SNT 1197-2003 油菜籽中转基因成分定性PCR检测方法

ProtocolofthepolymerasechainreactionfordetectinggeneticallymodifiedcomponentsinrapeseedsI~;~~&S~~~HM%VM.%@TMEOB~,&B~B~C:%B~~$I%#~W.I~~~#&PCR@WA&.*6@SHT&9ttNiPCDfBl,%tTMR@&4.@B;l;Bff%!fdJle##fsel@BPCR&MeTPI*%.5!XW@B8BSHT*4%?Bo3.1t)Stransgene~~#;Q~if~a~,*a~~i~~~marnDNAB~I.~~&N+A+H,@~&BP~#+~Ei-S.Q1Yi,U@BalfJbR4~8ii#611jPP%~e32fir&L#*K&polymerasechainreaction,PCW#&SE4@3'15k&&S2f!k&%J%6%,fiDNA#$B#H#SaMEj&%#T,&B#&%9JRit~R&Cll$hJJ)41]-4#&DNAm&@k#flff~-BIjllj:.@3ilJ%%aIkThi&X@&DNASe~H#HTU~#~&BM~~(dN~~')%E~~@9I~l9U~i~*#G5~BB3~~~~Afi@f$&-m*,@BrMmsm%Bunfi%arme33a#i%3.3.1PCR;polymerescchainreact~on,iQ-%PCR.3.3.2DNArdeoxyribonucleic~cid,a#%&#l&@~3.3.3dNTP;deoxyribonucleos~detripl1osihate,A#R&BBZbf9@.3.3.4dATP,deoxyadenos~netriphosphatc,8#$lMfi7@i!#ie3.3.5dCTPIdeoxycytid~netr~phosphnte,H##H%~.ljlPaQ.3.3.6dCTPtdeoxyguanos~netr1phos~hatc,A#$l4%3@@.3.3.7dTTP;deoxythymidinetriphospllate,A#~!jQH#:a&(ft.3.38dUTPldeoxyuridinetripho~phatc,R#$t@-ff'Z7#@!~3.3.9UDGIuracilDNAglycosylase,!3!@/@DNA-%#@.3.3.10bpIbasepair,@&XQ.33.11EDTAleth~lenediaminetetraaceticacid,&f@fl&@41.3.3.12TagITaqDNAPolymerase,DNASeIBB),3.3.13TEITris-HC1,EDTA$@#%.3.3.14SDS~sodiumdodecyls~lfate,+=~~~@~~3.3.15Tris;tris(hydroxymethyl)aminomethanevZ(BtB#)$2&$;Sm3.3.16PEPIpho~phoenolpyruvatecarboxylasegene,?i@l@#lBSWRm%.cI:@.33.17FMV35S135Spromoterfromamodifiedfigwortmosaicvirus(caulimovirusgroup),-k;$aRt.%f*35sB33.3.3.18CP4EPSPSl5-enolpyruvylshikimate3-phosphatesynthasegene,5-%Q:@-3-@@fi&&€%I.33.19CTPIchloroplasttransitpeptidegene,Utb3#BM!lkSF4a3.3.20G0X:glyphosateoxidoreductasegenerWE8P)tSfi%RWSH,33.21E93',3'sequenceofsmallsubunitofrbcS(ribu10se-I,5-bisphosphatecarboxylase)E9gene(frompea),S@T]BPtlXb9tlGH4$-1,5=RBB%.cI:BE9SB/J\YE2&3'MBY!!.3.3.22CaMV35S135Spromoterfromcauliflowermosaicvirus,a##%$E,Rt.#jSj35Sm23;bTJ..3.3.23PATIphosphinothricinacetyltransferasegenefromBacillusamyloliquefaciens.SHTRRBacillusamyloliquefaciensJ$JR#Zi%$$&i%S~.3.3.24BARNASE,ribonucleasegenefromBacillusumyloliquefaciens,MTRBacillusarnyloiiquefaciensrnribonucleaaegm.33.25BARSTARlspecificinhibitorofthebarnasegenefromBariliuaarnyloliquefaciens,%WTfiHBacillusamyioliquefa~ien~i%barnase&4$SFNfl)ll3&fZI.3.3.26NPTII~neomycin-31-phosphotransferasegene,&Ji4$#-3'-@j4fl%%ASB$.3.3.27NOSrprornoterofnopalinesynthasegenefromAgrobacteriumtumefaciens,s%T!&fia&~'rrtla.fta~sa~a~~3,3.28NOS3';terminatorofnopalinesynthasegene,E!488lBM&&@SH4%k3.3.3.29OCS3'octopinesynthasegeneterminator,33&@idt-ft&W%iZl~kT.3.3.30SsuAralribulose-1,s-bisphosphatecarboxylasesmallsubunitatslApromoterfromArabi-dopsisthallana,*BlT#l$j#F#ribuloae-l,5-~7#@%4fia$~J~BHatslA4il24h9G%Pe3.3.31TA29developmentallyregulatedpromoterfromanther-specificTA29genefromNicotianatabacum~~1EtR(T#lICTtYJ(J~%F#~TA29%C61&%$ffiTtf3.3.32TR73'13'regulatoryregionfromAgrobactcriumtumefuciensoftheT-DNAtranscript7,*BlT!&R@sclT-DNA@@?7M3'MTE@.5.1hh#&ASN/T1194*%$i!rnk&&Fj.5.2fill#@8kH20ga%H@fi,@F%XB(150CF%m&a2h)&120'C,30min&J3#js$&SEI(J@2SNIT1197-20036.1BZl+w,~swttDNA~~+txtg~mu~~~r,~trl~~as.m~~~~nlazt~~~.~~PCRB*.$$Wf&r#BblSBfVJDNA#8[9i,&@PCRVWW%,PIkBffda$EAXGRSBd5).62iiSH%lHlWRH4BSfi,##igBl%%3)%8%4kBfflJ,*%BbZ.#ecGH/'r6682#e~~-%&.6.2.11mn!/LTris-H~lB#@Bi(pH8.0)1#Fllk121.1~,Tris,#fT800ml.7JCql~#,B~Ai$!&&42m~.,~t;llS~B,M~&@%biWBpH28.0,J)%fiRLEXi!49H,6220.5mol/LEDTA(pH8.O)I&ROOml.I~+~IIA186.IgNAZ-ED'rA2H20,&ajla##@JflB#,H$R&~fiVB@PH8.O(W%20y%klff4kMBiN)~%%iZ?$!Z1I.9%%Gfi6.&XB*H.623DNAm@&@j@I%J&!100mI.Inlol/I.Trib-CI,50ml.0.5mol/LE:I)TA,#JIY5.0gSDS,16.848RRM,Z~E1~.,%wamma-$rrn.624Tris4'&;F5lB.6.2.5?arflB06266.2.75#WZPP.6.2.870%tBOP.629'1'Eg@#(pH8.0):-j%@I0n~l,In\ol/l.Tr~s-CI(pH8.0)4U21nL0.5rnol/l.ED'I'A(pH8.0),Ba&B%SI000rnL,%%6i!&BiXB-&M6.2.10IoXPCR$4#@3500mrnol/l.n-f&@,100rnnlol/l.'L'r1.s-HCl(p1-I8.3),15mmol/L&Rf3(MgClZ),0.1%Ma.6.211lox125mmol/L.621210XdNTPl32.5mmol/lddATI',dUTP,dC:l'I),d(;TP.6213Taqf#5Unit/pl,.6214Ejl1411@@$21IYJf9~8~itdi;l%,B~B%7kBi!%l&100pmol/l.#fij.,&@FATPCRBli-3W~I%%BYJ5pmo1/lde3t1@lftS$1~rBRhFIJ~SB%#B(Ji;lQB9J6.2.15Xrtff*,6.2.16RNaaeAl2mg/pL.6.2.17?&&ZI@I10mg/mL.0.2.18DNAteFf632100bp-2000bp.6.2.19%jh#.4;la~WaJBlNOSNPTIIFMV35SGOX(Bt$)CP4-EPSPSC#ikPATBARBARNASEaJBAlllfigfin9bHfiHfimfia'Im'Im31BRBJ5'-#~tcctpttg~~ggt~tt~'5'-ttrtcctsgtttgcgcgcta-3'5'-atcgttcaan~tttgpcn-3'5'-att#cgggactctaatcdta-3'5'-t1ggatctc~tcgtgaacat-3'5'-gcrcgapgaagcggtca-3'5'-pgatctcctptcatct-3'5'-gatcatcctpatcpar3'5'-mgc~tcaacaappcap3'5'-ctpctcpatpttgncaa~'5'-aap~catccaccpupactta-3'5'-aggacagctcttttccb.cptt3'5'-gt~ttcgt~tt~ctgp~accgtt-3'5'-garctppcappagqagapt-3'-5'-~t~tt#ttt~#t~gttt~Bte3'5'-gaaacccatccacttgpagts-3'5'-pacttpcgtpttcpttcttc-3'5'-mca~cpttpa~cttpapae3'5'-cscg~ttBtgatatcgttr,a~3'5'-tcttgcarcctctcta~atcatcrs3'5'-ptcpaclltptctccppagap3'5'-#~B0~~48~~~~#tat~3'5'-accatcptccraccrct~catcp3'5-~ctgccapaaacccacgtcst-3'6'-acugcacpptc~cttcc3'5'-actcppccgtccaptcste-3'5'-ctg(yptpgcatcaaaagpp~nce3'~'-tccggtct~aatt.tp~pcctg-3~5'-tcagaaptatcagcpacctccac~~'5'-aagtatgatpptpatptcpcagce~'rtl~Ijllbp180186183173186210121450204108191430175161236I~A.555555555560606555608055606060a%iWm&&flPRrB