Abstract: Objective To establish a new method for analyzing the polymorphism of mtDNA control region based on DHPLC (denaturing high performance liquid chromatography) without sequencing or hybridizing. Methods The fragment of mtDNA control region (including HVI, HVII and HVIII) was divided into 4 amplicons. Conditions for PCR and DHPLC (including the melt temperature and gradient, etc) were optimized. 100 pairs of mixed samples were analyzed using DHPLC to evaluate the system′s ability to detect different sequences. Results Only 1 elutive peak was obtained for the 10 pairs of samples mixed with identical sequences. More than 2 peaks were captured for the 90 pairs of samples mixed with sequence differences of 1～6 bp, 1 and 2 bases del (/ins), respectively. The capability of detecting different sequence is 100%. The least distinguishable ratio of the heteroplasmic DNAs is 10%. Conclusion The analytic system presented here is rapid, efficient and economic. It is more effective to detect the heteroplasmy than direct sequencing.

Key words: mitochondrial DNA (mtDNA), denaturing high performance liquid chromatography (DHPLC), polymorphism, heteroplasmy