A PCR-based paradigm for the analysis of DNA sequence variation

This thesis develops a paradigm of technologies and concepts for the systematic characterization of DNA sequence variation. The paradigm is based on in vitro amplification of defined genomic DNA segments via the polymerase chain reaction (PCR). The paradigm encompasses detection, sequencing, genotyping and molecular haplotyping of variant base pairs in homologous fragments within and between primate species.; To scan for sequence variation, DNA samples from five individuals or animals are pooled in Population tubes (Poptubes). Poptubes enhance the speed and sensitivity of scanning since they allow simultaneously amplifying a DNA segment from ten separate chromosomes while deliberately forming heteroduplexes among variant products. Variation is detected using denaturing gradient electrophoresis (DGE). Heteroduplexes have drastically reduced mobility during DGE.; Variants are sequenced using coupled amplification and sequencing (CAS). CAS proceeds directly from genomic DNA and generates simultaneously sequence ladders for both complementary strands of DNA segments, which facilitates sequence validation. For genotyping populations to assess frequency and distribution of DNA variants, the allelic sequence ladders are generated for each individual with one primer for the bases determined to be variant using CAS genotyping.; Strategies were also developed for haplotyping multiple polymorphisms molecularly without reliance on pedigrees. Single molecule dilution physically separates haploid equivalents from diploid genomic DNA by dilution to an expectation of one molecule of any given DNA segment per ampule. Biphasic booster PCR amplifies the template molecule 10{dollar}sp{lcub}12{rcub}{dollar}-fold for genotyping assays. Allele-specific amplification and competitive oligonucleotide priming in concert with CAS allow selective study of either the maternal or paternal chromosome directly from genomic DNA.; The paradigm has been applied to the first phylogenetic analysis which considers polymorphisms within each hominoid species. The study of a non-coding locus in the HOX2 cluster has demonstrated that polymorphisms may exceed inter-species differences among the hominoids. This fmding lends credence to the concept that ancestral polymorphic species are important in DNA sequence-based phylogenetic analysis of closely related species. The paradigm is also applicable to gene mapping, diagnostics, medical genetics and forensic science.