Digital MDA for enumeration of total nucleic acid contamination

被引：96

作者：

Blainey, Paul C.

Quake, Stephen R. ^{[1
]}

机构：

[1] Stanford Univ, Dept Bioengn, Stanford, CA 94305 USA

来源：

NUCLEIC ACIDS RESEARCH | 2011年 / 39卷 / 04期

基金：

美国国家卫生研究院;

关键词：

WHOLE-GENOME AMPLIFICATION; MULTIPLE DISPLACEMENT AMPLIFICATION; GENETIC-ANALYSIS; SINGLE CELLS; PCR; DNA; PRIMER;

D O I：

10.1093/nar/gkq1074

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by phi 29 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 phi 29 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.

引用

收藏

页数：9

相关论文

共 21 条

[1] Development of microbial genome-probing microarrays using digital multiple displacement amplification of uncultivated microbial single cells [J].

Chang, Ho-Won ;

Sung, Youlboong ;

Kim, Kyoung-Ho ;

Nam, Young-Do ;

Roh, Seong Woon ;

Kim, Min-Soo ;

Jeon, Che Ok ;

Bae, Jin-Woo .

ENVIRONMENTAL SCIENCE & TECHNOLOGY, 2008, 42 (16) :6058-6064

[2] Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification [J].

Dean, FB ;

Nelson, JR ;

Giesler, TL ;

Lasken, RS .

GENOME RESEARCH, 2001, 11 (06) :1095-1099

[3] Multiple mutation analyses in single tumor cells with improved whole genome amplification [J].

Dietmaier, W ;

Hartmann, A ;

Wallinger, S ;

Heinmöller, E ;

Kerner, T ;

Endl, E ;

Jauch, KW ;

Hofstädter, F ;

Rüschoff, J .

AMERICAN JOURNAL OF PATHOLOGY, 1999, 154 (01) :83-95

[4] Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease [J].

Handyside, AH ;

Robinson, MD ;

Simpson, RJ ;

Omar, MB ;

Shaw, MA ;

Grudzinskas, JG ;

Rutherford, A .

MOLECULAR HUMAN REPRODUCTION, 2004, 10 (10) :767-772

[5] Nanoliter scale PCR with TaqMan detection [J].

Kalinina, O ;

Lebedeva, I ;

Brown, J ;

Silver, J .

NUCLEIC ACIDS RESEARCH, 1997, 25 (10) :1999-2004

[6] RAPID-DETERMINATION OF 16S RIBOSOMAL-RNA SEQUENCES FOR PHYLOGENETIC ANALYSES [J].

LANE, DJ ;

PACE, B ;

OLSEN, GJ ;

STAHL, DA ;

SOGIN, ML ;

PACE, NR .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (20) :6955-6959

[7]

Lao Kaiqin, 2008, Biotechnol J, V3, P378, DOI 10.1002/biot.200700253

[8] Nanoliter reactors improve multiple displacement amplification of genomes from single cells [J].

Marcy, Yann ;

Ishoey, Thomas ;

Lasken, Roger S. ;

Stockwell, Timothy B. ;

Walenz, Brian P. ;

Halpern, Aaron L. ;

Beeson, Karen Y. ;

Goldberg, Susanne M. D. ;

Quake, Stephen R. .

PLOS GENETICS, 2007, 3 (09) :1702-1708

[9] Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth [J].

Marcy, Yann ;

Ouverney, Cleber ;

Bik, Elisabeth M. ;

Loesekann, Tina ;

Ivanova, Natalia ;

Martin, Hector Garcia ;

Szeto, Ernest ;

Platt, Darren ;

Hugenholtz, Philip ;

Relman, David A. ;

Quake, Stephen R. .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2007, 104 (29) :11889-11894

[10] A procedure for highly specific, sensitive, and unbiased whole-genome amplification [J].

Pan, Xinghua ;

Urban, Alexander Eckehart ;

Palejev, Dean ;

Schulz, Vincent ;

Grubert, Fabian ;

Hu, Yiping ;

Snyder, Michael ;

Weissman, Sherman M. .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2008, 105 (40) :15499-15504