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2、%( 6) ( i.( 2) 3 k4 RNA4 k4Trizol1 SInvitrogen ;yFDNA4 | k41?3; I c , RNAase4, SYBR Green Rea-lTimePCR mix (1 TOYOBA .( 3)* X sPDGFRA , 3 qPrimerPrimer510 !9, : 5c-AGCTGATCCGTGCTAAGGAA- 3c,/: 5c-TCCACTCAACATCAGGAAGC- 3c, 9167 bp.1. 2 ZE( 1)RT-PCR TrizolE4 |9RNA. I cQHq: 65 e 5min, 30 e 10min, 42 e 6
3、0m in,72 e 10min. PCRQHqM95 e 5m in;94 e 30 s, 53 e 30 s, 72 e 20 s, 32; 72 e %10min.8s 018% j k “H.( 2)4 |yFDNAF T,4 |F%CHO-K1yFDNAF pIRES-Neo3-sPDGFRA, sN_4 |DNA # i.( 3)S y vP Song11yS ZE,9 c101a102a103a104a105y J Vr8 , CHO-K1yF |10ng, T /:J cyVr8vl( 6 832 bp) yFDNA8vl(3 109 bp) =cyVr8 CHO-K1yF |
4、 Vr8(V J )CHO-K1yFDNA( 10 ng),yS .( 4) L H PCR8“ !Q8“: 2 LL, ( 10 pmol# L- 1 )018 LL,/( 10 pmol# L- 1 ) 018 LL, SYBR Green Rea-lTime PCRM ix 10LL, 614 LL.QHq:95 e 10min; 95 e 10 s, 60 e 20 s, 72 e 30 s, 32. wLs:55 e 95 e , sBQ. “ !3 “, | (.2 T2. 1 F%VrsPDGFRAyk|F% !,4 |RNA, RT-PCR9sPDGFRAy,8s 018% j
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6、S Cv ,V S !. Q+s, wLs(m2b),88 e OB,V 9+sdz.( 2)S wLT J 1 ( lgN ):US,CtUS,T S wL,i Z TY= - 01244 5X + 51731 3.m2 L H PCR_S J 2. 3 F%ysPDGFRA J k4 |F%yF,8s 018% j (m3),A UyFRNA .sY |10 ng L H PCR, 9sPDGFRAy(m4),i3Q L.V1 U,F%1sPDGFRAy3Q _CtSl, Lz. J 9Gv TY= - 01244 5X + 51731 39 .M: 100 bp DNAM arker;
7、1: CHO-K1yF;2 7:F%yFm3 yF mm4 F%1sPDGFRAy L H; PCRV1 L H PCR_F% J F%I|Ct1 2 3 (?SJ 1 18. 74 18. 61 18. 69 18. 68? 0. 06 14. 42 15. 21 15. 10 15. 17 15. 16? 0. 05 104. 73 18. 85 18. 98 18. 76 18. 86? 0. 11 13. 24 23. 06 23. 14 23. 12 23. 11? 0. 04 1. 25 21. 81 21. 86 21. 92 21. 86? 0. 05 2. 46 19. 90
8、 19. 82 19. 88 19. 87? 0. 04 7. 43 ) _y J ,; PCR/ Southern blots0ZEeL y, “ ,b _,( 120 LLQ8“ k42 3 ;),Y ( 1QQV_96“ ), BZEK X1B*zS ,NS X yyFDNA,i OSouth-ern blot y J . L=T , %, a a 3+y, 4“B*S .8D/ ,vP Song 1149Z, y J %yFDNAvl,B i1 qB*S, LC L H; PCRZEyJ 5 .ZE rF%1il, Vr J312 2v(1 S)30 r e, z 3,8= f12,v
9、 )M1, %Vr , y0 3 .1rVr%,“ Vr8Vr%/13, 91.7 S yVr J ,N f /,y V ?yF c u(Hot spot),rVr,7d$ 14- 15. L H; PCR/ F%1y J _,ZE 2 ,rF%41G . ID 1 FLAVELL R B. Inacrtivation of gene expression in plantsas a consequence of specific sequence duplication J.ProcNatlAcad SciUSA, 1994, 91( 9): 3490- 3496. 2 VAUCHERET
10、H, B*CLIN C, ELMAYAN T, et a.lTransgene-induced gene silencing in plants J. Plant J,1998, 16( 6): 651- 659. 3 YANG L, D ING J, ZHANG C, et a.l Estimating thecopy number of transgenes in transformed rice by rea-ltime quantitativePCR J. PlantCellRep, 2005, 23( 10- 11): 759- 763. 4 MASON G, PROVERO P,
11、VA IRA A M, et a.l Estima-ting the number of integrations in transformed plants byquantitative rea-l timePCR J. BMC Biotechno,l 2002,24( 2): 20. 5 MALAKHO S G, KORSHUNOV A, STROGANOVA AM, et a.l Fast detection ofMYCN copy number altera-tions in brain neuronal tumors by rea-l time PCR J. JClin LabAna
12、,l 2008, 22( 2): 123- 130. 6 W ILKE K, DUMAN B, HORST J. D iagnosis of haploidyand triploidy based on measurement of gene copy numberby rea-l timePCR J. Hum Mutat, 2000, 16( 5): 431- 436. 7 CANDELA M, V ITALIB, MATTEUZZID, et a.l Evalu-ation of the rrn operon copy number inB ifidobacterium u-sing re
13、a-l timePCR J. LettApplM icrobio,l 2004, 38( 3): 229- 232. 8 OMAR A A, GRAHAM JH, GROSSER JW. Estimationof transgene copy number in transformed citrus plants byquantitative multiplex rea-l time PCR J. Biotechno.lProg, 2008, 24( 6): 1241- 1248. 9 MORILLO JM, LAU L, SANZM, et a.l Quantitative re-a-l t
14、ime polymerase chain reaction based on single copygene sequence for detection of periodontal pathogens J.J Clin Periodonto,l 2004, 31( 12): 1054- 1060. 10 ANHUF D, EGGERMANN T, ZERRESK, et a.l Deter-m ination of SMN1 and SMN2 copy number using Taq-M anTM technology J. Hum M utat, 2003, 22( 1): 74- 7
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