Quantitative allele-specific PCR: demonstration of age-associated accumulation in human tissues of the A-->G mutation at nucleotide 3243 in mitochondrial DNA.
We have developed an improved allele-specific polymerase chain reaction (AS-PCR) procedure that can selectively amplify mutant DNA sequences (which differ from the normal sequences by a single base pair) in the presence of large excess of normal sequences. We applied this procedure to quantification of mutant molecules of human mitochondrial DNA (mtDNA). Conditions for AS-PCR have been systematically varied, encompassing DNA template input, annealing temperature, and PCR cycle number. Adjustment of these three reaction parameters to optimal conditions, using plasmids containing cloned segments of mutant and normal mtDNA, enabled the reliable detection of as little as 0.01% of mutant mtDNA. By standardising the DNA input for AS-PCR, the percentage of mutant molecules can be accurately quantified. This improved procedure was used here to detect and quantify the base substitution at nucleotide position 3243 (A-->G) in mtDNA from total cellular DNA isolated from various tissues of both infants and adults. We observed a 5- to 10-fold higher mutant mtDNA (3243 A-->G) frequency in adult tissues than in infant tissues. The results are consistent with the hypothesis that the accumulation of mtDNA mutations is an important feature of the human aging process. The quantitative and sensitive allele-specific amplification system described here is applicable to the quantification of low levels of somatic mutations in oncogenes and tumour suppressor genes in the context of human mutation, and could be extended to any biological situation in which only a small proportion of a DNA molecular population is subjected to a particular base substitution.
Liu
V W
VW
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Zhang
C
C
Linnane
A W
AW
Nagley
P
P
D000328:Adult; D000368:Aged; D000375:Aging / Q000502:physiology*; D000483:Alleles; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D007223:Infant; D008875:Middle Aged; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial