ELIMINATING MITOCHONDRIAL-DNA COMPETITION FOR NUCLEAR-DNA PRIMERS

Mitochondrial DNA (mtDNA) sequences were synthesized with nuclear DNA (nucDNA) sequence-tagged site (STS) primers by mismatch priming in thr ee independent studies of the human nuclear genome. Mismatch primer bi nding sites on the mtDNA were identified with from 6- to 10-bp identit y at the 3' ends of the primers. In two of three cases, single-strande d mtDNA copies were gel-isolated with intended nucDNA PCR products. Du ring routine screening of the STSs, the radiolabeled gel-isolated prod ucts hybridized to polymorphic mtDNA restriction fragments. Intense si gnals after overnight exposure of radiolabeled PCR probes on Southern blots suggest contaminating mtDNA PCR products. The theoretical anneal ing temperatures of the mismatches were well below the annealing tempe ratures of the PCR primers, demonstrating annealing reactions driven b y the molar surplus of the primers, that is, mass action. The probabil ity that two primers (either one of a pair or both), designed to ampli fy nucDNA, will bind to and amplify mtDNA may be as high as 1 in 64, a ssuming that an identical match with only the 3' hexanucleotide is suf ficient for amplification. To circumvent this problem we have develope d OLIGFIND, a program that has identified the 104 of 4096 possible hex amers that are not present in human mtDNA. Our results suggest that ti me could be saved by designing STS primers with one of these 104 hexam ers at the 3' end. OLIGFIND can also evaluate primer 3' ends for poten tial PCR products from mtDNA.