Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography

Abstract

Aim: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA).

Methods: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen.

Results: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with ApaI. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5% mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e.g., Pfu) in eliminating the false positive produced by Taq DNA polymerases.

Conclusion: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.

Figure 1

The minimal ratio of hetero-: homoduplex in mixture were identi-fied by DHPLC. The composition of mutant type in PCR product mixture are 0% (A, wild type), 1% (B), 5% (C), 10% (D), 20% (E), 30% (F), 40% (G) and 50% (H) respectively. Chromatogram B has no difference to chromatogram A. Heteroduplex peak start to change in sample containing 5% variant (C). The heteroduplex can be obviously discerned in sample with 10% variant (D). Chromatogram E to H is almost similar. It indicates that about 5% mutant composition in PCR product mixture can be detected by DHPLC.

Figure 2

The universality of a broad small peak proceeding the main peak induced by Taq DNA polymerase. An extra small peak is observed preceding the eluted chromatography profile of A and B but not in chromatogram of C, D and E. Chromatogram A is a representative of products amplified by Taq DNA polymerase. B is typical chromatography profile when Taq DNA polymerase matched with Pfu DNA polymerase buffer in PCR reaction. C and D respectively comes from amplimers amplified by Ampli-Taq Gold and Vent DNA polymerase. E is one of chromatograms coming from PCR amplification by using Pfu DNA polymerase.

Figure 3

The representative DHPLC chromatograms of variant fragments detected blindly for patients with gastric cancinoma in D-loop of mtDNA. The chromatogram of tumor tissue is visibly different to that of its counter-part non-tumor tissue. The variant cases are listed on the top of each DHPLC chromatogram. DF1, DF2, DF3 and DF4 represent the tested fragments of D-loop respectively. T: tumor tissues; N: non-tumor tissues.