DNA Detection Using Recombination Proteins
Olaf Piepenburg1, Colin H. Williams1, Derek L. Stemple2, Niall A. Armes1*
1 ASM Scientific Ltd, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom
DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.
Funding. The authors received no specific funding for this study.
Academic Editor: James Haber, Brandeis University, United States of America
Received: February 9, 2006; Accepted: April 18, 2006; Published: June 13, 2006
DOI: 10.1371/journal.pbio.0040204
Copyright: © 2006 Piepenburg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abbreviations: apoB, apolipoprotein B; Bsu, Bacillus subtilis Pol I; FAM, carboxyfluorescein; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus; PBDG, porphobilinogen deaminase; RPA, recombinase polymerase amplification; SCCmec, staphylococcal cassette chromosome mec; Sry, sex-determining region Y; THF, tetrahydrofuran abasic–site mimic
* To whom correspondence should be addressed. E-mail: niall@asm-scientific.co.uk
Citation: Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA Detection Using Recombination Proteins. PLoS Biol 4(7): e204 |
