Mitochondrial mutation detection using enhanced multiplex denaturing high‐performance liquid chromatography

Abstract

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.

Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC

“…Such GC clamps are incorporated into our primers. MtDNA fragments created by long-range PCR followed by restriction enzyme digestion [14,15] cannot harness the favourable effect of GC clamps.…”

Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC

“…Incubation of the PCR products with restriction enzyme DdeI results in the generation of 5 fragments of different sizes: 442, 342, 278, 210, and 126 bp (1 ). The mutation sites of interest lie in the 342-bp fragment containing the tRNA (leu1) sequence.…”

“…These primers can be obtained from the MitoScreen reagent set (Transgenomic) and have been used in a published study (1 ) restriction enzyme digestion and heteroduplex formation PCR products generated were digested with 1 unit of DdeI (New England Biolabs) at 37°C for 6 h. Digested PCR products were denatured at 95°C for 5 min and then slowly cooled to 25°C at a rate of 1°C/min to induce heteroduplex formation before DHPLC analysis. These conditions produce maximal heteroduplex formation-ϳ50% heteroduplex (see Results) in samples containing 50% heteroplasmy, indicating that the slow reannealing procedure is optimal.…”

“…It relies on the use of a hydrophobic column based on reversed-phase liquid chromatography for the separation of heteroduplex and homoduplex DNA at specific optimized temperatures and has recently been used by several research groups to study mitochondrial DNA (mtDNA) mutation (1)(2)(3)(4)(5)(6). In these studies, DHPLC has been used either to screen the whole mitochondrial genome (1, 2, 4 -6 ) or to study specific regions of the genome for mutation (3,4,25 ), followed by DNA sequencing to identify the mutation sites.…”

“…been developed to study mutations include denaturing HPLC (DHPLC) (1)(2)(3)(4)(5)(6), single-strand conformation polymorphism (7)(8)(9), denaturing gradient gel electrophoresis (10 -12 ), temperature gradient gel electrophoresis (13 ), temporal temperature gradient gel electrophoresis (14 -16 ), and pyrosequencing (2,17 ). A mutation detection method recently used in the identification of heteroplasmy relies on the use of Surveyor nuclease, which cleaves DNA at sites of base-substitution mismatch and short insertion/deletion (18,19 ).…”