Targeted and robust amplification of mitochondrial DNA in the presence of nuclear-encoded mitochondrial pseudogenes using Φ29 DNA polymerases.
Accidental co-amplification of nuclear-encoded mitochondrial pseudogenes (numts) in mitochondrial DNA (mtDNA) analyses is a common problem in biological and medical research alike [Yao et al., J Med Genet 45:769-772, 2008; Song et al., Proc Natl Acad Sci U S A 105:13486-13491, 2008]. A wealth of strategies have been developed to evade the contamination of mtDNA data sets with numts, but time- and cost-effective protocols of general applicability are scarce [Wolff et al., PLoS One 7:e37142, 2012]. The protocol presented here closes this gap by combining serial dilution with standard and rolling circle DNA amplification. This strategy leads to selective enrichment of mtDNA and removal of nuclear DNA (nuDNA) from template solutions, enabling mtDNA analyses in a virtually numt-free environment.
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington Campus, High Street, Sydney, 2052, Australia, jonciwolff@yahoo.com.
Jonci N
JN
Wolff
D000818:Animals; D001435:Bacteriophages / Q000201:enzymology; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D011544:Pseudogenes / Q000235:genetics*
D004272:DNA, Mitochondrial; D004259:DNA-Directed DNA Polymerase