Eliminating mitochondrial DNA competition for nuclear DNA primers.

Abstract

Mitochondrial DNA (mtDNA) sequences were synthesized with nuclear DNA (nucDNA) sequencetagged site (STS) primers by mismatch priming in three independent studies of the human nuclear genome. Mismatch primer binding sites on the mtDNA were identified with from 6-to 10-bp identity at the 3' ends of the primers. In two of three cases, single-stranded mtDNA copies were gel-isolated with intended nucDNA PCR products. During routine screening of the STSs, the radiolabeled gel-isolated products hybridized to polymorphic mtDNA restriction fragments. Intense signals after overnight exposure of radiolabeled PCR probes on Southern blots suggest contaminating mtDNA PCR products. The theoretical annealing temperatures of the mismatches were well below the annealing temperatures of the PCR primers, demonstrating annealing reactions driven by the molar surplus of the primers, that is, mass action. The probability that two primers (either one of a pair or both), designed to amplify nucDNA, will bind to and amplify mtDNA may be as high as 1 in 64, assuming that an identical match with only the 3' hexanucleotide is sufficient for amplification. To circumvent this problem we have developed OLIGFIND, a program that has

Distribution of nuclear mitochondrial DNA in cattle nuclear genome

“…In analysing mtDNA, the mtDNA fragments themselves will greatly predominate in copy number which will not influence the study results in routine applications. There are few specific situations under which the primers will match with numts better than mtDNA and cause contaminated mtDNA products, so that numts would get amplified preferentially (Collura & Stewart 1995): (i) a mutation in the authentic mtDNA sequence at the 3¢-most primer position (Zullo et al 1993), (ii) the attempt to amplify mtDNA from a cell/tissue types with lower mtDNA content (Greenwood & Paabo 1999), or ancient mtDNA, especially with samples contaminated with endogenous or exogenous DNA (Willerslev & Cooper 2005).…”

New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains

“…To further address the possible effects of DNA degradation over different time scales, we compare both quantitative and qualitative results obtained from permafrost preserved mammoth samples ranging between 12 and >48 ky BP to a year-old ‘modern’ elephant sample. Finally we review the generality and applicability of the repeatedly purported ‘1000 to 1 ratio of mitochondrial to nuclear DNA’ ( 20 , 23–25 ) and address the implications of our results for large-scale palaeogenomic studies.…”

Mitochondrial DNA insertions in the nuclear Capra hircus genome

“…When analyzing mtDNA, the mtDNA fragments themselves will greatly predominate the copy number, which should not influence the study results in routine applications. However, there are a few specific situations under which the primers may match numts better than the intended mtDNA, thus resulting in contaminated mtDNA products and a preferential amplification of the numts (Collura and Stewart, 1995;Liu and Zhao, 2007): i) a mutation in the authentic mtDNA sequence at the 3'-most primer position (Zullo et al, 1993), ii) the attempt to amplify mtDNA from cell/tissue types with a reduced mtDNA content (Greenwood and Pääbo, 1999), iii) or during the amplification of ancient mtDNA, especially samples contaminated with endogenous or exogenous DNA (Willerslev and Cooper, 2005). Therefore, the method using sequence-specific primers was developed in genotyping of mtDNA more than 20 years ago (Garritsen et al, 1997;Hori et al, 2003).…”