Mitochondrial mutation detection using enhanced multiplex denaturing high-performance liquid chromatography.
In this study, we investigated the presence of mutations within the mitochondrial genome in 40 Caucasian subjects using an enhanced multiplex denaturing high-performance liquid chromatography (DHPLC) approach. The enhanced DHPLC approach has increased sensitivity and throughput, and reduced analysis time per individual sample compared to conventional methods. This technique involved amplifying the mitochondrial genome in 18 fragments ranging in size from 300 to 2000 bp using a novel proofreading polymerase (Optimase, Transgenomic Inc., Omaha, NE) with a low misincorporation rate. Fourteen of these fragments underwent subsequent restriction digestion using a combination of five restriction enzymes to enable multiplex DHPLC analysis; the remaining four underwent conventional DHPLC. Using this complete mitochondrial genome-screening approach, we confirmed a number of previously reported mutations and additionally identified a large number of novel mutations using an enhanced DHPLC technique.
Bayat
A
A
Centre for Integrated Genomic Medical Research, University of Manchester, Manchester, UK. ardeshir.bayat@manchester.ac.uk
Walter
J
J
Lamb
H
H
Marino
M
M
Ferguson
M W J
MW
Ollier
W E R
WE
D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D005260:Female; D015894:Genome, Human; D006801:Humans; D008297:Male; D008875:Middle Aged; D015183:Restriction Mapping / Q000379:methods
D004272:DNA, Mitochondrial; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases