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COMMENT: See below for complete list of researchers available for comment

Rapid DNA probe could lead to low-cost tests for genetic diseases

STANFORD -- An international team of scientists has developed a chemical probe that rapidly detects single-gene mutations in human DNA. The researchers say that their method, reported in scientific correspondence to the March 21st issue of the journal Nature, could lead to a practical, low-cost method to screen for any genetic disease where the mutations that cause the disease are known, and occur at no more than a few positions in the DNA.

The probe takes advantage of the unique properties of peptide nucleic acid, or PNA, a laboratory-produced mimic of deoxyribonucleic acid, or DNA. Strands of the mimic molecule bind more tightly to DNA than DNA does itself. The researchers used that binding property to isolate fragments of DNA containing three different mutations of the gene responsible for cystic fibrosis, using a laboratory separation technique known as free-solution capillary electrophoresis.

"With this method we can find single base-pair substitutions and base-pair deletions," said Stanford chemistry Professor Richard Zare, a co-author of the Nature letter. "That means we now have a genetic probe."

The discovery unfolded over the course of two years of experiments at Stanford and several years of cooperation and conversation - often by e-mail - among scientists in five different countries: chemists Christina Carlsson, Mats Jonsson and Bengt Nordén of Chalmers University of Technology in Gothenburg, Sweden; chemists Maria Dulay and Richard Zare of Stanford; theoretical polymer physicist Jaan Noolandi of the Xerox Research Center of Canada in Mississauga; biochemist Peter Nielsen of the Panum Institute in Copenhagen, Denmark; and geneticists Lap-Chee Tsui and Julian Zielenski of the Hospital for Sick Children in Toronto, Canada.

Heat-tested match-ups

A single strand of peptide nucleic acid contains the same four amino acid bases that form the rungs in the double-stranded ladder of DNA: adenine, cytosine, thymine and guanine, or A-C-T-G. When the two halves of the ladder are split apart, PNA binds to DNA just as a strand of DNA would, with A always binding to T and G always binding to C to form base-pairs.

However, because PNA has a neutrally charged backbone instead of the negatively charged sugar phosphate backbone of DNA, it binds more tightly than a normal strand of DNA. It is much more difficult to separate the two by heating - the normal method used to separate two strands of genetic material.

The researchers found that when all the bases match up in the PNA-DNA hybrid, the temperature must be high before the two will separate. When only some of the bases match, the PNA and DNA will separate at a lower temperature.

To test this on human genes, the research team made a batch with many copies of single-stranded PNA, 15 bases long, to exactly mimic a known sequence of DNA. For their target sequence, they chose the section of the cystic fibrosis gene that contains the three
base-pair deletion that is responsible for most cases of the disease.

When they combined the CF-mimic PNA in a dish with many copies of human DNA from the same part of the CF gene, the molecules bound to one another. When they heated the solution and measured the results with capillary electrophoresis, they found that normal strands of the target DNA, with no mutations, bound completely to form a PNA-DNA hybrid.

Those strands of target DNA that contained the three base-pair CF deletion did not form a perfect PNA-DNA match. They began to separate at a lower temperature. The researchers had successfully detected a human genetic mutation.

"What is exciting," said Noolandi, "is that Carlsson and Dulay were also able to detect a single base substitution in the cystic fibrosis DNA. As far as I know, there is no other simple technique that allows you to do this."

A practical potential

More work needs to be done to test the value of this method as a genetic probe. Zare said that in practice, it might be possible to design a probe that would show how well the DNA and PNA match using some easily visible method. In the future, geneticists may have PNA probe libraries available to test for multiple mutations on one or many genes. As the research team wrote in their letter to Nature, "a PNA probe library . . . might form the basis for a universal screening strategy for any genetic disease with a known spectrum of mutations."

While many companies are working to develop gene tests, so far no rapid, low-cost tests are commercially available. Approximately 100 genes have so far been isolated with mutations linked to diseases such as cystic fibrosis, Huntington's disease and breast cancer. However, these links are complicated - cystic fibrosis may result from any of several hundred mutations on one gene.

"Genetic screening is going to be very complex unless we start to think about ways to make it simple," Noolandi said. "There will have to be tests not just for one specific mutation but for the whole range. Techniques like the PNA probe will make gene testing applicable to very large numbers of samples."

His colleagues credit Noolandi with being the intellectual catalyst who made the discovery possible. He began working on a related concept during a 1993 sabbatical at Stanford, working with postdoctoral fellow Maria Dulay in Zare's lab. They were aiming for a fast new way to isolate DNA, using a rapid molecule-sorting method called free-solution capillary electrophoresis.

On a visit to Sweden in 1994, Noolandi met with capillary electrophoresis researchers Nordén and Jonsson and suggested a collaboration that led Swedish graduate student Christina Carlsson to spend the summer working at Stanford with Dulay. At Noolandi's suggestion, they added a new aspect to the DNA isolation experiments: the mimic molecule PNA, which had recently been developed by Nielsen in Denmark.

Once it was clear that PNA would work very specifically to isolate and mark a known genetic mutation, Noolandi encouraged Tsui and Zielenski to join the collaboration. Tsui led the research team that first isolated the cystic fibrosis gene in 1989, and the Toronto researchers contributed samples of DNA from people with known CF mutations, as well as control samples known to have no mutations on that gene. Those samples were used to test the value of PNA as a genetic probe.

"This shows something about science," said Noolandi. "It's important not to get too bureaucratic at a stage like this. We could have waited for years for funding to come through from international agencies, and I think that would have killed the idea. This took trust that a crazy idea might be worth trying."

Zare noted another unique aspect of the intercontinental collaboration: "This is science conducted by e-mail and parcel post," he said. "Throughout all of this collaboration, never has the entire research team been together in the same room Many of us could pass each other on the street and not know it. The Internet is changing how science is done."



Researchers who may be available for comment include:
Richard Zare, Chemistry Dept., Stanford University: Zare is traveling March 21-31;
leave messages with Liz Edlund (415) 723-4329

Maria Dulay, Chemistry Dept., Stanford University: (415) 723-4318

fax: (415) 725-0259 e-mail:

Jaan Noolandi, Xerox Research Center of Canada: Noolandi is traveling March 25-31
will call in for messages at (905) 823-7091 ext 307 fax: (905) 822-7022
or e-mail at

Mats Jonsson, Chalmers University of Technology, Gothenburg, Sweden
phone: +46 31 772 3053 fax: +46 31 772 3858

Bengt Nordén, Chalmers University of Technology, Gothenburg, Sweden
phone: +46 31 772 3041 fax: +46 31 772 3858

Christina Carlsson, Chalmers University of Technology, Gothenburg, Sweden
phone: +46 31 772 3045 fax: +46 31 772 3858

Graphic by Maria Dulay


Simplified drawing shows how bases of the DNA mimic, peptide nucleic acid (PNA) bind to the bases of a single strand of DNA to form a double-stranded PNA-DNA hybrid. The black circles along the spine of DNA represent phosphates, which carry a negative charge; because PNA's spine carries no charge, its bases bind more tightly than DNA to DNA.

When heated, the PNA-DNA matched bases stay locked longer than others. When bases are not perfectly matched, they break apart at a lower temperature - thus sensitive detectors can find a single base mismatch.


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