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


CONCLUSIONS
DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.
Liu
Man-Ran
MR
Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, Peking University, Western District, Beijing 100034, China.
Pan
Kai-Feng
KF
Li
Zhen-Fu
ZF
Wang
Yi
Y
Deng
Da-Jun
DJ
Zhang
Lian
L
Lu
You-Yong
YY
D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D017241:MELAS Syndrome / Q000235:genetics; D017237:Mitochondrial Encephalomyopathies / Q000235:genetics; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics; D014407:Tumor Cells, Cultured
D004272:DNA, Mitochondrial