Efficient and specific amplification of identified partial duplications of human mitochondrial DNA by long PCR.
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 (delta-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 delta-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 delta-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 delta-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.
Fromenty
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B
Department of Neurology, Columbia University, New York, NY 10032, USA.
Manfredi
G
G
Sadlock
J
J
Zhang
L
L
King
M P
MP
Schon
E A
EA
D001483:Base Sequence; D015139:Blotting, Southern; D003573:Cytochrome b Group / Q000235:genetics; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D042967:Electron Transport Complex I; D003576:Electron Transport Complex IV / Q000235:genetics; D006801:Humans; D007625:Kearns-Sayre Syndrome / Q000201:enzymology / Q000235:genetics; D017240:Mitochondrial Myopathies / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D009886:Ophthalmoplegia / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017384:Sequence Deletion
D003573:Cytochrome b Group; D017931:DNA Primers; D004272:DNA, Mitochondrial; D009247:NADH, NADPH Oxidoreductases; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I