Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA 英文参考文献.docVIP

RapidAssemblyofMultiple-ExoncDNADirectlyfrom GenomicDNA XiaopingAn1.,JunLu2.,Jian-dongHuang3*,BaozhongZhang1,DabinLiu1,XinZhang1,JinhuiChen1,YusenZhou1,YigangTong1* 1State Key Laboratory of Pathogen and Biosecurity, Beijing Instituteof Microbiologyand Epidemiology,Beijing,China, 2Beijing YouAnHospital, CapitalMedicalUniversity,Beijing,China,3Department ofBiochemistry, TheUniversityof HongKong,HongKongSpecial AdministrativeRegion (SAR),China Background.Polymerasechainreaction(PCR)isextensivelyappliedingenecloning.Butduetotheexistenceofintrons,low copynumberofparticulargenesandhighcomplexityoftheeukaryoticgenome,itisusuallyimpossibletoamplifyandclone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open readingframe(ORF)withwildtypesequenceinanysplicedformfromasinglegenomicDNApreparation.Methodology.Our ‘‘GenomicDNASplicing’’techniquecontainsthefollowingsteps:first,allexonsofthegeneareamplifiedfromagenomicDNA preparation, using software-optimized, highly efficient primers residing in flanking introns. Next, the tissue-specific exon sequencesareassembledintoonefull-lengthsequencebyoverlappingPCRwithdeliberatelydesignedprimerslocatedatthe splicingsites.Finally,software-optimizedoutmostprimersareexploitedforefficientamplificationoftheassembledfull-length products.Conclusions.The‘‘GenomicDNASplicing’’protocolavoidsRNApreparationandreversetranscriptionsteps,andthe entireassemblyprocesscanbefinishedwithinhours.SincegenomicDNAismorestablethanRNA,itmaybeamorepractical cloning strategy for many genes, especially the ones that are very large and difficult to ge