Digital MDA for enumeration of total nucleic acid contamination.
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.
Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, 318 Campus Drive, Stanford, CA 94305, USA.
Paul C
PC
Blainey
Stephen R
SR
Quake
D059018:DNA Contamination; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction
D004259:DNA-Directed DNA Polymerase