2004级研究生现代免疫学实验技术结业报告

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1 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA PCR PCR DNA DNA Y (1 X) n Y DNA X (Y) n 100% DNA PCR DNA PCR DNA PCR PCR 5 DNA ( ) 5 3 PCR DNA PCR 1

2 PCR 10 10ul 4 dntp 200umol/L pmol DNA 0.1 2ug Taq DNA 2.5u Mg2+ 1.5mmol/L 100ul PCR PCR dntp Mg2+ 1 PCR PCR DNA DNA PCR DNA 18-24bp 20bp bp 10kb G+C 40-60% G+C G+C ATGC PCR 0.1 1umol pmol 2 Taq DNA PCR 2.5U( 100ul ) 3 dntp dntp PCR dntp dntp 1M NaOH 1M Tris HCL PH dntp PCR dntp umol/L 4 dntp ( ) ( ) PCR dntp Mg2+ Mg2+ 4 ( ) PCR DNA SDS K SDS SDS K DNA PCR PCR RNA 2

3 K RNase RNA TRIZOL RNA DNA protein 5 Mg 2+ Mg 2+ PCR PCR dntp 200umol/L Mg mmol/L Mg2+ Taq DNA PCR PCR 1 PCR - - DNA Taq DNA ( bp ) ( Taq DNA ) PCR lmin DNA 93 PCR PCR ( ) PCR DNA 20 G+C 50% 55 Tm ( )=4(G+C) 2(A+T) =Tm -(3 12 ) Tm PCR 20 40sec Taq DNA /S/ /S/ /S/ 90 DNA PCR PCR 500bp 20s bp 40s 3 4kb 3 4min 10Kb 15min 500bp/30s 2 PCR PCR DNA PCR 1 PCR DNA 3

4 Taq DNA Taq DNA ( ) 2 PCR (pg=10-12 g) (ug=10-6 g) 100 PCR 3 RFU( ) 3 3 PCR Taq DNA DNA DNA RNA DNA PCR PCR PCR 1 PCR EB PCR PCR 1 2% 6 10% 2 PCR 3 PCR PCR Southern ( ) PCR PCR PCR PCR 4 PCR PCR PCR 48h 48h 1 PCR PCR 4

5 Taq Taq PCR OD PCR Mg 2+ Mg 2+ PCR PCR PCR PCR PCR 20ul 30ul 50ul 100ul PCR 20ul PCR PCR PCR PCR 2 PCR PCR PCR PCR PCR 3 PCR Mg 2+ PCR 5

6 (93 65 ) 4 PCR dntp Mg 2+ dntp Mg 2+ 5 PCR PCR A ( ) ( )PCR PCR PCR. ( )PCR PCR PCR ( 1013 /ml) PCR PCR PCR ( ) B 1. PCR PCR PCR (100 ) PCR 2. PCR PCR DNA RNA PCR PCR C 6

7 ( ) PCR PCR PCR PCR PCR PCR PCR DNA RNA ( ) ( ) PCR PCR PCR -20 PCR PCR PCR ( ) ( ) ( ) PCR PCR ( ) Eppendorf RT-PCR RT-PCR RT-PCR PCR RNA cdna PCR RT-PCR cdna cdna RT-PCR Northern RNase S1 RNA RT-PCR RNA poly(a)+ RNA oligo(dt) GSP RT-PCR RT-PCR cdna 1/10 PCR RT-PCR PCR PCR RT-PCR RNA cdna RNA RNA EDTA SDS RNA cdna RNA Trizol RNA Trizol 100 1mg RNA RNase cdna RNase DTT RT-PCR protocol 7

8 H 2 O 10 MgCl 2 TaqDNA 5U/ul dntp( 2.5mmol/L) 10pmol/ul -20 DNA DNA0.1ug/ul, 0.1ng/ul RNA (RNasin) DTT RNasin DTT DTT Taq M-MLV Oligo(dT) ug/ml PCR 1 0.5ml PCR mrna(1ug/ul) 10ul Oligo(dT) (1ug/ml) 10ul Tris-HCl (1mol/L ph7.6) 2.5ul KCL (1mol/L) 3.5ul MgCl 2 (25mmol/L) 2ul dntp (5mmol each) 10ul DTT (0.1mol/L) 2ul RNA 45U 48ul 2 2ul M-MLV pharmacia, U/ml Triton X h min 5 0.5ml PCR 30ul 10 10ul dntp (2.5mmol/L) 8ul (10pmol/ul 10ul (10pmol/ul 10ul 5ul 100ul 1 10 ph 8.3, 2ul dntp (10mmol each) 2ul Oligo(dT) (100ug/ml) 1ul RNA 20U mrna 1-2ug MgCl 2 (50mmol/L) 1ul 20ul U MMLV 37 30min 8

9 3 95 5min pmol 10-50pmol 1 100ul Taq DNA 2.5U PCR PCR PCR RT-PCR 1. cdna *RNA * mrna * * 50 l 2. * PCR RT-PCR * RNA * RNA * PCR 1/10 * Oligo(dT) GSP RNA GSP SS * mrna a. 50 b. Oligo(dT) 3. * RT DNA RT PCR DNase I * PCR Tm 2 5 DNA * mrna RT-PCR 4. smear * PCR * * PCR / PCR * DNase DNA RNA 5. * * PCR cdna RNA * RNA DNA 9

10 DNA cdna DNA * 7. * PCR DNA 100 * PCR Tm 5 8. SS SS * 50 * 220 * PCR * *Tdt 9. SS ThermoScript ThermoScript SS 10. GSP GSP OligodT RNA mrna 11. RNase H PCR RNA/DNA 12. RT PCR PCR DNA RT-PCR RT-PCR DNA RT-PCR Platinum Taq RT-PCR Pfx Taq RT-PCR RT-PCR Elongase RT-PCR 10

11 dntp Amresco 0.5ml 10mmol each 60 rtaq DNA 500U Fermentas 140 M-MLV 10000U 200U/ul 350 Oligo(dT) 15 20ug Promega 300 RNase Inhibitor 2000 U/ul 40U/ul invitrogen PCR Optimizer Kit (K ) $128 PCR Reagent System ( ) $211 RT-PCR ThermoScript II RT-PCR System for First-Strand cdna Synthesis $117 ThermoScript II RT-PCR System plus Platinum Taq DNA Polymerase $121 11

12 RT-PCR SuperScript III One-Step RT-PCR System with Taq DNA Polymerase $115 PCR RT-PCR PCR real-time PCR 1 PCR? PCR PCR PCR real-time fluorescent quantitative PCR FQ-PCR PCR PCR PCR PCR PCR PCR FQ-PCR 2 FQ-PCR? PCR 1 PCR PCR 2 cdna(1) PCR mimic PCR 2 3 PCR PCR PCR PCR ( ) ( ) EB FQ-PCR 3? mrna mrna FQ-PCR PCR EB 4 FQ-PCR? Taqman Molecular Beacon 12

13 SNP SNP DNA DNA Laird Methylight DNA Taqman DNA PCR 5 FQ-PCR? mrna SYBR Green I RNA trizol Oligo(dT) mrna ( PCR FQ-PCR ) Taq dntp RT-PCR buffer [ SYBR Green I] FQ-PCR SYBR Green I TaqMan 6 FQ-PCR RNA mrna mrna mrna -20 FQ-PCR FQ-PCR 7 RNA TRIZOL RNA -5~-20 RNA -60~-70 RNA RNA ph 3.5 RNA degradation Tissues were not immediately processed or frozen after removal from the animal. Samples used for isolation, or the isolated RNA preparations were stored at -5 to-20 C, instead of -60 to -70 C. Cells were dispersed by trypsin digestion. Aqueous solutions or tubes were not RNase-free. 13

14 Formaldehyde used for agarose-gel electrophoresis had a ph below FQ-PCR Internal Control GAPDH -actin 9 FQ-PCR 1 threshold PCR (2)Ct C Cycle t threshold PCR Ct Ct DNA A 1 B 5 50 A B 50 threshold Ct Ct threshold Ct 10 SYBR Green I TaqMan SYBR Green I SYBR Green I DNA DNA DNA DNA DNA TaqMan Taq 5' 3' PCR PCR 11 FQ-PCR 2 - Ct Ct=(Ct target -Ct actin ) timex -(Ct target -Ct actin ) time0 timex time0 Ct c-myc GAPDH c-myc GAPDH SYBR Green I DNA c-myc GAPDH Ct Ct c-myc GAPDH PCR TaqMan 14

15 c-myc GAPDH cdna CT 2 - Ct 12 PCR PCR DNA Engine Opticon TM ml 470nm 505nm 523nm 543nm SYBR Green, FAM 540nm C C 0.4 C 30 C 105 C 15

荧光定量PCR

荧光定量PCR 荧光定量 PCR 荧光定量 PCR 原理荧光定量 PCR 最早称 TaqMan PCR, 后来也叫 Real-Time PCR, 是美国 PE(Perkin Elmer) 公司 1995 年研制出来的一种新的核酸定量技术该技术是在常规 PCR 基础上加入荧光标记探针或相应的荧光染料