because transplanted organs have genomes that are distinct from the recipient's genome, we used high throughput shotgun sequencing to develop a universal noninvasive approach to monitoring organ health. We analyzed cell-free DNA circulating in the blood of heart transplant recipients and observed significantly increased levels of cell-free DNA from the donor genome at times when an endomyocardial biopsy independently established the presence of acute cellular rejection in these heart transplant recipients. Our results demonstrate that cell-free DNA can be used to detect an organ-specific signature that correlates with rejection, and this measurement can be made on any combination of donor and recipient. This noninvasive test holds promise for replacing the endomyocardial biopsy in heart transplant recipients and may be applicable to other solid organ transplants.

  View details for DOI 10.1073/pnas.1013924108

  View details for Web of Science ID 000289413600060

  View details for PubMedID 21444804

• MOLECULAR AUTOPSY FOR SUDDEN CARDIAC DEATH USING WHOLE GENOME SEQUENCING 60th Annual Scientific Session and Expo of the American-College-of-Cardiology (ACC) / I2 Summit / ACCF/Herman K. Gold Young Investigator's Award in Molecular and Cellular Cardiology Dewey, F. E., Wheeler, M. T., Cordero, S., Perez, M. V., Pavlovic, A., Pushkarev, D., Freeman, J. V., Quake, S. R., Ashley, E. A. ELSEVIER SCIENCE INC. 2011: E1159–E1159

  View details for Web of Science ID 000291695101162

• Single-cell genomics NATURE METHODS Kalisky, T., Quake, S. R. 2011; 8 (4): 311-314

  Abstract

  Methods for genomic analysis at single-cell resolution enable new understanding of complex biological phenomena. Single-cell techniques, ranging from flow cytometry and microfluidics to PCR and sequencing, are used to understand the cellular composition of complex tissues, find new microbial species and perform genome-wide haplotyping.

  View details for DOI 10.1038/nmeth0411-308

  View details for Web of Science ID 000288940300017

  View details for PubMedID 21451520

• Heart Transplants Are Genome Transplants: Universal Noninvasive Detection of Solid Organ Transplant Rejection 31st Annual Meeting and Scientific Sessions of the International-Society-for-Heart-and-Lung-Transplantation (ISHLT) Khush, K. K., Snyder, T. M., Quake, S. R., Valantine, H. A. ELSEVIER SCIENCE INC. 2011: S186–S187

  View details for Web of Science ID 000288924300552

• Determinism and stochasticity during maturation of the zebrafish antibody repertoire PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jiang, N., Weinstein, J. A., Penland, L., White, R. A., Fisher, D. S., Quake, S. R. 2011; 108 (13): 5348-5353

  Abstract

  It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity.

  View details for DOI 10.1073/pnas.1014277108

  View details for Web of Science ID 000288894800043

  View details for PubMedID 21393572

• Digital MDA for enumeration of total nucleic acid contamination NUCLEIC ACIDS RESEARCH Blainey, P. C., Quake, S. R. 2011; 39 (4)

  Abstract

  Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom φ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.

  View details for DOI 10.1093/nar/gkq1074

  View details for Web of Science ID 000288019400001

  View details for PubMedID 21071419

• High-throughput single-molecule optofluidic analysis NATURE METHODS Kim, S., Streets, A. M., Lin, R. R., Quake, S. R., Weiss, S., Majumdar, D. S. 2011; 8 (3): 242-U83

  Abstract

  We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

  View details for DOI 10.1038/NMETH.1569

  View details for Web of Science ID 000287734800016

  View details for PubMedID 21297618

• COMMUNITY CORNER Opening the Pandora's box of prenatal genetic testing NATURE MEDICINE Wright, C., Quake, S. R., Bianchi, D., Wald, N. J. 2011; 17 (3): 250-251

  View details for DOI 10.1038/nm0311-250

  View details for Web of Science ID 000288070000017

  View details for PubMedID 21383717

• Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis PLOS ONE Blainey, P. C., Mosier, A. C., Potanina, A., Francis, C. A., Quake, S. R. 2011; 6 (2)

  Abstract

  Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ∼1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ∼70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles.

  View details for DOI 10.1371/journal.pone.0016626

  View details for Web of Science ID 000287656600009

  View details for PubMedID 21364937

  View details for PubMedCentralID PMC3043068

• Mapping Virus-Host Protein Interactions using the Ping Microfluidics Platform 55th Annual Meeting of the Biophysical-Society Gerber, D., Einav, S., Quake, S. R. CELL PRESS. 2011: 368–68

  View details for Web of Science ID 000306288603130

• Digital PCR provides absolute quantitation of viral load for an occult RNA virus J Virol Methods White, R. A., Quake, S. R., Curr, K. 2011
• High-Throughput Single Molecule Optofluidic Analysis Nature Methods Kim, S., Streets, A. M., Lin, R. R., Quake, S. R., Weiss, S., Majumdar, D, S. 2011; 3 (8): 242-245
• Dynamic Chromatin Localization of Sirt6 Shapes Stress - and aging - related Transcriptional Networks PLoS Genet Kawahara, T. L., Rapicavoli, N. A., Wu, A. R., Qu, K., Quake, S. R., Chang, H, Y. 2011; 6 (7)
• Genomic Analysis at the Single-Cell Level ANNUAL REVIEW GENETICS, VOL 45 Kalisky, T., Blainey, P., Quake, S. R. 2011; 45: 431-445

  Abstract

  Studying complex biological systems such as a developing embryo, a tumor, or a microbial ecosystem often involves understanding the behavior and heterogeneity of the individual cells that constitute the system and their interactions. In this review, we discuss a variety of approaches to single-cell genomic analysis.

  View details for DOI 10.1146/annurev-genet-102209-163607

  View details for Web of Science ID 000299299600019

  View details for PubMedID 21942365

• Whole-genome molecular haplotyping of single cells NATURE BIOTECHNOLOGY Fan, H. C., Wang, J., Potanina, A., Quake, S. R. 2011; 29 (1): 51-?

  Abstract

  Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ∼99.8% accuracy for up to ∼96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.

  View details for DOI 10.1038/nbt.1739

  View details for Web of Science ID 000286048900020

  View details for PubMedID 21170043

• Biocompatibility and Reduced Drug Absorption of Sol-Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications ANALYTICAL CHEMISTRY Gomez-Sjoberg, R., Leyrat, A. A., Houseman, B. T., Shokat, K., Quake, S. R. 2010; 82 (21): 8954-8960

  View details for DOI 10.1021/ac101870s

  View details for Web of Science ID 000283531200031

• Variability in G-Protein-Coupled Signaling Studied with Microfluidic Devices BIOPHYSICAL JOURNAL Bao, X. R., Fraser, I. D., Wall, E. A., Quake, S. R., Simon, M. I. 2010; 99 (8): 2414-2422

  Abstract

  Different cells, even those that are genetically identical, can respond differently to identical stimuli, but the precise source of this variability remains obscure. To study this problem, we built a microfluidic experimental system which can track responses of individual cells across multiple stimulations. We used this system to determine that amplitude variation in G-protein-activated calcium release in RAW264.7 macrophages is generally extrinsic, i.e., they arise from long-lived variations between cells and not from stochastic activation of signaling components. In the case of responses linked to P2Y family purine receptors, we estimate that approximately one-third of the observed variability in calcium release is receptor-specific. We further demonstrate that the signaling apparatus downstream of P2Y6 receptor activation is moderately saturable. These observations will be useful in constructing and constraining single-cell models of G protein-coupled calcium dynamics.

  View details for DOI 10.1016/j.bpj.2010.08.043

  View details for Web of Science ID 000283412500007

  View details for PubMedID 20959081

• De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis NATURE BIOTECHNOLOGY Fordyce, P. M., Gerber, D., Tran, D., Zheng, J., Li, H., DeRisi, J. L., Quake, S. R. 2010; 28 (9): 970-976

  Abstract

  Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.

  View details for DOI 10.1038/nbt.1675

  View details for Web of Science ID 000281719100024

  View details for PubMedID 20802496

  View details for PubMedCentralID PMC2937095

• Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing CLINICAL CHEMISTRY Fan, H. C., Blumenfeld, Y. J., Chitkara, U., Hudgins, L., Quake, S. R. 2010; 56 (8): 1279-1286

  Abstract

  Noninvasive prenatal diagnosis with cell-free DNA in maternal plasma is challenging because only a small portion of the DNA sample is derived from the fetus. A few previous studies provided size-range estimates of maternal and fetal DNA, but direct measurement of the size distributions is difficult because of the small quantity of cell-free DNA.We used high-throughput paired-end sequencing to directly measure the size distributions of maternal and fetal DNA in cell-free maternal plasma collected from 3 typical diploid and 4 aneuploid male pregnancies. As a control, restriction fragments of lambda DNA were also sequenced.Cell-free DNA had a dominant peak at approximately 162 bp and a minor peak at approximately 340 bp. Chromosome Y sequences were rarely longer than 250 bp but were present in sizes of <150 bp at a larger proportion compared with the rest of the sequences. Selective analysis of the shortest fragments generally increased the fetal DNA fraction but did not necessarily increase the sensitivity of aneuploidy detection, owing to the reduction in the number of DNA molecules being counted. Restriction fragments of lambda DNA with sizes between 60 bp and 120 bp were preferentially sequenced, indicating that the shotgun sequencing work flow introduced a bias toward shorter fragments.Our results confirm that fetal DNA is shorter than maternal DNA. The enrichment of fetal DNA by size selection, however, may not provide a dramatic increase in sensitivity for assays that rely on length measurement in situ because of a trade-off between the fetal DNA fraction and the number of molecules being counted.

  View details for DOI 10.1373/clinchem.2010.144188

  View details for Web of Science ID 000280501400016

  View details for PubMedID 20558635

• Single-cell NF-kappa B dynamics reveal digital activation and analogue information processing NATURE Tay, S., Hughey, J. J., Lee, T. K., Lipniacki, T., Quake, S. R., Covert, M. W. 2010; 466 (7303): 267-U149

  Abstract

  Cells operate in dynamic environments using extraordinary communication capabilities that emerge from the interactions of genetic circuitry. The mammalian immune response is a striking example of the coordination of different cell types. Cell-to-cell communication is primarily mediated by signalling molecules that form spatiotemporal concentration gradients, requiring cells to respond to a wide range of signal intensities. Here we use high-throughput microfluidic cell culture and fluorescence microscopy, quantitative gene expression analysis and mathematical modelling to investigate how single mammalian cells respond to different concentrations of the signalling molecule tumour-necrosis factor (TNF)-alpha, and relay information to the gene expression programs by means of the transcription factor nuclear factor (NF)-kappaB. We measured NF-kappaB activity in thousands of live cells under TNF-alpha doses covering four orders of magnitude. We find, in contrast to population-level studies with bulk assays, that the activation is heterogeneous and is a digital process at the single-cell level with fewer cells responding at lower doses. Cells also encode a subtle set of analogue parameters to modulate the outcome; these parameters include NF-kappaB peak intensity, response time and number of oscillations. We developed a stochastic mathematical model that reproduces both the digital and analogue dynamics as well as most gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-alpha-induced NF-kappaB signalling in various types of cells. These results highlight the value of high-throughput quantitative measurements with single-cell resolution in understanding how biological systems operate.

  View details for DOI 10.1038/nature09145

  View details for Web of Science ID 000279580800043

  View details for PubMedID 20581820

  View details for PubMedCentralID PMC3105528

• Sensitivity of Noninvasive Prenatal Detection of Fetal Aneuploidy from Maternal Plasma Using Shotgun Sequencing Is Limited Only by Counting Statistics PLOS ONE Fan, H. C., Quake, S. R. 2010; 5 (5)

  Abstract

  We recently demonstrated noninvasive detection of fetal aneuploidy by shotgun sequencing cell-free DNA in maternal plasma using next-generation high throughput sequencer. However, GC bias introduced by the sequencer placed a practical limit on the sensitivity of aneuploidy detection. In this study, we describe a method to computationally remove GC bias in short read sequencing data by applying weight to each sequenced read based on local genomic GC content. We show that sensitivity is limited only by counting statistics and that sensitivity can be increased to arbitrary precision in sample containing arbitrarily small fraction of fetal DNA simply by sequencing more DNA molecules. High throughput shotgun sequencing of maternal plasma DNA should therefore enable noninvasive diagnosis of any type of fetal aneuploidy.

  View details for DOI 10.1371/journal.pone.0010439

  View details for Web of Science ID 000277240300015

  View details for PubMedID 20454671

• Clinical assessment incorporating a personal genome LANCET Ashley, E. A., Butte, A. J., Wheeler, M. T., Chen, R., Klein, T. E., Dewey, F. E., Dudley, J. T., Ormond, K. E., Pavlovic, A., Morgan, A. A., Pushkarev, D., Neff, N. F., Hudgins, L., Gong, L., Hodges, L. M., Berlin, D. S., Thorn, C. F., Sangkuhl, K., Hebert, J. M., Woon, M., Sagreiya, H., Whaley, R., Knowles, J. W., Chou, M. F., Thakuria, J. V., Rosenbaum, A. M., Zaranek, A. W., Church, G. M., Greely, H. T., Quake, S. R., Altman, R. B. 2010; 375 (9725): 1525-1535

  Abstract

  The cost of genomic information has fallen steeply, but the clinical translation of genetic risk estimates remains unclear. We aimed to undertake an integrated analysis of a complete human genome in a clinical context.We assessed a patient with a family history of vascular disease and early sudden death. Clinical assessment included analysis of this patient's full genome sequence, risk prediction for coronary artery disease, screening for causes of sudden cardiac death, and genetic counselling. Genetic analysis included the development of novel methods for the integration of whole genome and clinical risk. Disease and risk analysis focused on prediction of genetic risk of variants associated with mendelian disease, recognised drug responses, and pathogenicity for novel variants. We queried disease-specific mutation databases and pharmacogenomics databases to identify genes and mutations with known associations with disease and drug response. We estimated post-test probabilities of disease by applying likelihood ratios derived from integration of multiple common variants to age-appropriate and sex-appropriate pre-test probabilities. We also accounted for gene-environment interactions and conditionally dependent risks.Analysis of 2.6 million single nucleotide polymorphisms and 752 copy number variations showed increased genetic risk for myocardial infarction, type 2 diabetes, and some cancers. We discovered rare variants in three genes that are clinically associated with sudden cardiac death-TMEM43, DSP, and MYBPC3. A variant in LPA was consistent with a family history of coronary artery disease. The patient had a heterozygous null mutation in CYP2C19 suggesting probable clopidogrel resistance, several variants associated with a positive response to lipid-lowering therapy, and variants in CYP4F2 and VKORC1 that suggest he might have a low initial dosing requirement for warfarin. Many variants of uncertain importance were reported.Although challenges remain, our results suggest that whole-genome sequencing can yield useful and clinically relevant information for individual patients.National Institute of General Medical Sciences; National Heart, Lung And Blood Institute; National Human Genome Research Institute; Howard Hughes Medical Institute; National Library of Medicine, Lucile Packard Foundation for Children's Health; Hewlett Packard Foundation; Breetwor Family Foundation.

  View details for Web of Science ID 000277655100025

  View details for PubMedID 20435227

  View details for PubMedCentralID PMC2937184

• A microfluidic oligonucleotide synthesizer NUCLEIC ACIDS RESEARCH Lee, C., Snyder, T. M., Quake, S. R. 2010; 38 (8): 2514-2521

  Abstract

  De novo gene and genome synthesis enables the design of any sequence without the requirement of a pre-existing template as in traditional genetic engineering methods. The ability to mass produce synthetic genes holds great potential for biological research, but widespread availability of de novo DNA constructs is currently hampered by their high cost. In this work, we describe a microfluidic platform for parallel solid phase synthesis of oligonucleotides that can greatly reduce the cost of gene synthesis by reducing reagent consumption (by 100-fold) while maintaining a approximately 100 pmol synthesis scale so there is no need for amplification before assembly. Sixteen oligonucleotides were synthesized in parallel on this platform and then successfully used in a ligation-mediated assembly method to generate DNA constructs approximately 200 bp in length.

  View details for DOI 10.1093/nar/gkq092

  View details for Web of Science ID 000277238900002

  View details for PubMedID 20176572

• Ostwald Ripening of Clusters during Protein Crystallization PHYSICAL REVIEW LETTERS Streets, A. M., Quake, S. R. 2010; 104 (17)

  Abstract

  Contrary to classical nucleation theory, protein crystals can nucleate via a two-step process in which the molecular arrangement of the ordered solid phase is preceded by nucleation of a dense amorphous phase. We study the growth of these precrystalline clusters in lysozyme using a combination of dynamic light scattering, optical microscopy, and microfluidics. Clusters display Ostwald ripening growth kinetics but deviate from this trend after nucleation of the crystal phase. This behavior arises from the metastable relationship between clusters and the ordered solid and is explained numerically using a population balance model.

  View details for DOI 10.1103/PhysRevLett.104.178102

  View details for Web of Science ID 000277210600053

  View details for PubMedID 20482145

• Microtechnology in the Clinical Laboratory: Will It Solve Analytical Problems, and When Will It Make an Impact? CLINICAL CHEMISTRY Wilding, P., Verpoorte, S., Northrup, A., Yager, P., Quake, S., Landers, J. 2010; 56 (4): 508-514

  View details for DOI 10.1373/clinchem.2009.138719

  View details for Web of Science ID 000277059900004

  View details for PubMedID 20167692

• Single molecule localization on self-assembled, semi-ordered nanoparticle arrays Stoltenberg, R. M., Schwartz, J. J., Quake, S. R., Bao, Z. AMER CHEMICAL SOC. 2010

  View details for Web of Science ID 000208189304608

• Static control logic for microfluidic devices using pressure-gain valves NATURE PHYSICS Weaver, J. A., Melin, J., Stark, D., Quake, S. R., Horowitz, M. A. 2010; 6 (3): 218-223

  View details for DOI 10.1038/NPHYS1513

  View details for Web of Science ID 000275024000024

• Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability NATURE NANOTECHNOLOGY Schwartz, J. J., Stavrakis, s., Quake, S. R. 2010; 5 (2): 127-132

  Abstract

  Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room-temperature observations, largely owing to the use of objective lenses with high numerical aperture, brighter fluorophores and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single-molecule level both in vitro and in vivo. However, it is difficult to study systems and phenomena at temperatures above 37 degrees C, because the index-matching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens, and sustained exposure to high temperatures can cause the lens to fail. Here, we report that TiO(2) colloids with diameters of 2 microm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 degrees C when placed in immediate proximity to an emitting molecule. The optical system is completed by a low-numerical-aperture optic that can have a long working distance and an air interface, which allows the sample to be independently heated. Colloidal lenses were used for parallel imaging of surface-immobilized single fluorophores and for real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 degrees C. Fluorophores in close proximity to TiO(2) also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.

  View details for DOI 10.1038/NNANO.2009.452

  View details for Web of Science ID 000275058500013

  View details for PubMedID 20023643

• Biocompatibility and Reduced Drug Absorption of Sol-Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications. Analytical chemistry Gomez-Sjoberg, R., Leyrat, A. A., Houseman, B. T., Shokat, K., Quake, S. R. 2010

  Abstract

  Poly(dimethyl siloxane) (PDMS)-based microfluidic devices are now commonly used for a wide variety of biological experiments, including cell culture assays. However, the porous, hydrophobic polymer matrix of PDMS rapidly absorbs small hydrophobic molecules, including hormones and most small-molecule drugs. This makes it challenging to perform experiments that require such substances in PDMS microfluidic devices. This study presents evidence that a sol-gel treatment of PDMS that fills the polymer matrix with silica nanoparticles is effective at reducing the absorption of drugs into the material while preserving its biocompatibility, transparency, and oxygen permeability. We show that the absorption of two anticancer drugs, camptothecin and a kinase inhibitor, is reduced to such an extent that on-chip microfluidic cell culture experiments can recapitulate the results obtained off-chip.

  View details for DOI 10.1021/ac101870s

  View details for PubMedID 20936785

  View details for PubMedCentralID PMC3032040

• Niocompatibility and Reduced Drug Absorption of Sol-Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications Ana Chem. Bao, X. R., Fraser, I. D., Wall, E. A., Quake, S. R., Simon, M. I. 2010
• Clinical assessment incorportating a personal genome Lancet Ashley, E. A., Butte, A. J., Wheeler, M. T., Chen, R., Klein, T. E., Dewey, F. E., Quake, S. 2010; 9725 (375): 1525:1535
• Variability in G-Protein-Coupled Signaling Studied with Microfluidic Devices Biophysical Journal Bao, X. R., Fraser, I. D., Wall, E. A., Quake, S. R., Simon, M. I. 2010; 99: 2414–2422
• Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing. Nature. Tay, S., Hughey, J. J., Lee, T. K., Lipniacki, T., Quake, S. R., Covert, M, W. 2010; 7303 (466): 267-71
• Whole Genome Molecular Haplotyping of Single Cells Nature Biotechnology. Fan, H. C., Wang, J., Potanina, A., Quake, S, R. 2010
• Comprehensive maternal-fetal pharmacogenomics - a novel pharmacogenomic thumbprint 30th Annual Clinical Meeting of the Society-for-Maternal-Fetal-Medicine Blumenfeld, Y., Fan, C., Sangkuhl, K., Altman, R., El-Sayed, Y., Quake, S. MOSBY-ELSEVIER. 2009: S254–S254

  View details for Web of Science ID 000279559500695

• Single molecule measurement of the "speed limit'' of DNA polymerase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schwartz, J. J., Quake, S. R. 2009; 106 (48): 20294-20299

  Abstract

  Although DNA replication is often imagined as a regular and continuous process, the DNA polymerase enzyme is a complicated machine and can pause upon encountering physical and chemical barriers. We used single molecule measurements to make a detailed characterization of this behavior as a function of the template's secondary structure and the sequence context. Strand displacement replication through a DNA hairpin by single DNA polymerase molecules was measured in real time with near single base resolution and physiological concentrations of nucleotides. These data enabled the measurement of the intrinsic "speed limit" of DNA polymerase by separating the burst synthesis rate from pausing events. The strand displacement burst synthesis rate for Escherichia coli DNA Polymerase I (KF) was found to be an order of magnitude faster than the reported bulk strand displacement rate, a discrepancy that can be accounted for by to sequence specific pausing. The ability to follow trajectories of single molecules revealed that the burst synthesis rate is also highly stochastic and varies up to 50-fold from molecule to molecule. Surprisingly, our results allow a unified explanation of strand displacement and single strand primer extension synthesis rates.

  View details for DOI 10.1073/pnas.0907404106

  View details for Web of Science ID 000272254400031

  View details for PubMedID 19906998

• Digital PCR provides sensitive and absolute calibration for high throughput sequencing (vol 10, pg 541, 2009) BMC GENOMICS White, R. A., Blainey, P. C., Fan, H. C., Quake, S. R. 2009; 10

  View details for DOI 10.1186/1471-2164-10-541

  View details for Web of Science ID 000272358000001

• Experimental determination of the evolvability of a transcription factor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maerkl, S. J., Quake, S. R. 2009; 106 (44): 18650-18655

  Abstract

  Sequence-specific binding of a transcription factor to DNA is the central event in any transcriptional regulatory network. However, relatively little is known about the evolutionary plasticity of transcription factors. For example, the exact functional consequence of an amino acid substitution on the DNA-binding specificity of most transcription factors is currently not predictable. Furthermore, although the major structural families of transcription factors have been identified, the detailed DNA-binding repertoires within most families have not been characterized. We studied the sequence recognition code and evolvability of the basic helix-loop-helix transcription factor family by creating all possible 95 single-point mutations of five DNA-contacting residues of Max, a human helix-loop-helix transcription factor and measured the detailed DNA-binding repertoire of each mutant. Our results show that the sequence-specific repertoire of Max accessible through single-point mutations is extremely limited, and we are able to predict 92% of the naturally occurring diversity at these positions. All naturally occurring basic regions were also found to be accessible through functional intermediates. Finally, we observed a set of amino acids that are functional in vitro but are not found to be used naturally, indicating that functionality alone is not sufficient for selection.

  View details for DOI 10.1073/pnas.0907688106

  View details for Web of Science ID 000271429800044

  View details for PubMedID 19841254

• Single-molecule sequencing of an individual human genome NATURE BIOTECHNOLOGY Pushkarev, D., Neff, N. F., Quake, S. R. 2009; 27 (9): 847-U101

  Abstract

  Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to approximately 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28x average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.

  View details for DOI 10.1038/nbt.1561

  View details for Web of Science ID 000269751400027

  View details for PubMedID 19668243

• Highly parallel measurements of interaction kinetic constants with a microfabricated optomechanical device APPLIED PHYSICS LETTERS Bates, S. R., Quake, S. R. 2009; 95 (7)

  View details for DOI 10.1063/1.3211382

  View details for Web of Science ID 000269288300081

• Automated microfluidic chromatin immunoprecipitation from 2,000 cells LAB ON A CHIP Wu, A. R., Hiatt, J. B., Lu, R., Attema, J. L., Lobo, N. A., Weissman, I. L., Clarke, M. F., Quake, S. R. 2009; 9 (10): 1365-1370

  Abstract

  Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.

  View details for DOI 10.1039/b819648f

  View details for Web of Science ID 000268227400008

  View details for PubMedID 19417902

• High-Throughput Sequencing of the Zebrafish Antibody Repertoire SCIENCE Weinstein, J. A., Jiang, N., White, R. A., Fisher, D. S., Quake, S. R. 2009; 324 (5928): 807-810

  Abstract

  Despite tremendous progress in understanding the nature of the immune system, the full diversity of an organism's antibody repertoire is unknown. We used high-throughput sequencing of the variable domain of the antibody heavy chain from 14 zebrafish to analyze VDJ usage and antibody sequence. Zebrafish were found to use between 50 and 86% of all possible VDJ combinations and shared a similar frequency distribution, with some correlation of VDJ patterns between individuals. Zebrafish antibodies retained a few thousand unique heavy chains that also exhibited a shared frequency distribution. We found evidence of convergence, in which different individuals made the same antibody. This approach provides insight into the breadth of the expressed antibody repertoire and immunological diversity at the level of an individual organism.

  View details for DOI 10.1126/science.1170020

  View details for Web of Science ID 000265832400053

  View details for PubMedID 19423829

• Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy 29th Annual Meeting of the Society-for-Maternal-Fetal-Medicine Fan, H. C., Blumenfeld, Y. J., El-Sayed, Y. Y., Chueh, J., Quake, S. R. MOSBY-ELSEVIER. 2009

  Abstract

  The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy.Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes.Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested.Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

  View details for DOI 10.1016/j.ajog.2009.03.002

  View details for Web of Science ID 000265253800029

  View details for PubMedID 19375573

• Association of reactive oxygen species levels and radioresistance in cancer stem cells NATURE Diehn, M., Cho, R. W., Lobo, N. A., Kalisky, T., Dorie, M. J., Kulp, A. N., Qian, D., Lam, J. S., Ailles, L. E., Wong, M., Joshua, B., Kaplan, M. J., Wapnir, I., Dirbas, F. M., Somlo, G., Garberoglio, C., Paz, B., Shen, J., Lau, S. K., Quake, S. R., Brown, J. M., Weissman, I. L., Clarke, M. F. 2009; 458 (7239): 780-U123

  Abstract

  The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

  View details for DOI 10.1038/nature07733

  View details for Web of Science ID 000265193600045

  View details for PubMedID 19194462

  View details for PubMedCentralID PMC2778612

• In Vitro Embryo Culture in Defined, Sub-microliter Volumes DEVELOPMENTAL DYNAMICS Melin, J., Lee, A., Foygel, K., Leong, D. E., Quake, S. R., Yao, M. W. 2009; 238 (4): 950-955

  Abstract

  The high attrition rate of in vitro human embryo culture presents a major obstacle in the treatment of clinical infertility by in vitro fertilization (IVF). Physical and genetic requirements are not well understood for human or mouse preimplantation embryo development. Group culture is an established requirement for optimal embryo development in the mouse model. However, conventional microdrop culture limitations hinder investigations of the effects of physical parameters on in vitro embryo development. We report a microfluidics platform that enables embryo culture in precisely defined, sub-microliter volumes (5-500 nl) which cannot be investigated using conventional methods. Groups of two embryos per microfluidic well successfully developed to the blastocyst stage, at a rate of over 80%, which is comparable to those cultured in 20-microl microdrops. This system can be used to dissect physical requirements of in vitro single or group embryo culture, and be made highly parallel to increase experimental throughput.

  View details for DOI 10.1002/dvdy.21918

  View details for Web of Science ID 000264921200015

  View details for PubMedID 19301395

  View details for PubMedCentralID PMC2678198

• Digital PCR provides sensitive and absolute calibration for high throughput sequencing BMC GENOMICS White, R. A., Blainey, P. C., Fan, H. C., Quake, S. R. 2009; 10

  Abstract

  Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

  View details for DOI 10.1186/1471-2164-10-116

  View details for Web of Science ID 000265791900001

  View details for PubMedID 19298667

• An in vitro microfluidic approach to generating protein-interaction networks NATURE METHODS Gerber, D., Maerkl, S. J., Quake, S. R. 2009; 6 (1): 71-74

  Abstract

  We developed an in vitro protein expression and interaction analysis platform based on a highly parallel and sensitive microfluidic affinity assay, and used it for 14,792 on-chip experiments, which exhaustively measured the protein-protein interactions of 43 Streptococcus pneumoniae proteins in quadruplicate. The resulting network of 157 interactions was denser than expected based on known networks. Analysis of the network revealed previously undescribed physical interactions among members of some biochemical pathways.

  View details for DOI 10.1038/NMETH.1289

  View details for Web of Science ID 000262370200027

  View details for PubMedID 19098921

• DIGITAL PCR ENABLES RAPID PRENATAL DIAGNOSIS OF FETAL ANEUPLOIDY 29th Annual Meeting of the Society-for-Maternal-Fetal-Medicine Fan, C., Blumenfeld, Y., Chueh, J., El-Sayed, Y., Quake, S. MOSBY-ELSEVIER. 2008: S30–S30

  View details for DOI 10.1016/j.ajog.2008.09.089

  View details for Web of Science ID 000262205700066

• Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fan, H. C., Blumenfeld, Y. J., Chitkara, U., Hudgins, L., Quake, S. R. 2008; 105 (42): 16266-16271

  Abstract

  We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.

  View details for DOI 10.1073/pnas.0808319105

  View details for Web of Science ID 000260597400037

  View details for PubMedID 18838674

• PHARMACOLOGICAL INHIBITORS OF A NEW HEPATITIS C TARGET-RNA BINDING BY NS4B-DISCOVERED BY MICROFLUIDIC AFFINITY ANALYSIS 59th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases Einav, S., Gerber, D., Bryson, P. D., Sklan, E., Elazar, M., Maerkl, S., Dvory, H. S., Machlin, E., Gu, W., Quake, S., Glenn, J. S. WILEY-BLACKWELL. 2008: 356A–356A

  View details for Web of Science ID 000259757400105

• Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis NATURE BIOTECHNOLOGY Einav, S., Gerber, D., Bryson, P. D., Sklan, E. H., Elazar, M., Maerkl, S. J., Glenn, J. S., Quake, S. R. 2008; 26 (9): 1019-1027

  Abstract

  More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

  View details for DOI 10.1038/nbt.1490

  View details for Web of Science ID 000259074700025

  View details for PubMedID 18758449

• ANYL 350-Highly automated microfluidic system for cell biology 236th National Meeting of the American-Chemical-Society Gomez-Sjoeberg, R., Leyrat, A. A., Quake, S. R. AMER CHEMICAL SOC. 2008

  View details for Web of Science ID 000270256301171

• Single-molecule DNA sequencing of a viral genome SCIENCE Harris, T. D., Buzby, P. R., Babcock, H., Beer, E., Bowers, J., Braslavsky, I., Causey, M., Colonell, J., DiMeo, J., Efcavitch, J. W., Giladi, E., Gill, J., Healy, J., Jarosz, M., Lapen, D., Moulton, K., Quake, S. R., Steinmann, K., Thayer, E., Tyurina, A., Ward, R., Weiss, H., Xie, Z. 2008; 320 (5872): 106-109

  Abstract

  The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

  View details for DOI 10.1126/science.1150427

  View details for Web of Science ID 000254633000042

  View details for PubMedID 18388294

• A synthetic Escherichia coli predator-prey ecosystem MOLECULAR SYSTEMS BIOLOGY Balagadde, F. K., Song, H., Ozaki, J., Collins, C. H., Barnet, M., Arnold, F. H., Quake, S. R., You, L. 2008; 4

  Abstract

  We have constructed a synthetic ecosystem consisting of two Escherichia coli populations, which communicate bi-directionally through quorum sensing and regulate each other's gene expression and survival via engineered gene circuits. Our synthetic ecosystem resembles canonical predator-prey systems in terms of logic and dynamics. The predator cells kill the prey by inducing expression of a killer protein in the prey, while the prey rescue the predators by eliciting expression of an antidote protein in the predator. Extinction, coexistence and oscillatory dynamics of the predator and prey populations are possible depending on the operating conditions as experimentally validated by long-term culturing of the system in microchemostats. A simple mathematical model is developed to capture these system dynamics. Coherent interplay between experiments and mathematical analysis enables exploration of the dynamics of interacting populations in a predictable manner.

  View details for DOI 10.1038/msb.2008.24

  View details for Web of Science ID 000255551300007

  View details for PubMedID 18414488

• A microfluidic processor for gene expression profiling of single human embryonic stem cells LAB ON A CHIP Zhong, J. F., Chen, Y., Marcus, J. S., Scherer, A., Quake, S. R., Taylor, C. R., Weiner, L. P. 2008; 8 (1): 68-74

  Abstract

  The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.

  View details for DOI 10.1039/b712116d

  View details for Web of Science ID 000251771000017

  View details for PubMedID 18094763

• Transcriptional instability is not a universal attribute of aging AGING CELL Warren, L. A., Rossi, D. J., Schiebinger, G. R., Weissman, I. L., Kim, S. K., Quake, S. R. 2007; 6 (6): 775-782

  Abstract

  It has been proposed that cumulative somatic mutations contribute to the aging process by disrupting the transcriptional networks that regulate cell structure and function. Experimental support for this model emerged from a recent study of cardiomyocytes that showed a dramatic increase in the transcriptional heterogeneity of these long-lived postmitotic cells with age. To determine if regulatory instability is a hallmark of aging in renewing tissues, we evaluated gene expression noise in four hematopoietic cell types: stem cells, granulocytes, naïve B cells and naïve T cells. We used flow cytometry to purify phenotypically equivalent cells from young and old mice, and applied multiplexed quantitative reverse transcription-polymerase chain reaction to measure the copy number of six different mRNA transcripts in 324 individual cells. There was a trend toward higher transcript levels in cells isolated from old animals, but no significant increase in transcriptional heterogeneity with age was found in the surveyed populations. Flow cytometric analysis of membrane protein expression also indicated that cell-to-cell variability was unaffected by age. We conclude that large-scale regulatory destabilization is not a universal concomitant of aging, and may be of significance as an aging mechanism primarily in nonrenewing tissues.

  View details for DOI 10.1111/j.1474-9726.2007.00337.x

  View details for Web of Science ID 000250938400007

  View details for PubMedID 17925006

• Versatile, fully automated, microfluidic cell culture system ANALYTICAL CHEMISTRY Gomez-Sjoeberg, R., Leyrat, A. A., Pirone, D. M., Chen, C. S., Quake, S. R. 2007; 79 (22): 8557-8563

  Abstract

  There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.

  View details for DOI 10.1021/ac071311w

  View details for Web of Science ID 000250937500017

  View details for PubMedID 17953452

• Detection of aneuploidy with digital polymerase chain reaction ANALYTICAL CHEMISTRY Fan, H. C., Quake, S. R. 2007; 79 (19): 7576-7579

  Abstract

  The widespread use of genetic testing in high-risk pregnancies has created strong interest in rapid and accurate molecular diagnostics for common chromosomal aneuploidies. We show here that digital polymerase chain reaction (dPCR) can be used for accurate measurement of trisomy 21 (Down syndrome), the most common human aneuploidy. dPCR is generally applicable to any aneuploidy, does not depend on allelic distribution or gender, and is able to detect signals in the presence of mosaics or contaminating maternal DNA.

  View details for DOI 10.1021/ac0709394

  View details for Web of Science ID 000249871000052

  View details for PubMedID 17715994

• Nanoliter reactors improve multiple displacement amplification of genomes from single cells PLOS GENETICS Marcy, Y., Ishoey, T., Lasken, R. S., Stockwell, T. B., Walenz, B. P., Halpern, A. L., Beeson, K. Y., Goldberg, S. M., Quake, S. R. 2007; 3 (9): 1702-1708

  Abstract

  Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

  View details for DOI 10.1371/journal.pgen.0030155

  View details for Web of Science ID 000249767800013

  View details for PubMedID 17892324

• Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marcy, Y., Ouverney, C., Bik, E. M., Loesekann, T., Ivanova, N., Martin, H. G., Szeto, E., Platt, D., Hugenholtz, P., Relman, D. A., Quake, S. R. 2007; 104 (29): 11889-11894

  Abstract

  We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.

  View details for DOI 10.1073/pnas.0704662104

  View details for Web of Science ID 000248199200007

  View details for PubMedID 17620602

  View details for PubMedCentralID PMC1924555

• Lola regulates Drosophila olfactory projection neuron identity and targeting specificity NEURAL DEVELOPMENT Spletter, M. L., Liu, J., Liu, J., Su, H., Giniger, E., Komiyama, T., Quake, S., Luo, L. 2007; 2

  Abstract

  Precise connections of neural circuits can be specified by genetic programming. In the Drosophila olfactory system, projection neurons (PNs) send dendrites to single glomeruli in the antenna lobe (AL) based upon lineage and birth order and send axons with stereotyped terminations to higher olfactory centers. These decisions are likely specified by a PN-intrinsic transcriptional code that regulates the expression of cell-surface molecules to instruct wiring specificity.We find that the loss of longitudinals lacking (lola), which encodes a BTB-Zn-finger transcription factor with 20 predicted splice isoforms, results in wiring defects in both axons and dendrites of all lineages of PNs. RNA in situ hybridization and quantitative RT-PCR suggest that most if not all lola isoforms are expressed in all PNs, but different isoforms are expressed at widely varying levels. Overexpression of individual lola isoforms fails to rescue the lola null phenotypes and causes additional phenotypes. Loss of lola also results in ectopic expression of Gal4 drivers in multiple cell types and in the loss of transcription factor gene lim1 expression in ventral PNs.Our results indicate that lola is required for wiring of axons and dendrites of most PN classes, and suggest a need for its molecular diversity. Expression pattern changes of Gal4 drivers in lola-/- clones imply that lola normally represses the expression of these regulatory elements in a subset of the cells surrounding the AL. We propose that Lola functions as a general transcription factor that regulates the expression of multiple genes ultimately controlling PN identity and wiring specificity.

  View details for DOI 10.1186/1749-8104-2-14

  View details for Web of Science ID 000258981200001

  View details for PubMedID 17634136

  View details for PubMedCentralID PMC1947980

• At the interface of physics and biology BIOTECHNIQUES Quake, S. 2007; 43 (1): 19-19

  View details for Web of Science ID 000248207100005

  View details for PubMedID 17695250

• Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230 -> Asp obtained using microfluidic crystallization devices BIOCHEMISTRY Anderson, M. J., DeLaBarre, B., Raghunathan, A., Palsson, B. O., Brunger, A. T., Quake, S. R. 2007; 46 (19): 5722-5731

  Abstract

  The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 --> Asp (GKG230D) was determined to 2.0 A resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity.

  View details for DOI 10.1021/bi700096p

  View details for Web of Science ID 000246283600010

  View details for PubMedID 17441732

• Experimentally validated quantitative linear model for the device physics of elastomeric microfluidic valves JOURNAL OF APPLIED PHYSICS Kartalov, E. P., Scherer, A., Quake, S. R., Taylor, C. R., French Anderson, W. 2007; 101 (6)

  View details for DOI 10.1063/1.2511688

  View details for Web of Science ID 000245317700148

• A systems approach to measuring the binding energy landscapes of transcription factors SCIENCE Maerkl, S. J., Quake, S. R. 2007; 315 (5809): 233-237

  Abstract

  A major goal of systems biology is to predict the function of biological networks. Although network topologies have been successfully determined in many cases, the quantitative parameters governing these networks generally have not. Measuring affinities of molecular interactions in high-throughput format remains problematic, especially for transient and low-affinity interactions. We describe a high-throughput microfluidic platform that measures such properties on the basis of mechanical trapping of molecular interactions. With this platform we characterized DNA binding energy landscapes for four eukaryotic transcription factors; these landscapes were used to test basic assumptions about transcription factor binding and to predict their in vivo function.

  View details for DOI 10.1126/science.1131007

  View details for Web of Science ID 000243407400045

  View details for PubMedID 17218526

• Solvent resistant microfluidic DNA synthesizer LAB ON A CHIP Huang, Y., Castrataro, P., Lee, C., Quake, S. R. 2007; 7 (1): 24-26

  Abstract

  We fabricated a microfluidic DNA synthesizer out of perfluoropolyether (PFPE), an elastomer with excellent chemical compatibility which makes it possible to perform organic chemical reactions, and synthesized 20-mer oligonucleotides on chip.

  View details for DOI 10.1039/b613923j

  View details for Web of Science ID 000244616200005

  View details for PubMedID 17180201

• Lola regulates Drosophila olfactory projection meuron identity and targeting specificity Neural. Develop. Spletter, M. L., Liu, J., Liu, J., Su, H., Giniger, E., Komiyama, T., Quake, S. 2007; 14 (2)
• Crystal Structure of a Hyperactive Escherichia coli Gylcerol Kinase Mutand Gly230Asp Obtained Using Microfluidic Crystallization Devices Biochemistry Anderson, M. J., DeLaBarre, B., Raghunathan, A., Palsson, B. O., Brunger, A. T., Quake, S, R. 2007; 46: 5277-5731
• Dissecting biological "dark matter" with single cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth Marcy, Y., Ouverney, C., Blik, E. M., Losekann, T., Ivanova, N., Martin, H. G., Quake, S. 2007

  View details for DOI 10.1073/pnas.0704662104

• High density single molecule surface patterning with colloidal epitaxy Appl. Phys. Lett. Schwartz, J. J., Quake, S, R. 2007; 91: 83902
• The Digital Array Response Curve Preprint. Warren, L. A., Weinstein, J. A., Quake, S, R. 2007
• Microfluidic large-scale integration: The evolution of design rules for biological automation ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Melin, J., Quake, S. R. 2007; 36: 213-231

  Abstract

  Microfluidic large-scale integration (mLSI) refers to the development of microfluidic chips with thousands of integrated micromechanical valves and control components. This technology is utilized in many areas of biology and chemistry and is a candidate to replace today's conventional automation paradigm, which consists of fluid-handling robots. We review the basic development of mLSI and then discuss design principles of mLSI to assess the capabilities and limitations of the current state of the art and to facilitate the application of mLSI to areas of biology. Many design and practical issues, including economies of scale, parallelization strategies, multiplexing, and multistep biochemical processing, are discussed. Several microfluidic components used as building blocks to create effective, complex, and highly integrated microfluidic networks are also highlighted.

  View details for DOI 10.1146/annurev.biophys.36.040306.132646

  View details for Web of Science ID 000247773000011

  View details for PubMedID 17269901

• Microfluidic digital PCR enables multigene analysis of individual environmental bacteria SCIENCE Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. 2006; 314 (5804): 1464-1467

  Abstract

  Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.

  View details for DOI 10.1126/science.1131370

  View details for Web of Science ID 000242406100045

  View details for PubMedID 17138901

• Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Warren, L., Bryder, D., Weissman, I. L., Quake, S. R. 2006; 103 (47): 17807-17812

  Abstract

  We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.

  View details for DOI 10.1073/pnas.0608512103

  View details for Web of Science ID 000242464900042

  View details for PubMedID 17098862

• Phase knowledge enables rational screens for protein crystallization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, M. J., Hansen, C. L., Quake, S. R. 2006; 103 (45): 16746-16751

  Abstract

  We show that knowledge of the phase behavior of a protein allows one to create a rational screen that increases the success rate of crystallizing challenging proteins. The strategy is based on using microfluidics to perform large numbers of protein solubility experiments across many different chemical conditions to identify reagents for crystallization experiments. Phase diagrams were generated for the identified reagents and used to design customized crystallization screens for every protein. This strategy was applied with a 75% success rate to the crystallization of 12 diverse proteins, most of which failed to crystallize when using traditional techniques. The overall diffraction success rate was 33%, about double what was achieved with conventional automation in large-scale protein structure consortia. The higher diffraction success rates are achieved by designing customized crystallization screens using the phase behavior information for each target. The identification of reagents based on an understanding of protein solubility and the use of phase diagrams in the design of individualized crystallization screens therefore promotes high crystallization rates and the production of diffraction-quality crystals.

  View details for DOI 10.1073/pnas.0605293103

  View details for Web of Science ID 000241969500024

  View details for PubMedID 17075056

• Biological large scale integration Quake, S. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2006: S379–S379

  View details for Web of Science ID 000241506401456

• Developing optofluidic technology through the fusion of microfluidics and optics NATURE Psaltis, D., Quake, S. R., Yang, C. 2006; 442 (7101): 381-386

  Abstract

  We describe devices in which optics and fluidics are used synergistically to synthesize novel functionalities. Fluidic replacement or modification leads to reconfigurable optical systems, whereas the implementation of optics through the microfluidic toolkit gives highly compact and integrated devices. We categorize optofluidics according to three broad categories of interactions: fluid-solid interfaces, purely fluidic interfaces and colloidal suspensions. We describe examples of optofluidic devices in each category.

  View details for DOI 10.1038/nature05060

  View details for Web of Science ID 000239278900030

  View details for PubMedID 16871205

• The biological frontier of physics PHYSICS TODAY Phillips, R., Quake, S. R. 2006; 59 (5): 38-43

  View details for Web of Science ID 000237264200018

• Microfluidic single-cell mRNA isolation and analysis ANALYTICAL CHEMISTRY Marcus, J. S., Anderson, W. F., Quake, S. R. 2006; 78 (9): 3084-3089

  Abstract

  Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.

  View details for DOI 10.1021/ac0519460

  View details for Web of Science ID 000237456400031

  View details for PubMedID 16642997

• Microfluidic platform for production of 18F-radiolabeled PET imaging probes 231st National Meeting of the American-Chemical-Society Sui, G., Lee, C., Elizarov, A., Satyamurthy, N., Heath, J. R., Phelps, M. E., Quake, S. R., Tseng, H. AMER CHEMICAL SOC. 2006

  View details for Web of Science ID 000238125907256

• Anomalous vibrational dispersion in holographically trapped colloidal arrays PHYSICAL REVIEW LETTERS Polin, M., Grier, D. G., Quake, S. R. 2006; 96 (8)

  Abstract

  Colloidal spheres localized in an array of harmonic wells form a thermally excited, viscously damped dynamical system capable of supporting propagating elastic waves. Experimentally realized with micrometer-scale polystyrene spheres localized in a line of holographic optical traps, the hydrodynamically coupled array's behavior is quantitatively explained by a model based on the Oseen superposition approximation. The spheres' purely dissipative coupling is predicted to mediate a crossover to a regime of underdamped propagating elastic waves with uniformly negative group velocities that has yet to be verified experimentally.

  View details for DOI 10.1103/PhysRevLett.96.088101

  View details for Web of Science ID 000235736200074

  View details for PubMedID 16606228

• Enhanced signals and fast nucleic acid hybridization by microfluidic chaotic mixing ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Liu, J., Williams, B. A., Gwirtz, R. M., Wold, B. J., Quake, S. 2006; 45 (22): 3618-3623

  View details for DOI 10.1002/anie.200503830

  View details for Web of Science ID 000238068000009

  View details for PubMedID 16639763

• A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination J. Am. Chem. Soc. Hansen, C. L., Classen, S., Berger, J. M., Quake, S, R. 2006; 10 (128): 3142-3
• Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria Science. Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J, R. 2006; 314: 1464-67
• Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR Warren, L., Bryder, D., Weissman, I. L., Quake, S, R. 2006

  View details for DOI 10.1073/pnas.0608512103

• Parallel Picoliter rt-PCR Assays Using Microfluidics. Anal. Chem. Marcus, J. S., erson, W. F., Quake, S, R. 2006; 3 (78): 956-8
• Microfluidic Single-Cell mRNA Isolation and Analysis Anal. Chem. Marcus, J. S., erson, W. F., Quake, S, R. 2006

  View details for DOI 10.1021/ac0518506

• Molecular Biology on a Microfluidic Chip J. Cond. Matt. Phys . Hong, J. W., Chen, Y., Anderson, W. F., Quake, S, R. 2006; 18 (18): S691-S701
• Anomalous Vibrational Dispersion in Holographically Trapped Colloidal Arrays Phys. Rev. Lett. Polin, M., Grier, D. G., Quake, S, R. 2006; 8 (96): 88101
• Microfabricated rubber microscope using soft solid immersion lenses Appl. Phys. Lett. Gambin, Y., Legrand, O., Quake, S, R. 2006; 17 (88): 174102
• Fluorescence Near-Field Microscopy of DNA at Sub-10nm Resolution Phys. Rev. Lett. Ma, Z., Gerton, J. M., Wade, L. A., Quake, S, R. 2006; 26 (97): 260801
• Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation Thermophilic DNA Polymerases Nucleosides, Nucleotides & Nucleic Acids. Lacenere, C. J., Garg, N. K., Stoltz, B. M., Quake, S, R. 2006; 25: 9-15
• High-throughput multi-antigen microfluidic fluorescence immunoassays BIOTECHNIQUES Kartalov, E. P., Zhong, J. F., Scherer, A., Quake, S. R., Taylor, C. R., Anderson, W. F. 2006; 40 (1): 85-90

  Abstract

  Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.

  View details for DOI 10.2144/000112071

  View details for Web of Science ID 000234806900012

  View details for PubMedID 16454045

• Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS Lacenere, C. J., Garg, N. K., Stoltz, B. M., Quake, S. R. 2006; 25 (1): 9-15

  Abstract

  The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.

  View details for DOI 10.1080/15257770500377714

  View details for Web of Science ID 000234388200002

  View details for PubMedID 16440981

• Multistep synthesis of a radiolabeled imaging probe using integrated microfluidics SCIENCE Lee, C. C., Sui, G. D., Elizarov, A., Shu, C. Y., SHIN, Y. S., Dooley, A. N., Huang, J., Daridon, A., Wyatt, P., Stout, D., Kolb, H. C., Witte, O. N., Satyamurthy, N., Heath, J. R., Phelps, M. E., Quake, S. R., Tseng, H. R. 2005; 310 (5755): 1793-1796

  Abstract

  Microreactor technology has shown potential for optimizing synthetic efficiency, particularly in preparing sensitive compounds. We achieved the synthesis of an [(18)F]fluoride-radiolabeled molecular imaging probe, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), in an integrated microfluidic device. Five sequential processes-[18F]fluoride concentration, water evaporation, radiofluorination, solvent exchange, and hydrolytic deprotection-proceeded with high radio-chemical yield and purity and with shorter synthesis time relative to conventional automated synthesis. Multiple doses of [18F]FDG for positron emission tomography imaging studies in mice were prepared. These results, which constitute a proof of principle for automated multistep syntheses at the nanogram to microgram scale, could be generalized to a range of radiolabeled substrates.

  View details for DOI 10.1126/science.1118919

  View details for Web of Science ID 000234093600040

  View details for PubMedID 16357255

• Multi-step preparation of PET imaging probes in integrated microfluidic circuits 230th National Meeting of the American-Chemical-Society Sui, G., Lee, C., Satyamurthy, N., Heath, J. R., Phelps, M. E., Quake, S. R., Tseng, H. AMER CHEMICAL SOC. 2005: U2757–U2758

  View details for Web of Science ID 000236797305437

• Polydimethylsiloxane based microfluidic diode JOURNAL OF MICROMECHANICS AND MICROENGINEERING Adams, M. L., Johnston, M. L., Scherer, A., Quake, S. R. 2005; 15 (8): 1517-1521

  View details for DOI 10.1088/0960-1317/15/8/020

  View details for Web of Science ID 000231512500020

• Long-term monitoring of bacteria undergoing programmed population control in a microchemostat SCIENCE Balagadde, F. K., You, L. C., Hansen, C. L., Arnold, F. H., Quake, S. R. 2005; 309 (5731): 137-140

  Abstract

  Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of Escherichia coli carrying a synthetic "population control" circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours.

  View details for DOI 10.1126/science.1109173

  View details for Web of Science ID 000230212800077

  View details for PubMedID 15994559

• Programmed population control by cell-cell communication in microfluidic chemostats 49th Annual Meeting of the Biophysical-Society Balagadde, F. K., You, L. C., Arnold, F. H., Quake, S. R. CELL PRESS. 2005: 519A–519A

  View details for Web of Science ID 000226378502536

• Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science Balagadde, F. K., You, L., Hansen, C. L., Arnold, F. H., Quake, S, R. 2005; 5731 (309): 137-40
• Microfluidics: Fluid physics at the nanoliter scale Rev. Mod. Phys. Squires, T. M., Quake, S, R. 2005; 3 (7): 977-1026
• Multistep Synthesis of a Radiolabeled Imaging Probe Using Integrated Microfluidics Science. Lee, C. C., Sui, G., Elizarov, A., Shu, C. J., Shin, Y. S., Dooley, A. N., Quake, S. 2005: 1793-1796.
• Tip-enhanced fluorescence microscopy at 10 nanometer resolution PHYSICAL REVIEW LETTERS Gerton, J. M., Wade, L. A., Lessard, G. A., Ma, Z., Quake, S. R. 2004; 93 (18)

  Abstract

  We demonstrate unambiguously that the field enhancement near the apex of a laser-illuminated silicon tip decays according to a power law that is moderated by a single parameter characterizing the tip sharpness. Oscillating the probe in intermittent contact with a semiconductor nanocrystal strongly modulates the fluorescence excitation rate, providing robust optical contrast and enabling excellent background rejection. Laterally encoded demodulation yields images with <10 nm spatial resolution, consistent with independent measurements of tip sharpness.

  View details for DOI 10.1103/PhysRevLett.93.180801

  View details for Web of Science ID 000224799500014

  View details for PubMedID 15525147

• Systematic investigation of protein phase behavior with a microfluidic formulator PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hansen, C. L., Sommer, M. O., Quake, S. R. 2004; 101 (40): 14431-14436

  Abstract

  We demonstrated a microfluidic device for rapidly generating complex mixtures of 32 stock reagents in a 5-nl reactor. This "formulation chip" is fully automated and allows thousands of experiments to be performed in a single day with minimal reagent consumption. It was applied to systematically study the phase behavior of the protein xylanase over a large and complex chemical space. For each chemical formulation that demonstrated a pronounced effect on solubility, the protein phase behavior was completely mapped in the chip, generating a set of empirical phase diagrams. This ab initio phase information was used to devise a rational crystallization screen that resulted in 72-fold improvement in successful crystallization hits compared with conventional sparse matrix screens. This formulations tool allows a physics-based approach to protein crystallization that may prove useful in structural genomics efforts.

  View details for DOI 10.1073/pnas.0405847101

  View details for Web of Science ID 000224369600024

  View details for PubMedID 15452343

• Microfluidic device reads up to four consecutive base pairs in DNA sequencing-by-synthesis NUCLEIC ACIDS RESEARCH Kartalov, E. P., Quake, S. R. 2004; 32 (9): 2873-2879

  Abstract

  We have developed the first fully integrated microfluidic system for DNA sequencing-by-synthesis. Using this chip and fluorescence detection, we have reliably sequenced up to 4 consecutive bps. The described sequencer can be integrated with other microfluidic components on the same chip to produce true lab-on-a-chip technology. The surface chemistry that was designed to anchor the DNA to elastomeric microchannels is useful in a broad range of studies and applications.

  View details for DOI 10.1093/nar/gkh613

  View details for Web of Science ID 000221746900031

  View details for PubMedID 15155856

• A nanoliter-scale nucleic acid processor with parallel architecture NATURE BIOTECHNOLOGY Hong, J. W., Studer, V., Hang, G., Anderson, W. F., Quake, S. R. 2004; 22 (4): 435-439

  Abstract

  The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.

  View details for DOI 10.1038/nbt951

  View details for Web of Science ID 000220610100032

  View details for PubMedID 15024389

• A microfluidic rectifier: Anisotropic flow resistance at low Reynolds numbers PHYSICAL REVIEW LETTERS Groisman, A., Quake, S. R. 2004; 92 (9)

  Abstract

  It is one of the basic concepts of Newtonian fluid dynamics that at low Reynolds number (Re) the Navier-Stokes equation is linear and flows are reversible. In microfluidic devices, where Re is essentially always low, this implies that flow resistance in microchannels is isotropic. Here we present a microfluidic rectifier: a microscopic channel of a special shape whose flow resistance is strongly anisotropic, differing by up to a factor of 2 for opposite flow directions. Its nonlinear operation at arbitrary small Re is due to non-Newtonian elastic properties of the working fluid, which is a 0.01% aqueous solution of a high molecular weight polymer. The rectifier works as a dynamic valve and may find applications in microfluidic pumps and other integrated devices.

  View details for DOI 10.1103/PhysRevLett.92.094501

  View details for Web of Science ID 000220055400017

  View details for PubMedID 15089471

• Solvent-resistant photocurable liquid fluoropolymers for microfluidic device fabrication [corrected]. Journal of the American Chemical Society Rolland, J. P., van Dam, R. M., Schorzman, D. A., Quake, S. R., DeSimone, J. M. 2004; 126 (8): 2322-2323

  Abstract

  We report the first fabrication of a solvent-compatible microfluidic device based on photocurable "Liquid Teflon" materials. The materials are highly fluorinated functionalized perfluoropolyethers (PFPEs) that have liquidlike viscosities that can be cured into tough, highly durable elastomers that exhibit the remarkable chemical resistance of fluoropolymers such as Teflon. Poly(dimethylsiloxane) (PDMS) elastomers have rapidly become the material of choice for many recent microfluidic device applications. Despite the advantages of PDMS in relation to microfluidics technology, the material suffers from a serious drawback in that it swells in most organic solvents. The swelling of PDMS-based devices in organic solvents greatly disrupts the micrometer-sized features and makes it impossible for fluids to flow inside the channels. Our approach to this problem has been to replace PDMS with photocurable perfluoropolyethers. Device fabrication and valve actuation were accomplished using established procedures for PDMS devices. The additional advantage of photocuring allows fabrication time to be decreased from several hours to a matter of minutes. The PFPE-based device exhibited mechanical properties similar to those of Sylgard 184 before and after curing as well as remarkable resistance to organic solvents. This work has the potential to expand the field of microfluidics to many novel applications.

  View details for PubMedID 14982433

• Correlating AFM Probe Morphology to Image Resolution for Single-Wall Carbon Nanotube Tips Nano Lett. Wade, L. A., Shaprio, I. R., Ma, Z. 2004; 4 (4): 725-731
• Scaling properties of a low-actuation pressure microfluidic valve J. Appl. Phys. Studer, V., Hang, G., Pandolfi, A., Ortiz, M., Anderson, W. F., Quake, S, R. 2004; 1 (95): 393-398
• Solvent-resistant photocurable liquid fluoropolymers for microfluidic device fabrication J. Am. Chem. Soc. Rolland, J. P., Van Dam, R. M., Schorzman, D. A., Quake, S. R., DeSimone, J, M. 2004; 8 (126): 2322-3
• Nanometer-scale Fluorescence Resonance Optical Waveguides Nano Lett. Vyawahare, S., Eyal, S., Mathews, S., Quake, K., D. 2004; 6 (4): 1035-1039
• Behavior of complex knots in single DNA molecules PHYSICAL REVIEW LETTERS Bao, X. R., Lee, H. J., Quake, S. R. 2003; 91 (26)

  Abstract

  We used optical tweezers to tie individual DNA molecules in knots. Although these knots become highly localized under tension, they remain surprisingly mobile and undergo thermal diffusion with classical random walk statistics. The diffusion constants of knots with different complexities correlate with theoretical calculations of knot sizes. We show that this correlation can be explained by a simple hydrodynamical model of "self-reptation" of the knot along a polymer.

  View details for DOI 10.1103/PhysRevLett.91.265506

  View details for Web of Science ID 000187719300038

  View details for PubMedID 14754067

• Microfabricated fountain pens for high-density DNA arrays GENOME RESEARCH Reese, M. O., van Dam, R. M., Scherer, A., Quake, S. R. 2003; 13 (10): 2348-2352

  Abstract

  We used photolithographic microfabrication techniques to create very small stainless steel fountain pens that were installed in place of conventional pens on a microarray spotter. Because of the small feature size produced by the microfabricated pens, we were able to print arrays with up to 25,000 spots/cm2, significantly higher than can be achieved by other deposition methods. This feature density is sufficiently large that a standard microscope slide can contain multiple replicates of every gene in a complex organism such as a mouse or human. We tested carryover during array printing with dye solution, labeled DNA, and hybridized DNA, and we found it to be indistinguishable from background. Hybridization also showed good sequence specificity to printed oligonucleotides. In addition to improved slide capacity, the microfabrication process offers the possibility of low-cost mass-produced pens and the flexibility to include novel pen features that cannot be machined with conventional techniques.

  View details for DOI 10.1101/gr.623903

  View details for Web of Science ID 000185876400017

  View details for PubMedID 12975313

• Integrated nanoliter systems NATURE BIOTECHNOLOGY Hong, J. W., Quake, S. R. 2003; 21 (10): 1179-1183

  Abstract

  Microfluidic chip platforms for manipulating liquid volumes in the nanoliter range are slowly inching their way into mainstream genomic and proteomic research. The principal challenge faced by these technologies is the need for high-throughput processing of increasingly smaller volumes, with ever higher degrees of parallelization. Significant advances have been made over the past few years in addressing these needs through electrokinetic manipulation, vesicle encapsulation and mechanical valve approaches. These strategies allow levels of integration density and platform complexity that promise to make them into serious alternatives to current robotic systems.

  View details for DOI 10.1038/nbt871

  View details for Web of Science ID 000185647200029

  View details for PubMedID 14520403

• Microfluidics in structural biology: smaller, faster... better CURRENT OPINION IN STRUCTURAL BIOLOGY Hansen, C., Quake, S. R. 2003; 13 (5): 538-544

  Abstract

  Microfluidic technologies promise unprecedented savings in cost and time through the integration of complex chemical and biological assays on a microfabricated chip. Recent advances are making elements of this vision a reality, facilitating the first large-scale integration of microfluidic plumbing with biological assays. The power of miniaturization lies not only in achieving an economy of scale, but also in exploiting the unusual physics of fluid flow and mass transport on small length scales to realize precise and efficient assays that are not accessible with macroscopic tools. Diverse applications ranging from time-resolved studies of protein folding to highly efficient protein crystal growth suggest that microfluidics may become an indispensable tool in biology.

  View details for DOI 10.1016/j.sbi.2003.09.010

  View details for Web of Science ID 000186317900002

  View details for PubMedID 14568607

• Solving the "world-to-chip" interface problem with a microfluidic matrix ANALYTICAL CHEMISTRY Liu, J., Hansen, C., Quake, S. R. 2003; 75 (18): 4718-4723

  Abstract

  We report an effective solution to the macroscopic/microfluidic interface issue and demonstrate how microfluidics can achieve impressive economies of scale in reducing the complexity of pipetting operations. Using an N x N microfluidic matrix with N = 20, we performed N2 = 400 distinct PCR reactions with only 2N + 1 = 41 pipetting steps, compared with the 3N2 = 1200 steps required with conventional fluid handling. Each vertex of the matrix has a 3-nL reactor, and a single 2-microL aliquot of polymerase is amortized over all 400 independent reactions, thus dramatically reducing sample overhead and minimizing reagent consumption. Beyond PCR, the matrix chip provides a general method to perform chemical and biological experiments with precious reagents in a highly automated fashion.

  View details for DOI 10.1021/ac0346407

  View details for Web of Science ID 000185439300002

  View details for PubMedID 14674446

• Microfluidic memory and control devices SCIENCE Groisman, A., Enzelberger, M., Quake, S. R. 2003; 300 (5621): 955-958

  Abstract

  We demonstrate microscopic fluidic control and memory elements through the use of an aqueous viscoelastic polymer solution as a working fluid. By exploiting the fluid's non-Newtonian rheological properties, we were able to demonstrate both a flux stabilizer and a bistable flip-flop memory. These circuit elements are analogous to their solid-state electronic counterparts and could be used as components of control systems for integrated microfluidic devices. Such miniaturized fluidic circuits are insensitive to electromagnetic interference and may also find medical applications for implanted drug-delivery devices.

  View details for Web of Science ID 000182719800049

  View details for PubMedID 12738857

• Sequence information can be obtained from single DNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Braslavsky, I., Hebert, B., Kartalov, E., Quake, S. R. 2003; 100 (7): 3960-3964

  Abstract

  The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.

  View details for DOI 10.1073/pnas.0230489100

  View details for Web of Science ID 000182058400078

  View details for PubMedID 12651960

• Polyelectrolyte surface interface for single-molecule fluorescence studies of DNA polymerase BIOTECHNIQUES Kartalov, E. P., Unger, M. A., Quake, S. R. 2003; 34 (3): 505-?

  Abstract

  We report the use of polyelectrolyte multilayers in a stable robust surface chemistry for specific anchoring of DNA to glass. The nonspecific binding of fluorescently tagged nucleotides is suppressed down to the single-molecule level, and DNA polymerase is active on the anchored DNA template. This surface-chemistry platform can be used for single-molecule studies of DNA and DNA polymerase and may be more broadly applicable for other situations in which it is important to have specific biomolecular surface chemistry with extremely low nonspecific binding.

  View details for Web of Science ID 000181471500014

  View details for PubMedID 12661156

• Quantifying double-strand breaks and clustered damages in DNA by single-molecule laser fluorescence sizing BIOPHYSICAL JOURNAL Filippova, E. M., Monteleone, D. C., Trunk, J. G., Sutherland, B. M., Quake, S. R., Sutherland, J. C. 2003; 84 (2): 1281-1290

  Abstract

  Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels.

  View details for Web of Science ID 000183123700052

  View details for PubMedID 12547808

• Generation of uniform photonic balls by template-assisted colloidal crystallization Synth. Mat. Yi, G. R., Jeon, S. J., Thorsen, T., Manoharan, V. N., Quake, S. R., Pine, D. J. 2003; 3 (139): 803-806
• Microfluidics in structural biology: smaller, faster ... better. Curr Opin Struct Biol. Hansen, C., Quake, S, R. 2003; 5 (13): 538-44
• Single Molecule Fluorescence and Force Microscopy Employing Carbon Nanotubes Nanotech Wade, L. A., Shapiro, I., Ma, Z., Quake, S. R., Collier, C, P. 2003; 3: 317-320
• Generation of uniform colloidal assemblies in soft microfluidic devices Adv. Mat. Yi, G. R., Thorsen, T., Manoharan, V. N., Hwang, M. J., Jeon, S. J., Pine, D. J., Quake, S. 2003; 15 (15): 1300+
• A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hansen, C. L., Skordalakes, E., Berger, J. M., Quake, S. R. 2002; 99 (26): 16531-16536

  Abstract

  Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions.

  View details for Web of Science ID 000180101600008

  View details for PubMedID 12486223

• Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions JOURNAL OF NEUROSCIENCE Chiu, C. S., Jensen, K., Sokolova, I., Wang, D., Li, M., Deshpande, P., Davidson, N., Mody, I., Quick, M. W., Quake, S. R., Lester, H. A. 2002; 22 (23): 10251-10266

  Abstract

  GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

  View details for Web of Science ID 000179458100022

  View details for PubMedID 12451126

• Microfluidic large-scale integration SCIENCE Thorsen, T., Maerkl, S. J., Quake, S. R. 2002; 298 (5593): 580-584

  Abstract

  We developed high-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers. These fluidic devices are analogous to electronic integrated circuits fabricated using large-scale integration. A key component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs. We used these integrated microfluidic networks to construct the microfluidic analog of a comparator array and a microfluidic memory storage device whose behavior resembles random-access memory.

  View details for DOI 10.1126/science.1076996

  View details for Web of Science ID 000178634800035

  View details for PubMedID 12351675

• Significance and statistical errors in the analysis of DNA microarray data PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brody, J. P., Williams, B. A., Wold, B. J., Quake, S. R. 2002; 99 (20): 12975-12978

  Abstract

  DNA microarrays are important devices for high throughput measurements of gene expression, but no rational foundation has been established for understanding the sources of within-chip statistical error. We designed a specialized chip and protocol to investigate the distribution and magnitude of within-chip errors and discovered that, as expected from theoretical expectations, measurement errors follow a Lorentzian-like distribution, which explains the widely observed but unexplained ill-reproducibility in microarray data. Using this specially designed chip, we examined a data set of repeated measurements to extract estimates of the distribution and magnitude of statistical errors in DNA microarray measurements. Using the common "ratio of medians" method, we find that the measurements follow a Lorentzian-like distribution, which is problematic for subsequent analysis. We show that a method of analysis dubbed "median of ratios" yields a more Gaussian-like distribution of errors. Finally, we show that the bootstrap algorithm can be used to extract the best estimates of the error in the measurement. Quantifying the statistical error in such measurements has important applications for estimating significance levels, clustering algorithms, and process optimization.

  View details for DOI 10.1073/pnas.162468199

  View details for Web of Science ID 000178391700087

  View details for PubMedID 12235357

• Identification and confirmation of a module of coexpressed genes GENOME RESEARCH Thompson, H. G., Harris, J. W., Wold, B. J., Quake, S. R., Brody, J. P. 2002; 12 (10): 1517-1522

  Abstract

  We synthesize a large gene expression data set using dbEST and UniGene. We use guilt-by-association (GBA) to analyze this data set and identify coexpressed genes. One module, or group of genes, was found to be coexpressed mainly in tissue extracted from breast and ovarian cancers, but also found in tissue from lung cancers, brain cancers, and bone marrow. This module contains at least six members that are believed to be involved in either transcritional regulation (PDEF, H2AFO, NUCKS) or the ubiquitin proteasome pathway (PSMD7, SQSTM1, FLJ10111). We confirm these observations of coexpression by real-time RT-PCR analysis of mRNA extracted from four model breast epithelial cell lines.

  View details for DOI 10.1101/gr.418402

  View details for Web of Science ID 000178396400006

  View details for PubMedID 12368243

• Velocity-independent microfluidic flow cytometry ELECTROPHORESIS Eyal, S., Quake, S. R. 2002; 23 (16): 2653-2657

  Abstract

  Pressure-driven flow in microfluidic channels is characterized by a distribution of velocities. This distribution makes it difficult to implement conventional flow cytometry data analysis. We have demonstrated a method to measure velocity as an independent parameter when performing microfluidic flow cytometry. This method allows velocity-independent analysis of particles such as beads or cells, and allows flow cytometry analysis of extended objects, such as long DNA molecules. It allows accurate flow cytometry in transient and nonuniform flows. This general measurement method could be used in the future to measure the velocity of particles in a variety of existing microfluidic devices without the need for changes in their design.

  View details for Web of Science ID 000177888500013

  View details for PubMedID 12210169

• An integrated microfabricated cell sorter ANALYTICAL CHEMISTRY Fu, A. Y., Chou, H. P., Spence, C., Arnold, F. H., Quake, S. R. 2002; 74 (11): 2451-2457

  Abstract

  We have developed an integrated microfabricated cell sorter using multilayer soft lithography. This integrated cell sorter is incorporated with various microfluidic functionalities, including peristaltic pumps, dampers, switch valves, and input and output wells, to perform cell sorting in a coordinated and automated fashion. The active volume of an actuated valve on this integrated cell sorter can be as small as 1 pL, and the volume of optical interrogation is approximately 100 fL. Different algorithms of cell manipulation, including cell trapping, were implemented in these devices. We have also demonstrated sorting and recovery of Escherichia coli cells on the chip.

  View details for DOI 10.1021/ac0255330

  View details for Web of Science ID 000175995400008

  View details for PubMedID 12069222

• A nanoliter rotary device for polymerase chain reaction ELECTROPHORESIS Liu, J., Enzelberger, M., Quake, S. 2002; 23 (10): 1531-1536

  Abstract

  Polymerase chain reaction (PCR) has revolutionized a variety of assays in biotechnology. The ability to implement PCR in disposable and reliable microfluidic chips will facilitate its use in applications such as rapid medical diagnostics, food control testing, and biological weapons detection. We fabricated a microfluidic chip with integrated heaters and plumbing in which various forms of PCR have been successfully demonstrated. The device uses only 12 nL of sample, one of the smallest sample volumes demonstrated to date. Minimizing the sample volume allows low power consumption, reduced reagent costs, and ultimately more rapid thermal cycling.

  View details for Web of Science ID 000175992800021

  View details for PubMedID 12116165

• Gene expression analysis with universal n-mer arrays GENOME RESEARCH van Dam, R. M., Quake, S. R. 2002; 12 (1): 145-152

  Abstract

  Gene expression profiling is one of the many applications that have benefited from the massively parallel nucleic acid detection capability of DNA microarrays. Current expression arrays, however, are expensive and inflexible. They are custom-designed for each organism and they do not offer the possibility of incorporating updated genomic information without production of a new chip. One possible solution is the development of a universal chip, consisting of all 4n possible DNA sequences of length n. Studying different organisms or new genes would simply require modifications to the hybridization pattern analysis software. The key problem is to find a value of n that is large enough to afford sufficient specificity, yet is small enough for practical fabrication and readout. We developed an analytical model, supported by computer-assisted calculation with yeast and mouse transcript data, to argue that it is both practical and useful to fabricate n-mer arrays with 10 < or = n < or = 16.

  View details for Web of Science ID 000173064900015

  View details for PubMedID 11779839

• Microfluidic Large Scale Integration Science Thorsen, T., Maerkl, S. J., Quake, S. R. 2002; 298: 580-584
• Fundamental approach for optoelectronic and microfluidic integration for miniaturizing spectroscopic devices Adams, M., DeRose, G., Quake, S. R., Scherer, A, . 2002
• Velocity Independent Microfluidic Flow Cytometry Electrophoresis Eyal, S., Quake, S. R. 2002; 23: 2653-2657
• Dynamic pattern formation in a vesicle-generating microfluidic device PHYSICAL REVIEW LETTERS Thorsen, T., Roberts, R. W., Arnold, F. H., Quake, S. R. 2001; 86 (18): 4163-4166

  Abstract

  Spatiotemporal pattern formation occurs in a variety of nonequilibrium physical and chemical systems. Here we show that a microfluidic device designed to produce reverse micelles can generate complex, ordered patterns as it is continuously operated far from thermodynamic equilibrium. Flow in a microfluidic system is usually simple-viscous effects dominate and the low Reynolds number leads to laminar flow. Self-assembly of the vesicles into patterns depends on channel geometry and relative fluid pressures, enabling the production of motifs ranging from monodisperse droplets to helices and ribbons.

  View details for Web of Science ID 000168525900060

  View details for PubMedID 11328121

• Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems JOURNAL OF NEUROSCIENCE METHODS Chiu, C. S., Kartalov, E., Unger, M., Quake, S., Lester, H. A. 2001; 105 (1): 55-63

  Abstract

  Quantitative aspects of synaptic transmission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins. To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify histidine-tagged GFP molecules (His6-GFP) bound to Ni-NTA moieties on transparent beads (80-120 microm diameter) over a density range comprising nearly four orders of magnitude (to 30000 GFP/microm2). The procedures employ commonly available Hg lamps, fluorescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities; (2) known numbers of His6-GFP molecules are coupled quantitatively to the beads. Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with root mean square (rms) deviation of 11-21% over this range. These satisfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities > approximately 1 W/cm2. If a suitable GFP-tagged protein can be chosen and incorporated into a 'knock-in' animal, the density of the protein can be measured with an absolute accuracy on the order of 20%.

  View details for Web of Science ID 000166873400006

  View details for PubMedID 11166366

• A Microfabricated Rotary Pump Biomedical Microdevices Chou, H. P., Unger, M. A., Quake, S. R. 2001; 3: 323-330
• Single molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use in "knock-in" animals and other expression systems Journal of Neuroscience Methods Chiu, C. S., Kartalov, E., Unger, M. A., Quake, S. R., Lester, H. 2001; 105: 55-63
• From micro- to nanofabrication with soft materials SCIENCE Quake, S. R., Scherer, A. 2000; 290 (5496): 1536-1540

  Abstract

  Soft materials are finding applications in areas ranging from microfluidic device technology to nanofabrication. We review recent work in these areas, discuss the motivation for device fabrication with soft materials, and describe applications of soft materials. In particular, we discuss active microfluidic devices for cell sorting and biochemical assays, replication-molded optics with subdiffraction limit features, and nanometer-scale resonators and wires formed from single-molecule DNA templates as examples of how the special properties of soft materials address outstanding problems in device fabrication.

  View details for Web of Science ID 000165446200039

  View details for PubMedID 11090344

• Femtonewton force spectroscopy of single extended DNA molecules PHYSICAL REVIEW LETTERS Meiners, J. C., Quake, S. R. 2000; 84 (21): 5014-5017

  Abstract

  We studied the thermal fluctuations of single DNA molecules with a novel optical tweezer based force spectroscopy technique. This technique combines femtonewton sensitivity with millisecond time resolution, surpassing the sensitivity of previous force measurements in aqueous solution with comparable bandwidth by a hundredfold. Our data resolve long-standing questions concerning internal hydrodynamics of the polymer and anisotropy in the molecular relaxation times and friction coefficients. The dynamics at high extension show interesting nonlinear behavior.

  View details for Web of Science ID 000087114400058

  View details for PubMedID 10990855

• Monolithic microfabricated valves and pumps by multilayer soft lithography SCIENCE Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. 2000; 288 (5463): 113-116

  Abstract

  Soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate stamps and microfluidic channels. We describe here an extension to the soft lithography paradigm, multilayer soft lithography, with which devices consisting of multiple layers may be fabricated from soft materials. We used this technique to build active microfluidic systems containing on-off valves, switching valves, and pumps entirely out of elastomer. The softness of these materials allows the device areas to be reduced by more than two orders of magnitude compared with silicon-based devices. The other advantages of soft lithography, such as rapid prototyping, ease of fabrication, and biocompatibility, are retained.

  View details for Web of Science ID 000086387700044

  View details for PubMedID 10753110

• An Apertureless Near-Field Microscope for Fluorescence Imaging Applied Physics Letters Yang, T. J., Lessard, G. A., Quake, S. R. 2000; 76: 378-380
• Integrated Elastomer Fluidic Lab on a Chip - Surface Patterning and DNA diagnostics Chou, H. P., Unger, M. A., Scherer, A., Quake, S. R. 2000
• From Micro to Nano Fabrication with Soft Materials Science Quake, S. R., Scherer, A. 2000; 290: 1536-40
• Fluorescence apertureless near-field microscope: a step towards imaging information in DNA Quake, S. R., Yang, T. J., Lessard, G. A., Unger, A., Kartalov, E. 2000
• Single-molecule fluorescence observed with mercury lamp illumination BIOTECHNIQUES Unger, M., Kartalov, E., Chiu, C. S., Lester, H. A., Quake, S. R. 1999; 27 (5): 1008-?

  Abstract

  We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.

  View details for Web of Science ID 000087218400022

  View details for PubMedID 10572649

• A microfabricated fluorescence-activated cell sorter NATURE BIOTECHNOLOGY Fu, A. Y., Spence, C., Scherer, A., Arnold, F. H., Quake, S. R. 1999; 17 (11): 1109-1111

  Abstract

  We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (microFACS) for sorting various biological entities. Compared with conventional FACS machines, the microFACS provides higher sensitivity, no cross-contamination, and lower cost. We have used microFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors. Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E. coli cells and shown that the bacteria are viable after extraction from the sorting device. These sorters can function as stand-alone devices or as components of an integrated microanalytical chip.

  View details for Web of Science ID 000083428000030

  View details for PubMedID 10545919

• A microfabricated device for sizing and sorting DNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chou, H. P., Spence, C., Scherer, A., Quake, S. 1999; 96 (1): 11-13

  Abstract

  We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp.

  View details for Web of Science ID 000078004400004

  View details for PubMedID 9874762

• Single Molecule Fluorescence Observed with Mercury Lamp Illumination Biotechniques Unger, M. A., Kartalov, E., Chiu, C. S., Lester, H., Quake, S. R. 1999; 27: 1008-1013
• A Direct Measurement of Hydrodynamic Cross Correlations Between Two Particles in an External Potential Phys Rev Lett Meiners, J. C., Quake, S. R. 1999; 82: 2211-2214
• A Self-Assembled Microlensing Rotational Probe Appl Phys Lett. Brody, J. P., Quake, S. R. 1999; 74 (1)
• A Scanning Apertureless Fluorescence Microscope Lessard, G., Yang, T. J., Barritault, P., Quake, S. R. 1999
• Dynamic Properties of an Extended Polymer in Solution Phys Rev Lett Hatfield, J. W., Quake, S. R. 1999; 82: 3548-51
• Disposable Microdevices for DNA Analysis and Cell Sorting Quake, S. R., Chou, H. P., Spence, C., Fu, A. Y., Scherer, A. 1998
• Microfabricated devices for sizing DNA and sorting cells Chou, H. P., Spence, C., Scherer, A., Quake, S. R. 1998
• The dynamics of partially extended single molecules of DNA NATURE Quake, S. R., Babcock, H., Chu, S. 1997; 388 (6638): 151-154

  Abstract

  The behaviour of an isolated polymer floating in a solvent forms the basis of our understanding of polymer dynamics. Classical theories describe the motion of a polymer with linear equations of motion, which yield a set of 'normal modes', analogous to the fundamental frequency and the harmonics of a vibrating violin string. But hydrodynamic interactions make polymer dynamics inherently nonlinear, and the linearizing approximations required for the normal-mode picture have therefore been questioned. Here we test the normal-mode theory by measuring the fluctuations of single molecules of DNA held in a partially extended state with optical tweezers. We find that the motion of the DNA can be described by linearly independent normal modes, and we have experimentally determined the eigenstates of the system. Furthermore, we show that the spectrum of relaxation times obeys a power law.

  View details for Web of Science ID A1997XK10900043

  View details for PubMedID 9217154

• Fast Monte Carlo algorithms for knotted polymers Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics. Quake, S, R. 1997; 1 (52): 1176-1180
• The Zimm model applied to extended single polymers J. Chem Phys. Quake, S, R. 1997; 5 (101): 4307-4311
• Topological effects of knots in polymers Phys. Rev. Lett. Quake, S, R. 1997; 24 (73): 3317-3320
• POLYMER EXPERIMENTS ON SINGLE DNA-MOLECULES Chu, S., Quake, S., Perkins, T., Smith, D. AMER CHEMICAL SOC. 1995: 374-PHYS

  View details for Web of Science ID A1995QP23301127

• Fast Monte Carlo algorithms for knotted polymers. Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics Quake, S. R. 1995; 52 (1): 1176–80

  View details for PubMedID 9963522

• RELAXATION OF A SINGLE DNA MOLECULE OBSERVED BY OPTICAL MICROSCOPY SCIENCE Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. 1994; 264 (5160): 822-826

  Abstract

  Single molecules of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The molecules, attached by one end to a 1-micrometer bead, were manipulated in an aqueous solution with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (tau) increased with length (L) as tau approximately L 1.68 +/- 0.10. A rescaling analysis showed that most of the relaxation curves had a universal shape and their characteristic times (lambda t) increased as lambda t approximately L 1.65 +/- 0.13. These results are in qualitative agreement with the theoretical prediction of dynamical scaling.

  View details for Web of Science ID A1994NJ94900026

  View details for PubMedID 8171336

• Topological effects of knots in polymers. Physical review letters Quake, S. R. 1994; 73 (24): 3317–20

  View details for PubMedID 10057346

• Single molecule measurement of the "speed limit" of DNA polymerase. Schwartz, J. J., Quake, S. R. 2,009