Single, long molecule PCR for the detection of rare mutations in Mitochondrial DNA

Abstract

IntroductionOocytes contain the whole mitochondrial genomes of mammals, and the inheritance pattern of mitochondrial DNA in oocytes do not follow Mendel's rules of heredity. Mitochondrial DNA is maternally inherited in the oocytes, and only mother's mitochondrial genome can be passed to the next generation. The mitochondrial genome is a closed, circular piece of DNA which has a length of about 16kb, and there are hundreds to hundreds of thousands mitochondrial DNA in a single cell. Mitochondrial DNA mutation rate is generally difficult to detect and is often over‐estimated. This research is focusing on developing a sequencing strategy to accurately get mitochondrial DNA mutation rate of single, long molecules from a wild type mouse germline.Research ObjectivesThe research goal is to develop an efficient and accurate way to assess the mutation rate of mitochondrial DNA from wild type mouse oocytes.MethodsWe isolated and individually lysed single oocytes from a wild type female mouse. A serial dilution of the DNA sample was made to find its working concentration for generating single, long molecules, and prepared a PCR reaction by using the specific DNA sample concentration. A high‐fidelity DNA polymerase was used to generate 16kb PCR products originating from single template molecules. Sanger sequencing was used to sequence these DNA molecules.ResultsWe used single molecule PCR to amplify the mitochondrial DNA from three wild type mouse oocytes and sequenced all the PCR products, yielding a total coverage about 330,392bp. Twenty‐two mitochondrial genomes from three oocytes were sequenced, with an average length of about 15,017bp. Mouse mitochondrial DNA length is 16,299kb, so each of our mitochondrial DNA molecule has an average coverage about 92.13% of the whole mitochondrial DNA genome. Only two mutations were found in two separate mitochondrial genomes, and the resulting mutation rate of mitochondrial DNA in the oocytes was 6.05×10^‐6. This is a very low mutation rate across over 300,000bp.ConclusionThe mutation rate of mitochondrial DNA in wild type mouse germline is remarkable low in our research by using single, long molecule PCR. This PCR method allows for the accurate detection of mutations in the mouse mitochondrial DNA in oocytes. Our data indicates that, by using single, long molecule PCR combined with Sanger sequencing, we can accurately get informations from a molecule that has the most coverage of its genome. Additionally, we used a high‐fidelity enzyme to serve as DNA polymerase in the PCR reaction, thus preventing the chance of artificial mutations from the beginning of the PCR reaction. With the right procedure of our sequencing strategy, the rare mutations and the mutation rate in single germline cells can be accurately determined.Support or Funding InformationEllison Medical Foundation; NIH R37‐AG012279