Abstract
The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.
Highlights
Mammalian mitochondrial DNA is a double-stranded multicopy genome of approximately 16.5 kbp
Under denaturing condi tions the mitochondrial DNA (mtDNA) separated as a long smear along the length of the lane, indicating that it consisted of single-stranded DNA fragments ranging from below 250 to over 10,000 nucleotides in length (Fig. 1b)
Re-probing of the blots in Fig. 1a-b confirmed that the integrity of nuclear DNA (nDNA) fragments correlated with that of mtDNA (Fig. S1b-c). These findings demonstrate that mtDNA molecules in total DNA preparations prepared by a standard isolation procedure contain several single-stranded DNA (ssDNA) breaks per strand of mtDNA, most likely intro duced by endogenous nuclease activity during the isolation procedure
Summary
Mammalian mitochondrial DNA (mtDNA) is a double-stranded multicopy genome of approximately 16.5 kbp. Depending on the requirements dictated by downstream analysis methods, most re searchers opt for a DNA isolation method that belongs to one of two main categories: isolation of mtDNA from purified or enriched mito chondria, or, if the planned analysis can distinguish mtDNA from a pool of other nucleic acids, the isolation of total genomic DNA (i.e. nuclear DNA and mtDNA). Both approaches have their benefits and disadvan tages. A sys tematic comparison of four different isolation procedures with regard to the integrity, yield and enrichment of the resulting mtDNA identifies a preferred isolation method for applications that require tissue mtDNA of high integrity
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