DNA Detection Using Recombination Proteins 英文参考文献.docVIP

o PL SBIOLOGY DNADetectionUsingRecombinationProteins Olaf Piepenburg1,Colin H.Williams1,Derek L.Stemple2,Niall A.Armes1* 1ASMScientificLtd,Cambridge,UnitedKingdom,2WellcomeTrustSangerInstitute,Hinxton,Cambridge,UnitedKingdom DNAamplificationisessentialtomostnucleicacidtestingstrategies,butestablishedtechniquesrequiresophisticated equipmentorcomplexexperimentalprocedures,andtheiruptakeoutsidespecialisedlaboratorieshasbeenlimited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targetingoftemplatematerialwithstrand-displacementDNAsynthesis.Itachievesexponentialamplificationwithno 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/detectionprocessareillustratedbyatestforthepathogenmethicillin-resistantStaphylococcusaureus. 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 combinationofpropertiesisasignificantadvanceinthedevelopmentofportableandwidelyaccessiblenucleicacid– basedtests. Citation:PiepenburgO,WilliamsCH,StempleDL,ArmesNA(2006)DNAdetectionusingrecombinationproteins.PLoSBiol4(7):e204.DOI:10.1371/journal.pbio.0040204 single-stranded DNA binding protein necessary for the reaction. We found that a unique combination of T4 uvsY, Introduction The ampli?cation of DNA is an essential step in most nucleic acid–based testing strategies. Established ampli?ca- tion techniques rely on sophisticated instrumentation, such as temperature-regulating equipment or complex sample- handling procedures. While unproblematic for specialised laboratories, these requirements have hampered the uptake ofnucleicacidanalysesinpoint-of-useand?eldsettings.The technologypresentedinthis stu