SNT 1196-2003 玉米中转基因成分定性PCR检测方法.pdf

Protocol of the polymerase chain reaction for detecting genetically modified components in maize Td$iB.BXj#r#r$iEfB#3;f&HE0 3.1 R S B B transnene#N*%&A%N,%NSi-Ffbillofllhcsfi DNA#9 l&j i r p%A A&,$!W +,S C$I W l%B?$:E 3.2 Bbl#&JJi$!polymerase chain reaction,PCR&J?J Y i i%!%l?k&DNA fiH*M#S&HE&$T*&BPJg 2-t&1W+%143)9II%8$5 DNA H%#L&fiiW-BX$bIP9rJ2+BA#X%6,f&#&DNA R e B$8 l T I J A 4#s 4!(d N T P)%&t&5 1 l M t$*G ;f;I I&%A i#1 t -m6&rNtmsaLnlv 49-3.3 HMl%3.3 1 PCR polymerase chain reaction,j%j%PCR.3 3.2 DNA,deoxyribonucleic acid,%&#+&.3 3 3 dNTP,deoxyribonucIeosde trphosphte.H#gf(Z.3 3.4 ATP PI deoxyadeNOSine t i.i p h o s p l t e,I B 3.3.5 dCTP,deoxycytdlne triphosphte,a$f.fi#Z#a 3 3.6 dtiTP,deoxyguaNOSine triphoupliate,n#!&P5 8 3mZll.3 3.7 dTTP,deoxythymdine tr1plrosphate,jttlffw8:ii 3 3.8 dUTP,deoxyuridne triphosl)hate,MRtiirtfZ#3.3.9 bpl base pair,&4iH.3.3.10 EDTAI ethylene diaminetetraacetic a c i d t Z,Z Z,.3.3.1 1 Tagl Thermu aquaticu,&!&%#!%.3.3.12 Trisl tris(hydroxymethyl)aminomethanetFi(I&)$lBfe$2,3.3.13 TEITris-C1,EDTA#a,3.3.14 IVRinvertasel genefrommaize,3*&fiBl%m.3.3.15 Zeinlh*l#biPBSB$,3.3.16 CaMV 35S135S promoter from cauliflower mosaic virus,fi?g#%awJi$t#J 355 a3.3.3.17 NPTIIl neomycin-3-phosphotransferase genet%#*-3-8&H.3.3.18 NOS1 terminator of nopaline synthase gene,861813U&9ElSm&k+.3.3.19 IVS21intron from the maize alcohol dehydrogenase gene,3#B)rB#%#%ElW&BJfi E 2.3.3 20 PATt phosphinothricin acetyltranrferase gene*PTf#Z,B%#Sfl.3.3.21 HSPO;O.8 Kb intron from the hsp70 gene(heat-shock protein),A Q%#j&Q(hsp70)S$1BO 0.8KbLBk4LX.3.3-22 CryIA(b)1 a synthetic gene encodes the first 648 amino aidsinsecticidal-active ftruncated product identical to that of CrylA(b)gene of Bacillus thuringisnsi,s u b s p,%Z&f i&e crylA(b)Sa.3.3.23 CDPK I calcium-dependent protein kinase(CDPK)gene,$5%$&%&#S$1.3.3.24 Cry9CI insecticidal-active ftruncated product identical to that of CrgC gene of Bacillus thuringiensis s u b s p,Z&i$f i&l&l CrgC sm.3.3.25 CaMV35SI 135s terminator of CaMV g e n o m c r t E#i%R i-S H l 35s%kF.3.3.26 P actin 1 I the promoter derived from the rice(Oryza sativa)actin 1 g e n e,A%F h)/t$j El lLHI%&diStlf.3.3.27 OTPIoptimized transit peptide pene,fi#Si#&4i.3.3 28 mEPSPS,maize 5-enolypyruvylshikimate-3-phosphate synthase,$in*h*s(J#*mB&z-B%!.5.1 n w EM SN/T 1194 rfiB&I%&E.5.2 U#a f l 2 0 gh*#fi,LF%$II(lSOr?%PlfafiB 2 h)120C.30 min&Em3JfiSf&sN&6$W$llZ#fig3SisS&flO.5 mm%&A/.6.1 QIX4#&SBR D N A ,X S&H Q R A#N P B W J L R I#PCR fh*,43#VN5+&DNA%i*l H PCRrqSW*#R#t;#db+t.Bs%HSH&a.6.2 O I R l M M k4IP3RBWIBfi*XithBfPJ%J)MiPt&#k)iZN tl)c%BM GB/T 6682 iBBJ-87k.2 6.2.2 Taq DNA fiZ.$B 6.2.3 dNTPtdATP,dTTP,dCTP,dGTP,dUTP.6.2.4%1 1 1#I$%#.6.2.5 a4kZB(EB)idt#f&3k&hrd.6.2.6 5#V%e 6.2.7%am.6.2.8 SgJB.6.2.9 70%285.6.2.10 S)Fg Marker:50 bp-300 bp.6.2.11 CTAB SII)%613%CTAB(JB3if)91,4 401/L#4k4!10.2%(#N3)9k)189Z,#.20 mrnol/L EDTA,100 mmol/L TrirHCllpH8.0.6.2.12 TE M a Bit 10 mmol/L Tris-HCl lpH8.061 mrnol/L EDTAl pH8.0.6 2.13 10XP.CR aM?&1100 rnmol/L,160 mrnol/L a.20 mrnol/L,200 rnmol/L Tris-HCl(pH8.8)1%Triton X-1001 1 rng/mL BSA.6.2.14 5XTBE#*lTris 54 gl%275 g.0.5 mol/L EDTA(pH8.0)20 m L l$a a*31 000 mI,.6.2.15 lOXk#eirO.25%mlR,40%RM.6.2.16 RNA R(10 ug/mL).6.3 B#6.3.1 Mii#l8lR#.6.3.2 l J i f t b .f i S/I S G R 1 M i n i +Aff,L&.6.3.3*#%#IBWS9BC tWS#m#0 6.3.4 Xv l&f 0.001 g.6 3.5 REXH%ra.6.3.6 7L6WP#.6.3-7 f%Qllk#ls,Xi%?k%g.6.3.8*i4.*%#.6.3.9 fiJJbk#L.6.3.10 b%HS$6#.6.3.11 dgP.6.3.12%rWiX.6.3.13 IBW.6.3.14 PCR&MI$&.6.3.15%El3)#7l.X.6.3.16 if&l#,O.1 pL-2 pL.0.5 pL-10 pL,2 PI,-20 pL-10 pL-100 pL.20 pL-200 pL,200 pL-1 000 pL.6.3.17#&$!1$#&%.6.3.18%N%XPCRW.6.3.19 Eppendorf 1.5 rnI,-5 mL.6.3.20 PCR I 200 pI-,500 pI.n#B#.6.4 aWJI 6.4.1#fa DNA%2 g$#T 10 mL ddG$Fq,&A 5 mL CTAB?8YM%(?%BBt RNA B),?fi33,60CdcbB 4#B 1 hr2 000 r/min F1$lL-5 minrl8k.#%t):,b%#S#VB/%dtBj(#H3)91(1r24 1 l)tEft,f 5 min.8 000 r/min BIG 5/IJLRI.SB%R#RZNVG/#I#BS(#B)1 1)#3 Ql#R 5 min18 000 r/rnin Rbb 5 minr&bk#?&b 0.65 f$f$#J%i,133,12 000 r/rnin 4 C I$,L 10 minrfPk#%,&500 pL 70%Irf(LBP&$#-&,12 000 r/min 4CFIBbb 5 rninrBk.#,#$E I$rlf.,fmA 50 PI.TE,#FBB(4Caf&,d 37Cf%E!1 h)I&%,&DNA m.&H.)fl&Stifi#DNA&fetH&J&JBt DNA,6.4.2 PCR W#6 4.2.1 PCRE&#IR#rlhlwm%rn+fi,m-i x m PCP.izu 2.+m m s t i+f i g ussrnljte4;nrnpa*aafirntt,tsm#xt,ul)ct.etii DNA#%9$XQMWI.2 SB39ki4.9blaPi4&1M PCR EP#S 6.4.2 2 PCR Eh!lfRZ$#94Cfi493&2 mln.94C$#40 s,55f-58Cajk 60 s(R#Agj%j+Jil$!1,72C#60 S,35+3WC4;.72Cde4$5 mi.4Ci51.thSfl&!#;IHM2&BVWt!ZX4 PCR E i h?t 8 5 6 4 1 1 1#%5 3 Rd&.6 4.3 P C W rI#P#Jr$%&w 8&%!3!I+%(lXTBE&TAE)$+J$2%HR8$Rllbf($&?%55C-6oC&libflA EHd!Jti&!#$tPJH#t8%0.5 p g/m I.,-l&?%f 3 .14 10 11.-15 PI.PCR V#P%3)%l1#1 2 PI.k.#383a&,j&fifi#.81 100 bp I.addcr I)NA Marker&GesH DNA Marker#j).?S&ji Z.3 V/cm-5 V/cm tflEE,&$J 20 min-40 mln.Ji!2211R#3)EiZD 6 5 aR#gi 6 5.1 AIIEiN4bI 8et.S3i#fiNM IVR$M(ljt Zein&f4)i(;lih9l%lXX DNA BRkRkitt37 PCR NN,It!k Bflfl 8fkMJ!flFfB!#%&?&TY$Ib 226 bp($173 tp)b!J PCK PCIstiRITiA PCR P#!rN IJ#,f!Jl DNA B&BS.fiFlH,&DNA 4j!?&ltlG#d PCR E G U J?,$P&I DNA,BPJ B