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Nucleic Acids Res. 2011 Mar;39(4):e19. doi: 10.1093/nar/gkq1074. Epub 2010 Nov 11.

Digital MDA for enumeration of total nucleic acid contamination.

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  • 1Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, 318 Campus Drive, Stanford, CA 94305, USA.

Abstract

Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom φ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.

PMID:
21071419
[PubMed - indexed for MEDLINE]
PMCID:
PMC3045575
Free PMC Article
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