A new method for locating the positions of genes on chromosomes has been developed by LBL scientists. Called "random-breakage mapping," the technique is a quick and relatively easy way of mapping internal DNA sequences with respect to the ends of a chromosome.
Mapping the location of the sequence of nucleotides that makes up a gene within the long stretch of DNA that makes up a chromosome has traditionally been a grueling and time-consuming process. For example, in the 50 years that scientists have been mapping the human genome, out of a total of some 100,000 genes, less than 2,000 have been located.
Random-breakage mapping could speed things up significantly. Developed by John Game, a biologist in LBL's Cell and Molecular Biology Division, working with Maren Bell and Robert Mortimer of CMB, and Jeff King, of UC Berkeley's Department of Molecular and Cell Biology, this technique reveals the distance of a gene, or other specific sequence of DNA base-pairs, from the end of its chromosome. By comparing these distances for each gene within a given chromosome, the respective positions of those genes is easily determined.
"The technique is particularly useful when there is little information known other than which chromosome the DNA sequence is on," says Game. "Although it only works for sequences that are not repeated more than a few times, that should cover most of the genes people will want to map."
Random-breakage mapping is best understood by considering a chromosome that is broken only once. For a once-broken chromosome, the shortest fragment that can contain the DNA sequence of a specific gene must be a piece with the gene on the end.
"No fragment shorter than the distance from the sequence to the nearest end of the chromosome can contain that sequence without at least two breaks in the chromosome," says Game.
Game and his colleagues begin their technique by irradiating with low energy x-rays a sample containing a number of molecules of a specific type of chromosome. The irradiation is done at an intensity that will result in most of the molecules being broken only once at random locations. Propelling the fragments by an alternating electrical field through a gel -- a technique known as gel electrophoresis -- separates them according to size. Another standard technique -- called "Southern blotting" -- is then used to transfer the fragments onto a special paper that makes those containing a selected gene visible when the gene is hybridized with a radioactively labeled probe.
"The size distribution of these fragments is such that two threshold changes in the intensity of our probe signal are seen in the pattern formed below the unbroken chromosome molecules," says Game. "The positions of the changes represent the distances in base-pairs from the gene to the each end of the chromosome."
Game and his colleagues developed the random-breakage mapping technique for their research with yeast DNA. They verified the accuracy of their findings using four different yeast genes that had previously been mapped.
Random-breakage mapping cannot be used on whole human chromosomes at this time because even a once-broken human chromosome is still far too large to be resolved in today's electrophoresis gels. Even if a gel could resolve such large molecules, the probe signal might be too weak.
However, once a human gene known to be on a particular chromosome has been cloned, random-breakage mapping could be used to pinpoint its location. Restriction enzymes -- enzymes that cut DNA at specific sites -- would first be used to selectively fragment the chromosome. These restriction fragments, whose position along the chromosome is known, could then be handled in the same manner as yeast chromosomes in order to map the gene.
"I see no fundamental technical barriers to applying random- breakage mapping to the human genome," says Game. "Right now we are trying to map some probes on chromosome 21 to get around the problem of noise from too much DNA. Our preliminary results look good."
Game says the technique should also be well-suited to the analysis of mammalian DNA that has been introduced into yeast chromosomes (called YACs or yeast artificial chromosomes) for replication purposes, and to studies of DNA breakage and repair.