Molecular analysis of human minisatellites.

Tandem-repetitive hypervariable minisatellites detected in a DNA fingerprint provide highly informative genetic markers. To identify and localize specific loci represented in a DNA fingerprint, it is necessary to clone individual minisatellites. This thesis is concerned with the characterization of single locus minisatellite probes cloned from DNA fingerprints. Seven single locus human minisatellite probes have been cloned by screening ? libraries with DNA fingerprint probes 33.6 and 33.15. Each locus consists of a minisatellite, with repeat units ranging in length from 9 to 47 base pairs depending on the locus. These autosomal loci are amongst the most variable loci characterized to date. The heterozygosity values of D1S7, D1S8, D5S43, D7S21, D7S22 and D12S11 range from 85% to >99%. Clustering of minisatellites was initially detected at the D12S11 locus. This observation led to the subsequent discovery of minisatellites showing close physical linkage as well as a tendency for minisatellites to be localized in proterminal chromosomal regions. An association of a minisatellite with a dispersed repetitive element was identified when studying the organization of cloned D7S22. This phenomenon was later found to be common amongst minisatellites. Pedigree analysis revealed a high level of instability of the locus detected by D1S7. This manifestation of detectable mutant alleles demonstrated the feasibility of direct estimation of mutation rates at minisatellite loci. The hypervariability of loci detected by minisatellites and their sensitivity in blot hybridizations make minisatellites a powerful tool in genetic analysis. These probes have already proved instrumental in many genetic and clinical studies. The high degree of individual specificity and the relatively simple banding pattern generated make these probes invaluable in forensic medicine. D1S7 and D7S21 were used in the first example of DNA-based identification in a rape and murder enquiry. One minisatellite probe was found to detect two loci, DNF21S1 and DNF21S2, on chromosomes 6 and 16 respectively. The 39 base pair repeat unit of this minisatellite is itself repetitive. The heterozygosity values of DNF21S1 and DNF21S2 are 61% and 16% respectively. Genomic mapping and sequence analyses revealed close similarity between these loci. Human population and pedigree studies showed that some individuals carry two alleles at DNF21S2, some carry one allele, some carry a duplicated allele while some are devoid of this locus. A model of duplication of a large proterminal segment of chromosome 6 DNA containing a minisatellite and transposition into an interstitial region of chromosome 16 in some human individuals is suggested. This is, to my knowledge, the first report of a human DNA polymorphism arising via transposition of DNA. The duplication unit on chromosome 16 is large (>15 kb) and has inserted into a member of a target site family present in 5-10 copies per genome. This sequence family represents a novel class of human repetitive DNA