Isolation of highly purified genomic material from mitochondria of muscle tissue cells

Abstract

In this work, we adapted a method for isolation of a highly purified fraction of mitochondrial DNA from muscle tissues suitable for further sample preparation of libraries without an amplification step for sequencing tasks on various NGS platforms and a method for evaluating the purity of DNA from contamination by nuclear genome regions. We optimized several techniques for enrichment of the mitochondrial fractions and purifying mtDNA. Here, we describe a protocol that allows getting from 80-100 mg of muscle tissues up to 1000 ng mtDNA, almost free from impurities of RNA and fragments of the nuclear genome.To assess the degree of purity of human mtDNA fraction from impurities of the nuclear genome, we adapted the PCR-screening technique for the beta-actin gene region and AluSx-repeats in the human genome. This methodology avoids false-heteroplasmy calls (PCR biases or NUMT contamination) that occur when long-range PCR amplification is used for mtDNA enrichment.