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Efficient and specific amplification of identified partial duplications of human mitochondrial DNA by long PCR

1996; Elsevier BV; Volume: 1308; Issue: 3 Linguagem: Inglês

10.1016/0167-4781(96)00110-8

1879-2634

Bernard Fromenty, Giovanni Manfredi, James E. Sadlock, Lee Zhang, Michael P. King, Eric A. Schon,

Molecular Biology Techniques and Applications

The use of PCR to identify mtDNAs containing a partial duplication (dup-mtDNA) in the presence of a heteroplasmic population of mtDNAs harboring the corresponding deletion (Δ-mtDNA) leads to ambiguous results: when the primers anneal in the duplicated portion of the dup-mtDNA (which is also the non-deleted region of the Δ-mtDNA) and point towards the abnormal breakpoint junction, both templates are amplified indiscriminately. We have developed two different ‘long PCR’ approaches to amplify dup-mtDNA even in the presence of Δ-mtDNA and wild-type mtDNA (wt-mtDNA). Long PCR with two primers annealing in the non-duplicated region in dup-mtDNA (equivalent to the region missing in Δ-mtDNA) and whose 3′ ends pointed towards the duplicated area amplified both dup-mtDNA and coexisting wt-mtDNA. We observed, however, a preferential amplification of the wt-mtDNA over that of the longer dup-mtDNAs. This problem was partly overcome by modifying the PCR conditions (extension time, amplicon length, amount of template). In order to overcome the problem of co-amplification, we developed a novel PCR method to amplify specifically dup-mtDNAs. A forward primer annealing across the breakpoint junction was used in conjunction with a backward primer annealing in the non-duplicated region. For those duplication breakpoints flanked by direct repeats, we designed a ‘breakpoint loop-out’ primer whose sequence omitted the repeated region, in order to avoid the annealing of this primer to wt-mtDNA. This second approach was able to amplify specifically and efficiently the dup-mtDNA in all samples analyzed, irrespective of the size of the duplication or its proportion in the samples.