第三节常用的目的基因制备方法: 1.聚合酶链式反应( polymerase chain reaction, pcr): 2反转录PCR( retrotranscription PCR,RT -PCR) 3.基因文库筛选: 基因组文库: cDNA文库 4.化学合成:
第三节 常用的目的基因制备方法： 1.聚合酶链式反应（polymerase chain reaction,PCR）： 2.反转录PCR（retrotranscription PCR, RT －PCR）： 3.基因文库筛选： 基因组文库： cDNA文库： 4.化学合成： 26
Pcr Denaturing Template DNA Heat causes dna strands to separate Denaturation of dna at 94oC
Denaturing Template DNA Heat causes DNA strands to separate 3′ 5′ 5′ 3′ Denaturation of DNA at 94oC 5′ 3′ 3′ 5′ PCR 27
PCR Annealing primers primers bind to the template sequence Taq polymerase recognizes double-stranded substrate 5 3 ↓ Primers anneal at 55oc
Annealing Primers •Primers bind to the template sequence •Taq polymerase recognizes double-stranded substrate 3′ 5′ 5′ 3′ Primers anneal at 55oC 3′ 5′ 5′ 3′ 3′ 5′ 5′ 3′ PCR 28
PCR Tag polymerase extends ●●6●● Tag polymerase extends primer DNA is replicated 5 3 Extend at 72oC 国国国国国国回回回回回国国国国圆_■■■■□ 3~ Repeat denaturing, annealing, and extending 30 cycles
5′ 3′ 3′ Taq polymerase extends...... 3′ 5′ 5′ Extend at 72oC 5′ 3′ •Taq polymerase extends primer •DNA is replicated Repeat denaturing, annealing, and extending 30 cycles 5′ 3′ 5′ 3′ 3′ 5′ 5′ 3′ PCR 29
RT-PCR AAAAAAAAA 3 mRNA reverse transcriptase anchor primer (VTTTT )V=G, C or A AAAAAAAAA 3 mRNA 3 TTTT CDNA RNase or RNase activity of reverse transcriptase TTTT 5′cDMA Taq DNA polymerase sequence-specific primer(-) anchor primer ds cdNA TTTT 5′ 5 3 5
RT-PCR 30