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PMID: 21071419 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Digital MDA for enumeration of total nucleic acid contamination.

Nucleic acids research ·Vol. 39 ·No. 4 ·2011-03-00 ·Pages e19

Blainey PC, Quake SR

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.

MeSH Terms
DNA Contamination DNA-Directed DNA Polymerase/metabolism Nucleic Acid Amplification Techniques Polymerase Chain Reaction
Chemicals
DNA-Directed DNA Polymerase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Blainey Paul C
Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, 318 Campus Drive, Stanford, CA 94305, USA.
Quake Stephen R
References (21)
21 references, click to expand
  1. Ancient DNA forces reconsideration of evolutionary history of Mediterranean pygmy elephantids.
    Biol Lett. 2006 Sep 22;2(3):451-4 PMID: 17148428
  2. Digital PCR provides sensitive and absolute calibration for high throughput sequencing.
    BMC Genomics. 2009 Mar 19;10:116 PMID: 19298667
  3. Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease.
    Mol Hum Reprod. 2004 Oct;10(10):767-72 PMID: 15322224
  4. Whole genome amplification using single-primer PCR.
    Biotechnol J. 2008 Mar;3(3):378-82 PMID: 18293327
  5. Multiple mutation analyses in single tumor cells with improved whole genome amplification.
    Am J Pathol. 1999 Jan;154(1):83-95 PMID: 9916922
  6. Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.
    Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):11889-94 PMID: 17620602
  7. Nanoliter scale PCR with TaqMan detection.
    Nucleic Acids Res. 1997 May 15;25(10):1999-2004 PMID: 9115368
  8. Whole genome amplification from a single cell: implications for genetic analysis.
    Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847-51 PMID: 1631067
  9. A procedure for highly specific, sensitive, and unbiased whole-genome amplification.
    Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15499-504 PMID: 18832167
  10. Whole genome amplification and de novo assembly of single bacterial cells.
    PLoS One. 2009 Sep 02;4(9):e6864 PMID: 19724646
  11. Development of microbial genome-probing microarrays using digital multiple displacement amplification of uncultivated microbial single cells.
    Environ Sci Technol. 2008 Aug 15;42(16):6058-64 PMID: 18767666
  12. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses.
    Proc Natl Acad Sci U S A. 1985 Oct;82(20):6955-9 PMID: 2413450
  13. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification.
    Genome Res. 2001 Jun;11(6):1095-9 PMID: 11381035
  14. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.
    Genomics. 1992 Jul;13(3):718-25 PMID: 1639399
  15. Digital PCR.
    Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41 PMID: 10430926
  16. Nanoliter reactors improve multiple displacement amplification of genomes from single cells.
    PLoS Genet. 2007 Sep;3(9):1702-8 PMID: 17892324
  17. Quantitation of targets for PCR by use of limiting dilution.
    Biotechniques. 1992 Sep;13(3):444-9 PMID: 1389177
  18. A novel real-time quantitative PCR method using attached universal template probe.
    Nucleic Acids Res. 2003 Oct 15;31(20):e123 PMID: 14530456
  19. One bacterial cell, one complete genome.
    PLoS One. 2010 Apr 23;5(4):e10314 PMID: 20428247
  20. Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.
    BMC Genomics. 2006 Aug 23;7:216 PMID: 16928277
  21. Whole-genome amplification of DNA from residual cells left by incidental contact.
    Anal Biochem. 2004 Jan 15;324(2):312-4 PMID: 14690700
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkq1074
Published
2011-03-00
Epub
2010-00-11
Pages
e19
Language
English
Region
England
NLM ID
0411011
PMCID
PMC3045575
Subset
IM
Grants
NHGRI NIH HHS · 5R01HG004863-02 · United States
Howard Hughes Medical Institute · United States
External Links
PubMed source DOI: 10.1093/nar/gkq1074 PMC Full Text View journal details
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