An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences 英文参考文献.docVIP

AnIntegratedPCRColonyHybridizationApproachto ScreencDNALibrariesforFull-LengthCodingSequences JacobPollier1,2.,MiguelGonza′lez-Guzma′n1,2.¤,WilsonArdiles-Diaz1,2,DannyGeelen3 ,Alain Goossens1,2 * 1Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Gent, Belgium, 2Department of Plant Biotechnology and Bioinformatics, Ghent University,Gent,Belgium,3DepartmentofPlantProduction,FacultyofBioscienceEngineering,GhentUniversity,Gent,Belgium Abstract Background: cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is acommonlyused technique for genome- wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequenceinformationareobtainedforalargenumberofdifferentiallyexpressedgenetags.However,mostofthegene tagsdonotcorrespondtofull-length(FL)codingsequences,whichisaprerequisiteforsubsequentfunctionalanalysis. Methodology: A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incompletecDNAs.ThemethodwasappliedtoscreenfortheFLopenreadingframesofaselectionof163cDNA-AFLPtags fromthreedifferentmedicinalplants,leadingtotheidentificationof109(67%)FLclones.Furthermore,theprotocolallows fortheuseofmultipleprobesinasinglehybridizationevent,thussignificantlyincreasingthethroughputwhenscreening forraretranscripts. Conclusions:Thepresentedstrategyoffersanefficientmethodfortheconversionofincompleteexpressedsequencetags (ESTs),suchascDNA-AFLPtags,toFL-codingsequences. Citation:PollierJ,Gonza′lez-Guzma′nM,Ardiles-DiazW,GeelenD,GoossensA(2011)AnIntegratedPCRColonyHybridizationApproachtoScreencDNALibraries forFull-LengthCodingSequences.PLoSONE6(9):e24978.doi:10.1371/journal.pone.0024978 Editor:Ju¨rgenBrosius,UniversityofMu¨nster,Germany ReceivedMay12,2011;AcceptedAugust25,2011;PublishedSeptember15,2011 Copyright: ? 2011 Pollier et al.