SNT 1198-2003 马铃薯中转基因成分定性PCR检测方法.pdf

Protocol of the polymerase chain reaction for detecting genetically modified components in potatoes*&BB74%S+H%SBB3)HBtk PCR U1k?kO;4&#&3;B 7 q 9 a pa$(NEW L E A P POTATO LINES BT-06,ATBT 04-06,ATBT 04-31,ATBT 04-36 AND SPBT 02-05;(NEW L E A P PLUS POTATO LINES PBMT 21-129,PBMT 21-350 AND PBMT 22-82 i(NEW LEAFY)POTATO LINES RBMT 15-101,SEMT 15-02 AND SEMT 15;15%&HH%SB4S&%-.tf&4S0iFA,4SiF8HUStle 7;PJ$tESZX(SB)3H;f;46.3.1 transgene#Hr4W6WHsff%TJ%fi!iHHrJ%DNA BFU*a3&H%APR,XBB&#+a?74&9 RE%lWIR.?f)8JiHpa$f&E.3.2 ff&*$EBi polymerase chain reaction,PCR#i&iSBFFPJ5C8!iB!Etk&%#DNA&WFHi3BHlZG%#7;,i#&iJYPJi2 itHH&51B3)%1-4&DNA H%Pk#GH-&X+E89J84Bkmi#4:%+,#&S?!E DNA Se IH+H7;RDlWI%SB%(dNTP)%liii,5I%Uj,#G6WfiP#kI#Ij$ia-#w,arMHsm%vALm#%r*o 3.3 P%ia 3.3.1 Pata:4%lWIMfRl!k DNA 31%,Hl!H%3%4%?ASafllEHEl Patatin SB#.3.3.2 CaMV 35S:promoter from cauliflomer mosaic virus,&$35s g 33.3.3.3 NPT-1:neomycin phosphotransferase-fl,-3 -%m 3.3.4 N0S:nopaline synthase terrninator,lflB%6Bm 3%3%k 3.3.3.5 PVYcp:potato Y virus coat protein,4+$%Y YS9ESEPQ.3.3.6 cry 3A:-+A B.thuringiensis subsp,Tcnebrionis(B.t.t.strain BI 256-82 Hgh.j&/Fcg EI W8%&%tk,ft$EEJ j&#t k%+4;C 4 4 S T&(Colorado potato beetle larvae).1 3.3.7 CP4 EPSPS:5-enolpyrulshikimate3-phosphate synthase 5-enolpyrul shikimate3-phosphate synthase,*aT$!fi Agrobacterium sp.Strain CP4 H 5-*-3-g&j 3.3.8 PLRVrep:potato leafroll virus,$#S%W#3SSBFFPJ 3.3.9 E93:the 3 non-translated region of the pea ribulosel,5-bisphosphate carboxylase small subunit(rbcS)E9 gene.,SRBTWiZHBM#-l,5-I.#t%j E9 SBi/JEi&3%lJ?PJ 3.3.10 ArabSSU1 A:arabidopsis thaliana ribulosel 5-bisphosphate carboxylase(Rubisco)small subunit ats 1A promoter,arabidopsis thaliana&#-1,5-1#t%B J E 1A mB37b3.3.3.11 aad:(resides outside T-DNA)?ZffB I3 fin 7 rfrSi%SAjeSa%E(S)ffjk%jH SBiO 3.3.12 oriV:(resides outside T-DNA)-+$!RE Agrobacterium H RK2 1.3 kb MBBJ EM0 3.3.13 ori 322t(resides outsideT-DNA)-+%iG pBR322BBm 1.8 kbH#&,#rfr&-$g$djEj&%l4%4$%&#LA bom.3.3.14 FMV 35s:a promotor derived from figwort mosaic virus(FMV),gzw$S 35s a?.I 5.2 q*mW#5.2.1 ISZ.&ISB.&aR.;(sHS#O-&ess.*s.*s#,$+#aa%,aa/ntn7;1 cm3 M#&%Sf&HTf JBt DNA.X#fM-f&Mq%sB#,z$+#&M-#q#%T#,H/Jntn7;2 cm2 MW#%Ct&H ff JBt DNA.v P 5.2.2 9#aStl)S#Hr$l&ESJBt&M 120 g BRfE3)3FE,M+120 g+z$100 mg%X#.6.1#&Pa?lBz$DNA G,HH%S$14&fiHAM9EIS$1 MS$1JF9Jiflit5l#,S?l PCR B t$9#FF#VM#%S$1H DNA fipa,&B PCR VMSa*;rJlBEfiB&+ES*g%SB&3).6.2 Sfl#%WgSB9E,g#XB1%3)6%8&fiX5PJ,*%If?%GB/T 6682%BM-%*.6.2.1 3l%r SHS 3#M I#I&I#u A%*E&100 pmol/pL P2Cf9Bil&BB R T PCR E&H 20 pmol/pI HIfF#iE.!i!,bffZk A.3 f t i H q#?a PCR l!&arlMH5lIbPlr9JB PCRPlblkl 31 4 8 PATA CaMV 35s FMV 35s NOS NPTII PLRVrep PVYCP Cry3A)+l?2 216 bp 195 bp 365 bp 180 bp 173 bp 172 bp 161 bp 112 bp*S nm fim 9 b m 9bS na 9bEi%&F9NM Ni!&.SB&W NEW L E A P%ii!&.PB&W NEW L E A P PLUS NEW L E A P Y R%&W NEW L E A P PLUS NEW L E A P Y NEW L E A P%a NEW LEAP PLUS NEW LEAP Y NEW L E A P PBLlW NEW LEAP PLUS Saua NEW LEAP Y Palla NEW LEAP PLUS NEW LEAP Y NEW L E A P 31 F 5-tga cct gga cac cac agt tat-3 R 5-gtg gat ttc agg agt tct tcg-3 F 5-gct cct aca aat gcc atc a-3 R 5-gat agt ggg att gtg cgt ca-3 F 5-agt cca aag cct caa caa ggt c3 R 5-cat tag tga gtg ggc tgt cag g-3 F 5-gaa tcc tgt tgc cgg tct tg-3 R 5-tta tcc tag ttt gcg cgc ta-3 F 5-gga tct cct gtc atc t-3 R 5-gat cat cct gat cga c3 F 5-tcg tca tta aac ttg acg ac-3 R 5-ctt ctt tca cgg agt tcc ag-3 F 5-gaa tca agg cta tca cgt cc-3 R 5-cat ccg cac tgc ctc ata cc-3 5-aga gcc gtc gca aac acc aat c-3 5-tct ggg tgc tgg cct cat cg-3 6.2.2 Tag DNA#t$B 6.2.3 dNTP:dATP.dTTP.dCTP,dGTP,dUTP.6.2.4 BflBM:Q%o 6.2.5 4kZ(EB)8)c.1b!&dJ 6.2.6 3.gRTR-6.2.7;WEB.6.2.8 70%ZB.6.2.9*?-1&Marker:50 bp-300 bp.6.2.10,DNA slg%:sl 100 mL lmol/L Tris-Cl,SO mL 0.5 mol/L EDTA,5.0 g+-ESQB?H(SDS)916.8 48 g.g R k g C t J ,$E 1 L,*%Xi%le/-$H.6.2.11 TE 8 10 mmol/L Tris-HC1,pH 8.Of 1 mmol/L EDTA9pH8.0.6.2.12 lOXPCRS*%:lOO mmol/L.gRkQrfl(KCl,160 mm0l/LfjilE(NH)SO,20 mmol/I.jB(MgS04 1,200 mmol/L Tris-Hcl(H8.8)1%Triton X-100,l mg/mL BSA.6.2.13 5XTBE:Tris,54g,E27.5g,0.5mol/LEDTA(pH8.0)20mL,ffU 2 1 000 mL.6.2.14 IOXMS%:O.2 s x s m 1 4 0%a a.6.2.15 RNAE(10 pg/mL).6.2.16 is%Z#Pk%BM(PVP)6.3 R 9 1 1 6.3.1 H#8#t&236ff#.6.3.2 Ra%&Lbt&,&S/laBllz&,Mini-?-ABJL$L.6.3.3;rk%#f3#jlET,B8i%3,8iRB%30 6.3.4*y:&f 0.001 g.6.3.5&EXi%.6.3.6&BT#jlET.6.3.7 8ti;rkl,XX%7Jcl 6.3.8 E?S.?9 1&%j l E T o 6.3.9 BJ%&.6.3.10 RR#B%!6.3.11#Po 6.3.12 SBVWiR.6.3.13 Qt&i!Xm 6.3.14 PCR&#If$fi.6.3.15&iBiB%fiiR.6.3.16#it.&#:O.1 pL-2 pL.0.5 PI.-10 pL92 pL-20 pL,10 pL-100 pL,20 pL-200 pL9 200 pL-1 000 pL.6.3.17 E&#?5%o 6.3.18%El$%*PCR a.6.3.19 Eppendorf :1.5 mL-5 mL.6.3.20 PCR lZ&:200 pL,500 pL m#j4!#!ia 6.4 6.4.1#iB;DNA#!SRR T .&.&.&#+#*E E T .f f u%*#H&4 Se%100 me fBM&SB&#lS!h%?1.5 mL M eppendorf+,MA 600 PL Bi%MBSlrP%,Afih 200 pL20%m PVP,%,65C#l20 min,#lMiila&3&e MA#lRI-3.blVBtgZ$2M lSQ.10 000 r/min$Sib 10 mine 1;bA33)21#lRbjcMSCEgPr,S3J.10 000 r/min P f l b l o i n.*H13R%+mWEfjBP9&NXE8B$RB.ltiJB$R$Z+fRA 10 PL M R N A E (10 mg/mL),37 30 min.fiUA 1 mL.Z98(5 mol/L.F 10 000 r/min 10 min.&#-tJAT%,