Every person usually carries two copies of the genes they possess (except for sex-linked genes), but the human genome is highly polymorphic and a given person may possess an alternative DNA sequence at a particular chromosomal site that determines the usefulness of that site for genetic studies. Sites that exhibit alternative sequences are termed DNA polymorphisms, which are useful as genetic markers for the site, or for the chromosome bearing the site. From a pharma-cogenetic standpoint, such sites may be useful for identifying individuals who are predisposed to peculiarities in drug response. When these sites are tracked within the context of family studies and in larger populations, inheritance patterns and estimates of their prevalence and significance to humans can be determined. By taking advantage of recombinant DNA techniques, polymorphic sites can be studied at two levels, one employing Southern analysis to survey chromosomal sites for polymorphism and the other employing DNA sequencing to determine the precise location of base changes that define the polymorphism. The latter approach is, in essence, a refinement of the first.
Consider the application of Southern analysis for such a purpose. Restriction enzymes cleave DNA at recognition sites of four to eight bases. An eight-base cutter enzyme, for example, will recognize an appropriate sequence every 48 = 65,536 base pairs (the probability that a specific base will occur at each of the eight positions); similarly, a six-base cutter will see its sequence every 4096 (= 46) base pairs, and a four-base cutter will see its sequence every 256 (44) base pairs. If one of the bases at the recognition site of a given restriction enzyme is polymorphic, a panel of six different enzymes of the eight-base cutter type will sample the DNA sites at every 65,536/6 % 9400 base pairs, a six-base cutter will sample the genome at every 4096/6 % 680 base pairs, and a four-base cutter will sample it at every 256/6 % 43 base pairs. Hence, a panel of six of the eight-base cutters would sample a gene of say approximately 30,000 base pairs only about three times (% 30,000/9400), the panel of six-base cutters would sample it 44 times (% 30,000/680), and the panel of four-base cutters would sample it about 700 times (=30,000/43).
These concepts are illustrated by two studies of the N-acetyltransferase NAT2 gene that is responsible for the isoniazid acetylation polymorphism and is polymorphic in rabbits and humans. In both species the NAT2 gene has a coding region of 870 bases and encodes a protein of approximately 33,000 daltons; additionally, NAT2 is devoid of introns in the coding region and contains 15,000 to 20,000 base pairs. A panel of five six-base cutter restriction enzymes (EcoRI, Hindlll, XbaI, Bglll, and ApaI) was employed to survey the rabbit genome for NAT2 polymorphism,45 and a panel of three six-base cutter enzymes (KpnI, BamHI, and EcoRI) was employed for the human polymorphism.46 The restriction fragment length polymorphism (RFLP) patterns indicated that the NAT2 of rabbits and humans were both polymorphic; the polymorphism in rabbits was due to a gross deletion of the slow acetylator gene, while the human RFLP patterns suggested that a small genic lesion, possibly a single base substitution, could account for the human polymorphism. Additional studies confirmed this conclusion for human NAT2 poly-morphism.47
The human genome also contains a variety of short variable DNA repetitive sequences called variable number tandem repeats (VNTR) that contribute to its polymorphic character. Each of these sequences may be repeated 100 times or more in different persons. If a restriction enzyme cuts the DNA on either side of a VNTR, the size of the fragment produced will be proportional to the number of repeats in the VNTR, and the different-sized fragments will migrate differently on an electrophoresis gel.
Gene amplification is another mechanism that generates different-sized DNA fragments that can be observed by Southern analysis. A well-known example of this phenomenon is provided by the study of two families in which amplification of the gene for the debrisoquine hydroxylation polymorphism (CYP2D6) was observed. Restriction analysis with EcoRI showed that a single copy of the gene that had undergone amplification was 12.1 kb in length. The RFLP patterns of XbaI-cleaved genomic DNA prepared from members of these families revealed restriction fragments of 175 and 42 kb. The 175-kb fragment from the father, daughter, and son of family 1 represented 12 copies of the amplified gene, and the 42-kb fragment from two sons of family 2 represented two copies of the amplified
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