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【】“”08BJZ07【】510602458GCDNAPCＲ【】GCDNAPCＲ。15GC66．0%～84．0%DNAPCＲTm≥79．7℃Tm△Tm≤1℃。。26GC35．2%～53．5%DNAPCＲ。15GCDNATm△TmGCDNA。Tm△TmGCDNAPCＲ。DNA。【】GC【】Ｒ34【】Adoi10．3969/j．issn．1009-2595．2014．07．005PrimerDesignMethodforPCＲofGC-richDNATamplatesGAOMeiLIUShu-renLIQiangZHANGLing-feng．ClinicalLaboratoryNo．458HospitalofthePeople＇sLiberationArmyGuan-gzhouGuangdong510602China【Abstract】ObjectiveToestablishaprimerdesignmethodforamplificationofGC-richDNAsequences．MethodsAgroupof15pairsofprimerswithhigherTm≥79．7℃andlower△Tm≤11℃weredesignedtoamplifyGC-richsequences66．0%～84．0%．ThestatisticalanalysisofprimerparametersandGCcontentofPCＲproductswasperformedandcomparedwithliteratures．Agroupof26pairsofprimersweredesignedtotesttheapplicabilityofthisprimerdesigningmethodinamplifi-cationsofnon-GCrichsequences35．2%～53．5%．ＲesultsAlltheDNAsequencesinthisstudyweresuccessfullyampli-fied．TmandΔTmwerethemainfactorsinfluencingamplifications．ConclusionsThisprimerdesigningmethodofferedaperfecttoolforamplificationofGC-richsequences．Itprovesthatthesecondarystructurescannotbeformedathigherannealingtempera-tureconditions．【Keywords】PolymerasechainreactionGC-richsequencesPrimerdesignPCＲ。GCDNAGCDNAPCＲ1-2。3%DNAGC、GCDNA3。、40%GCDNA4。GCDNAPCＲ。GCDNAPCＲPCＲ、、、、、7--2-5-11AmpliTaqTMTaqTMKOD4OptimaseDNAPlatinumDNA12DNA。NaOHDNAPCＲstep-downPCＲslowdownPCＲGCDNAPCＲ13-17。Mg2+、pH、、PCＲ。PCＲ。PCＲ18。15GCDNAMeltingTemperatureTm△TmDNA·336·2014728287MilMedJSChinVol．28No．7July282014。DNAPCＲ。PCＲGCDNAPCＲ。Tm△Tm。15GCDNAPCＲ26GCDNAPCＲ。11．1DNA15GC26GCDNA。15GCDNA8HBA2NC_000016．9FMＲ1NC_000023．10APOENC_000019．9HＲES1NC_000001．9CSTBNC_000021．8IN-SＲNC_000019．9AＲNC_000023．10GATA4NC_000008．10GC66．0%～84．0%。26GCDNAXq28F826GC35．2%～53．5%。http//．ncbi．nlm．nih．gov/nucleotide/-DNA。1．215GCDNAPCＲDNA5’3’。PCＲGCTm△Tm、Oligo6．64。26GCDNAPCＲGCDNAPCＲ。INVITＲOGEN。GCDNAPCＲ12。1GCDNA15PCＲTable1ParametersoffifteenpairsofprimersforGC-richDNAamplificationsinthisstudy5’－3’merTmΔTmkal/molPCＲGC%bpINSＲFCGCGGCCCCCAGCGCCTCTTＲGCCCGCGGCGCCCAGTAGCA202086．586．60．110．316．513．42．61．082．8180HＲES1aFGGCCCGGGCTGGCGGGGCGＲCCCCCGCGCCCCGCGCAC191891．390．31．022．210．322．75．55．184．0206HＲES1bFGGAAACACATCCCCAGCTGAGGGCＲGCAAGAGGAAACTTGAAAAGGCGGATCAC242981．080．50．510．15．46．22．11．768．91226HＲES1cFGGAAACACATCCCCAGCTGAGGGCCGGGAGＲGCAAGAGGAAACTTGAAAAGGCGGATCACGCCAATGC303790．190．00．110．19．86．72．95．468．91226FMＲ1FGCCCCGCACTTCCACCACCAGCTCＲGGCGCTCAGCTCCGTTTCGGTTTCACTTC242985．485．80．46．39．86．701．580．9283HBA2aFCCCCACAGACTCAGAGAGAACCCACCAＲCAGGTAAACACCTCCATTGTTGGCACATTCC273181．482．00．63．26．05．02．01．566．3885HBA2bFCCCCACAGACTCAGAGAGAACCCACCAＲGGGGAGGCCCAAGGGGCAAG272081．481．40．03．29．35．02．04．168．0764APOEaFCCCGGTGGCGGAGGAGACGＲGTCGCGGCCCTGTTCCACCAG192181．281．70．59．810．39．42．20．875．5322APOEbFGCCTGGGGCAGGGGGAGAACAGCＲGCCCGACCCCGAGTAGCTCTCCTGAGAC232884．684．40．26．66．39．32．12．067．4282HBA2cFCGCGGGTTGCGGGAGGTGTAGＲCCGGGATAGAGAGAACCCAGGCACAC212681．280．50．710．39．89．40066．2343CSTBaFCGGCGCCCGGAAAGACGATACＲCGGGGCCAAAGCGGCTTCTT212080．079．90．116．09．39．81．51．876．7473·436·2014728287MilMedJSChinVol．28No．7July2820145’－3’merTmΔTmkal/molPCＲGC%bpCSTBbFGCCCCCACCCAGCCTGGAGＲCGGGGCCAAAGCGGCTTCTT192080．279．90．36．69．312．40．91．876．0786gata4aFGCGGGTGCCCTCCTCCGTGＲCCCTCGCGCTCCTACTCACCGAGA192481．381．80．56．710．36．72．4078．8510gata4bFGAGCCTAGAGCCCTTTGCTCAATGCTGＲGGGGTGTAAGCGGCTCCGTCG272179．780．40．76．36．79．41．9066．5500AＲFTCGGCCGCCGTCCAAGACCTACＲCGGCGGCTCCAGGCTCTGG221981．681．40．216．56．619．62．31．766．03802GCDNA26PCＲTable2Parametersoftwenty-sixpairsofprimersfornon-GC-richDNAamplificationsinthisstudyF85’-3’TmΔTmPCＲGC%bp11FCCAGTGGGTAAAGTTCCTTAAAATGCTCＲCCTTCCTCCAAGCAGACTTACATCC74．073．70．344．436522FCCAGCTGCTTTTTGAAGTGTCCACＲGCAGTTGGCATAGACCAAGCAGAG74．774．80．142．038133FGGGCATGCTTCTCCACTGTGACＲCGCCACCATTACAAAGCACACA74．974．60．345．030244FGAGGCGGACGTTGCTGTGAＲGGCAGAGGACAGTAGAAATCACTACTGATAAAG74．574．30．241．672155FAGCCAAGTTATCAAGAGACAGAAGACTACATTＲCCCATCTCCTTCATTCCTGAACAGT73．074．11．135．232466FCGGTCATTCATGAGACACATGCTTAGＲGAGGCCAGAGATCTTCAAGTATTCAGTG74．373．80．547．430477FGGTGATTGGTGACCTTGGCAAGＲCGGGATCTTGGCTGAGGTCTAATAC74．473．50．939．95498+98FCAGCCTAATATAGCAAGACACTCTGACATTGTＲCTTCTTACCTGACCTTAAATCTTTTCTTCAACTTAC74．573．31．240．1805109FGTCCTGGAACCAATCCTCCATGＲGGCCAACAGCTGGAGAAAGGA73．474．41．035．63931110FGTGCGACTTTAGCTTCCACTTGCTＲCCAGCACTTGGAAAGGCAAGA73．573．20．940．83801211FGCCATCGCTTTCATCATAGACTGCＲCACCACCCACTGGACTTAAGTGCT74．574．10．444．52721312FCCTAATTGTATCATGACAATCACAATCCAAＲGGGCAAAAGAGCATACGAATGG73．873．20．639．236214131FGGGAATGGGAGAGAACCTCTAACAGA1ＲGGGGAGATGACTTTTTGCCAAATAGA74．274．50．341．9810142FGGACCCCCAAGTATGCCAGTTC2ＲGAGGCAAAACTACATTCTCTTGGATTAATGTT74．374．20．136．6925153FGGGTGAATTTACAAAGGACGTAGGACTC3ＲGGGAGCCAACCTCTCTTTGATCA74．174．30．238．9923164FGGAGATGACTGGTGATCAAAGAGAGGT4ＲGGTGTCATCATCTGGTAAAGTCAAATGTC74．474．00．443．88801517FGGCAAGAGTATTTCAAGGAAGATGAAGTGＲCCAAAAGTGGGAATACATTATAGTCAGCAAG74．374．40．138．537416+1718FGGGATGTAAACCCTAAGGACCTTAAGATCＲCCCTGGATCAAGTCTCATTTGTCAA73．273．90．739．78801819FGGGCTCCCTGCAATATCCAGATＲGGTGCCCTATGGGATTTGAGATG74．074．10．136．775419+2020FGGGGTCCAAAAGTAGTGGAACTACCAＲCAGCCCAGGTTCTTGGAGCA74．574．40．137．4960·536·2014728287MilMed