PCR Induced Pseudoheteroplasmy Of MtDNA D-loop Repeating Region And Its Possible Mechanism

Heteroplasmy of D-loop region of mammalian mtDNA revealed site mutation and repeat number variation. Compelling studies reported that some species exhibited very high heteroplasmy, indicating low ability of error correction in the process of DNA replication in mitochondria, which conflicts with existing knowledge. In order to confirm the truth of heteroplasmy as reported, we studied repeating sequences of D-loop region of several carnivore species namely blue fox (Alopex lagopus), black bear (Selenarctos thibetanus), brown bear (Ursus arctos), dog (Canis familiaris), wolf (Canis lupus) and tiger (Panhera tigris) by using PCR strategy that is commonly used in those reports. Results indicated that PCR products always appeared as ladder-liked pattern on 8% PAGE gel, despite templated by whole DNA, purified or cloned PCR product. The length difference between adjacent bands was one repeat motif. It suggested that the high heteroplasmy might be a fack result induced by PCR. Further experiments confirmed such pseudoheteroplasmy was not related to pimer, but may be associated with the type of DNA polymerase to a certain extent, but not totally eliminated. Tiger did not produce ladder-liked pattern in all experiments. The analysis of repeat motifs of these species using software RNAstructure v3.7 revealed different motif might generate different periodic secondary structure where double stranded structures were available for binding DNA polymerase. Based on above results and analysis, we proposed a possible mechanism inducing the pseudoheteroplasmy during PCR process. The nascent strand as the primer, DNA polymerase lost one or several of repeating motifs, or transferred the other same or homologous strand. Finally, our results suggested that there be a risk to study heteroplasmy of repeating region of mtDNA D-loop region using PCR strategy.