Mitochondrial DNA depletion analysis by pseudogene ratioing.
The mitochondrial DNA (mtDNA) depletion status of rho(0) cell lines is typically assessed by hybridization or polymerase chain reaction (PCR) experiments, in which the failure to hybridize mtDNA or amplify mtDNA using mtDNA-directed primers suggests thorough mitochondrial genome removal. Here, we report the use of an mtDNA pseudogene ratioing technique for the additional confirmation of rho0 status. Total genomic DNA from a U251 human glioma cell line treated with ethidium bromide was amplified using primers designed to anneal either mtDNA or a previously described nuclear DNA-embedded mtDNA pseudogene (mtDNApsi). The resultant PCR product was used to generate plasmid clones. Sixty-two plasmid clones were genotyped, and all arose from mtDNApsi template. These data allowed us to determine with 95% confidence that the resultant mtDNA-depleted cell line contains less than one copy of mtDNA per 10 cells. Unlike previous hybridization or PCR-based analyses of mtDNA depletion, this mtDNApsi ratioing technique does not rely on interpretation of a negative result, and may prove useful as an adjunct for the determination of rho0 status or mtDNA copy number.
Swerdlow
Russell H
RH
Department of Neurology, University of Virginia Health System, Box 394, Charlottesville, VA 22908, USA. rhs7e@virginia.edu
Redpath
Gerard T
GT
Binder
Daniel R
DR
Davis
John N
JN
VandenBerg
Scott R
SR
D045744:Cell Line, Tumor; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D018628:Gene Dosage; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011544:Pseudogenes; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D004996:Ethidium