Primer effect in the detection of mitochondrial DNA point heteroplasmy by automated sequencing

Abstract

The correct detection of mitochondrial DNA (mtDNA) heteroplasmy by automated sequencing presents methodological constraints. The main goals of this study are to investigate the effect of sense and distance of primers in heteroplasmy detection and to test if there are differences in the accurate determination of heteroplasmy involving transitions or transversions. A gradient of the heteroplasmy levels was generated for mtDNA positions 9477 (transition G/A) and 15,452 (transversion C/A). Amplification and subsequent sequencing with forward and reverse primers, situated at 550 and 150 bp from the heteroplasmic positions, were performed. Our data provide evidence that there is a significant difference between the use of forward and reverse primers. The forward primer is the primer that seems to give a better approximation to the real proportion of the variants. No significant differences were found concerning the distance at which the sequencing primers were placed neither between the analysis of transitions and transversions. The data collected in this study are a starting point that allows to glimpse the importance of the sequencing primers in the accurate detection of point heteroplasmy, providing additional insight into the overall automated sequencing strategy.

Reappraising the human mitochondrial DNA recombination dogma

“…Positive reactions from the smPCR were subjected to nested PCR. Independent amplifications were performed for each of the heteroplasmic positions (Z141-position 189: primers L2-16485 5′-gaactgtatccgacatctgg-3′(19) and H2-481 5′- gattagtagtatgggagtgg-3′(19); Z141-position 15496: primers 15416for 5′- tacacaatcaaagacgccctc-3′(18) and 15825rev 5′-gtgaagtatagtacggatgct-3′(18); Z176- position 8307: primers 8196for 5′-acagtttcatgcccatcgtc-3′(20) and 8600rev 5′- agaatgatcagtactgcggcg-3′(18); Z176-position 15908: 15721for 5′-ttgactcctagccgcagac-3′(21) and 16042rev 5′-ctgcttccccatgaaagaacag-3′(21).…”