Comparison of different quantitative PCR procedures in the analysis of the 4977-bp deletion in human mitochondrial DNA.
Four quantitative PCR procedures were compared to analysing the level of the 4977 bp deletion in human mitochondrial DNA. A single preparation of total cellular DNA from the heart of a 69-year-old female subject was used for all four methods. We estimated the deletion to represent 0.005% of total mtDNA molecules using the serial dilution procedure and two internal standard methods with two separate sets of reference recombinant plasmids. However, the value obtained with the kinetic PCR analysis was 0.5%, two orders of magnitude higher. The internal standard procedures are likely to be the most accurate among the four methods used, but the more technically convenient serial dilution method would also be an appropriate choice.
Zhang
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C
Centre for Molecular Biology and Medicine, Monash University, Clayton, Victoria, Australia.
Peters
L E
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Linnane
A W
AW
Nagley
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P
D000368:Aged; D001345:Autoradiography; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D003845:Deoxycytosine Nucleotides; D005260:Female; D006801:Humans; D008929:Mitochondria, Heart; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D017384:Sequence Deletion
D017931:DNA Primers; D004272:DNA, Mitochondrial; D003845:Deoxycytosine Nucleotides; D010761:Phosphorus Radioisotopes; C024107:2'-deoxycytidine 5'-triphosphate