A novel method to quantify deleted mitochondrial DNA in a real time PCR

Abstract

The quantification of 4977 bp deleted mitochondrial DNA (dmtDNA) may be of interest for the forensic as well as the clinical pathologist. However, the determination of dmtDNA in two separate PCR reactions may lead to imprecise if not false results due to pipetting inaccuracies, deviant PCR conditions, etc. A conventional duplex PCR with subsequent fragment analysis yields only relative quantities of dmtDNA based on the analysis of PCR end products. To eliminate these factors, we established a real time duplex PCR using FAM- and VIC-labeled MGB probes. The PCR was carried out on an ABI Prism 7000 Sequence Detection System using standard chemistries. Amplicon sizes were 123 bp for deleted and 113 bp for total mitochondrial DNA. Serial dilutions showed a detection limit of 100 fg for total mtDNA. Application of this method to clinically and forensically relevant samples proved the suitability of this duplex PCR for quantification of minute amounts of mtDNA as well as highly degraded material.