Mutation screening of the mitochondrial genome using denaturing high-performance liquid chromatography.

摘要: Over 170 known mutations of the mitochondrial genome are responsible for disease. Due to unique features genetics, such patients clinically diverse and difficult diagnose. As pathogenic DNA (mtDNA) mostly heteroplasmic, denaturing high-performance liquid chromatography (DHPLC) could be used detect these heteroplasmic species therefore act as a rapid screening test mtDNA mutations. The entire was amplified by PCR in 40 overlapping regions. In addition, mutants were constructed each regions using PCR-based site-directed mutagenesis approach. These positive controls showed detection limit 3-10% heteroplasmy DHPLC (depending on specific mutation) compared 40% conventional sequencing. To further validate test, from 17 with seven different compare mutation had sensitivity 88% 82% This increased 100% when excluding m.8993T>G mutation. analysis is sensitive, cost-effective method screen genome. role pyrosequencing quantitation mutant load highlighted m.3243A>G an illustrative example. Pyrosequencing able discriminate samples containing little 5% proved accurate reproducible estimation load.