Quantification of mitochondrial DNA (mtDNA) damage and error rates by real-time QPCR.
Mitochondrial dysfunction has reported in several diseases including diabetes, cancer, skeletal muscle disorders and neurodegenerative diseases such as Wolfram syndrome. Several different methods have evolved to study mtDNA damage including Southern blotting, 8-oxoG damage, or a comprehensive scanning of the mitochondrial genome by RFLP or TTGE analyses. However these approaches require large amounts of DNA or are labor intensive. The use of polymerase amplification of long DNA products (LRPCR) has been described by several groups and more recently summarized by Van Houten's group. The underlying basis use of DNA polymerases capable of generating long DNA products and the rationale is that any lesion (strand breaks, base modifications, apurinic sites) will stop a thermostable DNA polymerase. In this method, band density of the PCR product is quantified either by Southern blotting or binding of a fluorescent dye. Although the latter approach still has some limited use in the study gene expression, it is semi-quantitative and realtime PCR analysis has largely supplanted it. Direct application of real-time PCR to LRPCR has been made difficult because of low processivity and polymerization rates of the DNA polymerases used and SYBR green inhibition of DNA amplification. We have modified the LRPCR protocol to use the commercially available PfuUltra() II Fusion HS DNA Polymerase for real-time determination of mitochondrial DNA amplification as a means to simplify and improve of the accuracy for quantification of mtDNA damage.
Edwards
John G
JG
Department of Physiology, New York Medical College, Valhalla, New York 10595, United States. jgedwards@nymc.edu
D000080242:8-Hydroxy-2'-Deoxyguanosine; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D014466:Ultraviolet Rays
D017931:DNA Primers; D004272:DNA, Mitochondrial; D005456:Fluorescent Dyes; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine