Directed-termination PCR and multilevel analyses on mitochondrial DNA mutations.

摘要

Mitochondrial DNA (mtDNA) has been a focal point in studies of genetic effects at several levels of biological organization, but little is known about the causal linkages between genetic effects at different biological levels. My thesis addressed a technological advance and two conceptual approaches to the study of mtDNA mutations at multiple biological levels.; I developed a PCR-based method (DT-PCR) that integrates both DNA amplification and directed termination into a single reaction. This method exploits unbalanced nucleotide concentrations to induce the polymerase chain reaction to terminate at specific nucleotide sites, leading to the generation of nested termination fragments from a complex DNA mixture. These termination fragments can be examined for sequence variation in either denaturing or non-denaturing polyacrylamide gels. I demonstrated that DT-PCR provides a single-step and highly effective technique for the detection of both insertions/deletions and single nucleotide substitutions in sequences up to 1 kb in length; I subsequently developed a novel approach for the identification of newly arisen mtDNA variants at the population level, which are designated as terminal branch haplotypes (TBHs). This approach examines the patterns of genetic diversity in a rapidly evolving segment of mtDNA to determine the incidence of recent germline mutations present in the gene pool of natural populations. I discovered that TBHs are prevalent in brown bullhead (Ameiurus nebulosus) populations, but that contaminant exposure played a negligible role in their generation. Instead, these TBHs appear to reflect the high spontaneous mutation rate of mtDNA. The variation in incidence of TBHs among populations is likely due to demographic factors.; I also investigated the incidence of mutational events at an intraindividual level. By coupling cloning and DT-SSCP analysis, I detected three different types of somatic mutations in the D-loop region of mtDNA from A. nebulosus , producing an in vivo somatic mutation rate (1.29 × 10−5 nucleotide/site) which is 2 to 3 orders of magnitude higher than those for selectable nuclear genes. By analyzing spectral shifts of nucleotide variation from cellular to population levels, I demonstrated shifts in the type and distribution of mtDNA mutations at different biological levels, suggesting the need to recognize three different fixation rates of mtDNA mutations from cellular to population levels.