1、Protocol of qualitative PCR for the detection of genetically modified components in tobacco a3=.3.2.13 TrisI tris(hydroxymethyl)arninomethane,Z(%4 S)JfilSEfl ti2 a 3.2.14 DNA I dexribonucleic acid,li#$I.3.2.15 dNTPs 1 dTTP,dATP,dCTP,dGTP.3.2.16 dTTP I deoxyribonucleoside triphosphate,M#VZ#l 3.2.17 d
2、ATPl deoxyadenosine triphosphate,ll#BP3Ra 3.2.18 dCTPl deoxycytidlne triphosphate,W#lekB3fEl#l.3.2.19 dGTPl deoxyguanosine kphosphate,%4 3.2.20 dUTPI deoxyuridine triphoaphate,lR#R%Z119B!.3.2.21 bptbase pair,&Xlf.fQaH&#s&At 7SH#&#tlP#$d&W7&sb.k9rcH&dBflBA,#&BJ&$8.$!N#aH&*&?&#BekQ,#&.&#,BR*&fl,P;f*I%
3、#.RH&3244fi&R s&a&*A m *&m H m m m&s&#s w R-+%s m B#+B k?u A#m&%f i RMB#&ESHaHRSBBM&N&SHBHHnBaF*&k?,B#BeSMSfi&B#ESMbC#LiS H&;J$BBUWa NF#SBM5619S8H83iMJ3=91%i 35s G&?,NOs k P N B#SESB;NPT-I1 r#!&!,i$l bb k rtfal$&I EI&S iA TMV-CP,CMV-CP,TMV-54KD,RT,PVY-CP.6.1*B?miH$&M8 GB/T 6682 437JC(Z%*)M)5#5.6.2-F
4、nBl*jifQ#N 6,2.1 akB,ZB.#r#gt,CTAB,%,EDTA,Tris,BQ,P%iFiLe 6.2.2 dNTPa*Taq DNA fltf3sBC,bl#ft#ZBi!,DNA j).?.it6lE.&%4&#?4%.6.3 X#ZPEYlllkn(%1).1 BR90SBatarllA,*ZRSIJiR3I#Afla 1 2 6 R 35s NOS NPT-I1 B t CMVCP 91 b P J 5-gctcctacaaatgccatca-3 5-grtagtgggattgtgcgtca-3 5-gaatcctgttgccggtcttg3.5-ttatcctag
5、tttgcgcgcta-3 5-ggatctcctgtcatct-3 5-gatcatcctgatcgac-3 5-ccactagttaacaatttgattga-3-5-ccggaagtttaggacttagt-3 5-aagacgttggcagctggtcg3 5-ctcgaatttgaatgcgcgaa-3&8 5 1?R H 9 C n)hi#9b M fim m u Ftll#&.tf;B 19%l8obp 173bp 299bp 202bp bB%Blk39t;llW%B!hl MIY1111%Yr$l!&M PS!Sbtl Tris I tris(hydroxymethyl)am
6、inomethane,3(%q go DNA I deoxyribonucleic acid,li#.$I#.dNTPs I dTTP,dATP,dCTP,dGTP.dTTPI deoxyribonucleoaide triphosphate,NRll$%f:3.dATP I deoxyadenosine triphosphate,j3jt%#l$ZB.dCTPt deoxycytidine triphosphate,W#$Mft:ZBp.dCTPI deoxyguanosine tl*iphosphate,W#&$P3.dUTPsdeoxyuridine triphosphate,N#RG.
7、bp,base pair,MSX$.REQ%T$BrH#WBrJ&PR*SH&+BE1SBB3AXSHS+.$QbE NSiri&J)*#SBBQ*%?#C1I#C#i,%&%bW%l RR#C&iB,Pf%tk#*EU&%+?a R B a m B A m U*B m H M M l a J M s&m$f i B MSH8%-+%SHSM&%R*#42&B3F%kU2%fi2B%RHfi M 3&#S *Rsm&BHw&a*Nm*9JB3F,%kF,%#z&a3fia B*#BRNR#MHMSBUfo RIfeQSBn%#HMMBJYPjjm 35SG3F,NOS%JkFjjll 1$#6
8、 E S B NPT-I1 r#!4&i B fi B l f EI&BB$j TMV-CP,CMV-CP,TMV-54KD.RT,PVY-CP.6.1 B?BmiH*&#&GB/T 6682 EP%dC(3#*)#3!4#.6.2-FNlje#PlB*gift#e%fi*%?*fi%HtpJ*6.2.1 ak#.3)&Efl,%&tft,CTAB,%Pi.811,EDTA.Tris,lJQ.%&FiZBe.6.2.2 dNTPs,Taq DNA flt&BBC,lMfiZ.DNA%F.it68.$3dr#$il#%.6.3 XH3BBB3l4(%$!S 1).1 1W9PiiBetsrVA.
9、9EiBIJiR3I#AN*&tdaBl 35s NOS NPT-11 B t CMVCP 91 4 8 8;9 4 5-gctcctacaaatgccatca-3 5-gatagtgggattgtgcgtca-31 5-gaatcctgttgccggtcttg3 5-ttatcctagtttgcgcgcta-3 5-gga tctcctgtcatct-3 5-gatcn tcctgatcgac-3 5-cccactagttaacaatttgnttgga-3 5-ccgaagctttaggactgtaggt-3 5-aagacgttggcagctggtcg-3 5-ctcgbatttgaatc
10、gcgu-3*88?4M 9t.a 9t.m 9 b t!l fim 4tdM trN#&Eb 195bp leobp 173bp 299bp 202hp 83%Bm&#z815 RBW!l%&Y1R PgBM P&UI 7.1%.7.2 PCR N.7.3 M%QiYR.7.4#la&$IIRlG&.7 5 *%+A.7 6 l$tlPll&#%fE.7 7&*I#i!?a 7.8 Q88f.7 9&E%B b 1 4 W 1.7.10 WPT#BL.7 11%7k#.7 12&fi#$j#(O.5 pL.-I OOO pI-),+$A#fts;(IOO PI.-1 OOO ILL),&3#
11、$L (2 5 0 pL-1 500 pl.),f#$.8.1 W#B b i l W SN/T 1194 qB%H-fi&8?io 8.2(8#fiURB%;t$lt*;t$lN&%lEXi8:AtljFb(8:(HRi.13;JR%!%IBIJ&BIJ%IIE fi,L 27C-28CtPC%#)&ji-k*S,a;trIrH#NKl#NRA#alt-Zoo mg YfT&EXB?8src:W#h*Ltt;#B9YfiM=EZ*HBfiS*tRfibA4t%&*%BjrrfiWBat-#&;W.8.3 DNA H#!&8 3.1&?#I%DNA B(J*%iSt2Clifld*4%flil
12、SBcJ#tk&Bfi DNA.8.3 2#FfjFll&#lBt+DNA&I%.8.3.2 1#i)i%t&HliBll%WFEGAC1%)ik%91 l1 5 m1.Rlh4FP,#$IS%1 pI./rng bU A 65Cm&l x X I A B#P P%Z&!A M A)*%Z%(#W3)B)fiIIA P-%SZz,BP,R3)R*J ,65C;r)C%irtt-bn$&1 tnn-3 min,Ltt;?&+tkL!lIftM,Mf!#lt-NB.8.3.2 2 hA$,#&IXI3.Wfr/#fift(24 l,#8!3)&),$&%,&a&,13 OOO r/mn,L85 m
13、in.3 1l12&81 PVY-CP TMV 54KD TMV-CP-C h l o r o p l a s t o n-coading DNA trtl)#BQEB 362bp 295ph 3 2 7 b 550bp 1 5%$!&till#Z#iM gnus hI#Rl(SB41IR+I-m,$1 i n fim ha$1 I?Jd 51-gatalttcaestaclcgeeca-3 5-gcataacgcgtaaaccne3 5-gagttgtct1(ctcattga-3 5-acaatggtcaasgccgggta-3-5-tgtctttcacagcggggc-3 5-caccgt
14、tgcgtcgtcttrcrctacg-3-.5-cgeaatcggtagacgcracg-3 5-ggggaregegggactrpaae-3 8.3.2.3#L E*A-#&R J L%v A T R%#B u A )X-#10%CTAB(6 5 t W B),a$=&fRA3#RHG%FB/%aM(24 1 1,#3)323)?l3,13 OOO r/min FI(IJ 5 min.lO%CTAB a)r/lfisCJ&#!l%NI#%k A.8.3.2.4#k_f:*a%saia-%JistJLviPruAqiiHH CTAB s m t,s s m ,15 000 r/minffL
15、10 min,%E#B.CTABE$i!&!)r/l%l&!#J#!&gijk A.8.3.2.5$RE+fRA 200 rL%&k TE#,65CB#310 minp#25i.#!E m#A.8.3.2.6 bUA=.gb;lFRb)(F?M t Bp V;5 000 r/rnin at 10 rnin,Srl.Ej$m.8.3.2.7 M 8o%Zs5r%B-M3g9T&hS%TPR6Afi3)%&1.t.MHB DNA%BjeoDzw/0480 ttI;.Mt?Z 11.7-1.9 kMfl,Re PCR I&NS!r)t.8.3.3 T#lw-B#9a*DNA M&%8.3.3.1#8
16、.2+ilflM#5iC%100 me%h-%h!J 1.5 m1.abl!F+,fin 700 p I.1 XCTAR M&Fb313itEi#l%,65cPB 10 min.0.3.3.2 fiAA#$R5$IYB/FF&lS(24 l,#RB$3%l!&Hj#r 913 OOO r/min s t J t 5 min.8.3.3.3 IQR&;SI%A-%KIRJL$TP*fiAffiX-#%JIlo%CTAB RtlBB#%,sE/Q,SbnA 7B;#$R=gl%-WBB(24 1,&B)B,%3)?&,13 000 r/min R J 5 min.8.3.3.4%kElk41+-%iliH#L14:+BZI:-$#Sb;liAH CTAB tKB#%li#i,15 000 r/min,Rt 10 rnin,B.8.3.3.5&EiiPfitlA 200 pL B&k T E HFb,65CB%10 rnin,#%Re 8.3.3.6 bnA=f$fHBkF?H ZiI$,-70c#4$30 minV15 000 r/min 10 m i n,#.a.3.3.7 a3 80%
