- 2 - Identification of Dna Polymorphisms

Three approaches were used to identify DNA polymorphisms in humans by examining the DNAs of a number of individuals within Huntington's Disease pedigrees as well as random individuals for restriction fragment length polymorphisms (RFLPs). The first approach used randomly chosen, cloned single-copy DNA segments from four types of human recombinant DNA libraries as hybridization probes to detect by Southern blot analysis, RFLPs generated by digestion of total human DNA with Eco RI, Sac I, or Hind III. 14 hybridization probes were used to analyze the DNA from panels of 6 or 8 individuals. 133 enzyme sites per individual were examined accounting for 798 base-pairs (bp) of DNA sequence. A total of 3 variant restriction enzyme patterns were found with 3 different hybridization probes: 2 variant patterns result from differences in Hind III cleavage sites, one variant pattern results from the elimination of an Eco RI cleavage site. This Eco RI variant is polymorphic with at least two alleles present and an allele frequency for the absence of an Eco RI site of 0.56. The second approach used restriction enzymes as probes for DNA polymorphisms by examining cloned homol.ogous DNA sequences isolated from human genome libraries made from two individuals. DNA segments of 13-17 kilobase-pairs (kb) of Eco RI-digested human genomic DNA from two individuals were inserted into the bacteriophage lambda vector CH4A*. One recombinant phage clone from each library was randomly chosen. A single-copy DNA subfragment was isolated and used as a hybridization probe to isolate from the other library the homologous cloned DNA sequence (matching clone). Two sets of matching clones were isolated and analyzed with 22 restriction enzymes for restriction fragment length differences. One set of matching clones is identical in all 86 restriction enzyme sites examined, accounting for a total of 407 bp of DNA sequence. A total of 99 restriction enzyme sites (459 bp) in the other set of matching clones was analyzed. Four variant restriction enzyme patterns were found: One with Alu I and Taq I, and two with Hae III. The Taq I variant pattern results from the elimination of a Taq I cleavage site. This Taq I cleavage site variant is polymorphic with at least two alleles present with an allele frequency for the absence of a Taq I site of 0.7. The third approach used cloned middle repetitive DNA probes to detect variant restriction fragment length patterns, by Southern blot analysis, in specific size classes of DNA from two individuals. Two repetitive probes were used to examine patterns generated by 3 different