Consideration of heteroduplexes and homoduplexes for the quantification by competitive PCR of human mitochondrial DNA deletions with ageing of tissues and cells.
The purpose of this work was first to construct two internal standards for human mitochondrial DNA mt DNA corresponding respectively to the fragment resulting from the 4,977 bp common deletion (H2del) and a fragment which was never reported to be deleted (H1). Secondly, we wished to consider the possible effect of annealing between the target and corresponding internal standard which forms heteroduplexes. These experiments show that the correction of the number of copies found by competitive PCR by considering the percentage of heteroduplexes allows a more accurate quantification of the number of target copies present in mt DNA samples. The design of internal standards specific to the fragment resulting from other deletions could also help a more accurate quantification of the frequency of other mt DNA deletions as well, and reconsideration of the role of mt DNA deletions in ageing.
Frippiat
C
C
Department of Biology, Unit of Cellular Biochemistry and Biology, University of Namur (FUNDP), 61, rue de Bruxelles, B-5000 Namur, Belgium.
Dierick
J F
JF
Remacle
J
J
Toussaint
O
O
D000375:Aging / Q000235:genetics*; D016922:Cellular Senescence; D004272:DNA, Mitochondrial / Q000032:analysis*; D006801:Humans; D008931:Mitochondria, Muscle; D009692:Nucleic Acid Heteroduplexes; D017246:Ophthalmoplegia, Chronic Progressive External / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods
D004272:DNA, Mitochondrial; D009692:Nucleic Acid Heteroduplexes