Method for in situ investigation of mitochondrial DNA deletions.
A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells.
Kovalenko
S A
SA
Centre for Molecular Biology and Medicine, Austin and Repatriation Medical Centre, Victoria, Australia.
Harms
P J
PJ
Tanaka
M
M
Baumer
A
A
Kelso
J
J
Ozawa
T
T
Linnane
A W
AW
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D018482:Muscle, Skeletal / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion / Q000235:genetics*
D004272:DNA, Mitochondrial