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Gene-Finding Method May Speed Search For Disease-Causing Mutant Forms

A new research tool -- developed at the University of Southern California by a collaboration between mathematicians and laboratory molecular biologists --promises to speed dramatically the hunt for disease-causing genes.

Coupled with the use of DNA chips, the ExonPCR technique created by gene researcher Norman Arnheim, Ph.D., and computational biologist Pavel A. Pevzner, Ph.D., allows speedy reconstruction of the way genes are written on human chromosomes.

The problem addressed is posed by the curious and still unexplained way in which the genetic message is concealed on chromosomes within a huge volume of nonmessage-bearing "junk" DNA.

Genes consist of a length of DNA roughly 10,000 "letters" long in the four-letter A-C-G-T chemical alphabet of heredity.  Unfortunately for researchers, the message is not written in a single, continuous stretch, but divided up into as many as 50 segments called "exons," spaced out over a stretch of chromosome DNA 1 million letters long or even longer.

Such exons are fused into a continuous message when the gene goes into actual use, creating what is called an "mRNA" or "cDNA" form of the gene.

New DNA chip techniques have recently made it feasible to zero in on all cDNA from large regions on chromosomes suspected of carrying a disease-causing gene.  This  enables researchers to isolate a group of suspect genes for further testing.

But knowledge of the exon borders from the original chromosomal form of these genes is still required to quickly compare genes from large numbers of people in order to find disease-causing mutant forms.  Researchers first must know how the genetic message actually appears on the chromosomes -- that is, into how many exons the message is broken up, and where the breaks occur.

Since with existing techniques this information is invisible in the cDNA form, biologists are now forced to do time-consuming chromosomal DNA sequencing to reconstruct the "hidden" boundaries.  (The cDNA form is too elusive and expensive to detect in mass screenings.)

And this is where USC's new technique comes into play.  Dr. Pevzner, a  professor of mathematics and computer science specializing in computational biology, will discuss the new technique Friday, Nov. 7, at the Gene Discovery in Silicon Conference in Atlanta, Ga.

According to Pevzner, the technique resembles the old parlor game of "20 Questions" in which a series of "yes" or "no"answers is used to narrow the possibilities and solve a riddle.

In the molecular biological form of the game, questions are asked using a technique known as the Polymerase Chain Reaction (PCR).  Dr. Arnheim, who heads the Molecular Biology Program in the USC College of Letters, Arts and Sciences, was one of the original developers of PCR.  The ExonPCR experimental protocol was developed in Arnheim's USC laboratory.

Every query in the "game" measures the distance between two PCR primers -- specified pairs of "words" (groups of letters long enough to be unmistakably recognized) in the genetic message.  However, this distance may differ in cDNA and  chromosomal DNA since chromosomal DNA contains junk DNA between PCR primers.

If the chromosomal DNA distance is greater, reasoned Pevzner, it's because  the two words are separated by junk DNA on the chromosomes and must therefore be carried on different exons.

By repeating the process and starting at different words, the boundaries of the exons can be narrowed down.  Pevzner and computer science graduate student Sing-Hoi Sze have demonstrated mathematically that, on average, 30 such word queries can reveal an approximate map of the exons in cDNA.  Then, another technique, "ligation mediated PCR," can establish  the exact boundaries.

The technique, which the researchers describe as "gene hunting without DNA sequencing," avoids expensive and time-consuming efforts to sequence the entire million-base-pair length of chromosomal DNA. 

Working on the "20 Questions" technique in the Arnheim laboratory were  postdoctoral researchers Guorong Xu and Cheng-Pin Liu, with computer scientist Sze coaching the ExonPCR software to  play the "20 Questions" game

"We believe that the technique will greatly speed the identification of mutations, with direct applications for research into genetically based human disease," says Arnheim, who holds USC's George and Louise Kawamoto Chair in Biological Sciences.

The research was funded by grants from the National Institutes of Health, the U.S. Department of Energy and the National Science Foundation.

Source: University Of Southern California. November 1997.

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