Locate a disease gene by screening for markers linked to the gene.

HI! One of the first steps in locating a disease gene is screening families with the disease for markers that are linked to the gene. Scientists use short tandem repeats (STR) as markers. These repeats can vary from ten to hundreds of base pairs, and are usually found in multiple copies. Different people will have different numbers of these repeats. In this example, A has two copies and B has four copies of the tandem repeat. The DNA sequences flanking the repeats are unique sequences found in everyone. PCR primers can be made to the unique flanking sequences and the intervening fragments can be amplified. These fragments are different sizes because of the number of repeats present in the individuals. The size difference can be seen when the fragments are electrophoresed on a gel. Assuming that A and B are homozygous for the length of an STR, what would the gel pattern look like for their progeny C? No, if C is the progeny of A and B, then it should have both bands. No, if C is the progeny of A and B, then it should have two bands. No, if C is the progeny of A and B, then it should not have a different size band. A progeny from a cross between A and B will be heterozygous for the length of the STR. In other words, C will have the smaller-size fragment from A and the larger-size fragment from B. The size differences of STRs are markers that can be associated with the occurrence of a disease or genetic trait. Which of the following gels and associated pedigrees shows an STR linked with an autosomal, recessive disease gene? No, two affected individuals have different STR patterns; there is no linkage. No, two unaffected individuals have different STR patterns; there is no linkage. No, both the affected and unaffected parent have the same STR pattern; there is no linkage. In this example, the STR is linked to an autosomal, recessive disorder. Carriers and the affected individual all have the same STR fragment. This is only a very small sample size. Larger, multi-generational analysis can confirm the linkage of this STR with the disease trait. Gene hunters try to find two markers linked to one gene. Which two markers (labeled 1 and 2) will be most useful? Gene hunters look for markers that are tightly linked to the disease gene. This indicates that the gene is nearby. By finding two flanking markers, gene hunters narrow their search to a defined stretch of DNA. Though the flanking markers are relatively close together, there may be a million base pairs and a hundred genes to wade through. Your next step is to find the coding sequences in this region. First you need to clone the DNA between the two markers, and order them with additional markers. If your biggest clone is 150,000 bp, which of the following "clone maps" is most useful in your hunt? No, there are gaps between the clones. No, the clones are too big. Remember, you don't know where the gene is, so you have to clone the entire region. Overlapping clones are needed to put each clone in the proper order. Now, you need to locate the coding sequences on the clones. What method CANNOT be used to identify coding sequences? A) Look for hybridization between cloned DNA and a human cDNA library. (No, human cDNA will identify coding sequences by hybridization.) B) Look for hybridization between cloned DNA and DNA from other species. (No, DNA from other species will identify coding sequences by hybridization if the genes are conserved.) C) Sequence the clones and look for tell-tale signs of coding sequences. (No, common promoter sequences can be used to find the beginnings of coding sequences.) D) Look for hybridization between cloned DNA and DNA from patients with the disease. B and D. (No, only one of these is incorrect.) When using DNA instead of cDNA, hybridization will reveal noncoding as well as coding sequences. Suppose you get lucky, and your analysis only reveals three candidate genes. For each, you compare sequences of unaffected people and disease patients. Candidate Gene #1 No differences found. Candidate Gene #3 Disease patients are missing an exon. Candidate Gene #2 Single nucleotide polymorphisms found in some disease patients. Which candidate is most likely the disease gene? Candidate #1 No differences found. (No, that is incorrect.) Candidate Gene #2 Single nucleotide polymorphisms found in some disease patients. (No, that is incorrect.) Candidate Gene #3 Disease patients are missing an exon. (That is correct) Single nucleotide polymorphisms between the two groups — unaffected and disease individuals — do not always point to a disease gene. Remember, there are redundancies in the codons and some changes are tolerated. However, if an exon is missing, chances are the protein has been altered and this could lead to a disease phenotype . CONGRATULATIONS! YOU'RE SO SMART!

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