PCRPolymerase Chain Reaction Three steps denaturation primer annealing

The PCR cycle • 95℃ step to denature the duplex DNA, • An annealing

procedures • template(DNA or RNA), • primers, • Taq enzyme, • 10×PCR buffer, •

Primer design Most imp. (1)length: 20～ 30 bp (2)G+C contents: ATCG random distributed (3)primers

Primer designed with the help of computer DNA database conservative region comparision primers or

PCR reaction components and their functions template：ss. DNA, ds. DNA m. RNA needed to

primes If the PCR product is to be cloned, it is sensible to include

buffer 10 -50 mmol/L Tris-HCl(p. H 8. 3 -8. 8, 20℃)。 Mg 2+：for Taq

Taq DNA polymerase： from thermophilic bacteria. Taq polymerase from Thermus aquaticus. othermostable DNA polymerase

thermostable DNA polymerases ①Taq DNA polymerase ②Tth DNA polymerase—from T. thermophilus ③VENT DNA polymerase—from

RAPD(Random Amplified Polymorphism DNA) 1990: Williams(Du Pont)：RAPD; Welsh(Cal. Biology Inst. )：AP-PCR(Arbitrary Primer-PCR) =Random Primer-PCR

其他 • 简单序列重复区间（Inter-simple Sequence Repeat, ISSR) • 序列标记位点（Sequence-Tagged Sites, STS) • 小卫星DNA（Minisatellite DNA) •

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PCR(Polymerase Chain Reaction) Three steps: denaturation, primer annealing, polymerization. Peoples Choice Reaction 生命经纬 www. biox. cn

The PCR cycle • 95℃ step to denature the duplex DNA, • An annealing step of around 55℃ to allow the primers to bind, • 72℃ polymerization step. • Mg 2+, d. NTPs are required in addition to template, primers, buffer and enzyme 生命经纬 www. biox. cn

procedures • template(DNA or RNA), • primers, • Taq enzyme, • 10×PCR buffer, • Mg++, • 5 mmol/L d. NTPs • pre-denaturation-denature completely • Denaturation • annealing • Elongation • cycles: 25 -30 生命经纬 www. biox. cn

Primer design Most imp. (1)length: 20～ 30 bp (2)G+C contents: ATCG random distributed (3)primers : no complementary sequence (4)3‘end of the primer: no modification (5)5‘end of the primer: product length，can be modified (6) specificity: less than 70% homology with nonspecific amplification sequence, or 8 bp continuous complementary base 生命经纬 www. biox. cn

Primer designed with the help of computer DNA database conservative region comparision primers or blast 生命经纬 www. biox. cn

PCR reaction components and their functions template：ss. DNA, ds. DNA m. RNA needed to be RT as c. DNA samples：from blood, sperm or any other tissue, from older forensic specimens, from ancient biological samples or in the lab. From bacterial colonies or phage plaques. DNA：very stable 生命经纬 www. biox. cn

primes If the PCR product is to be cloned, it is sensible to include the sequence of unique restriction enzyme sites within the 5’-ends of the primers. Can amplify fragments over 10 kb in length Store: 纯化引物在 25％乙腈溶液中 4℃；冻干引物于-20℃可保存 1 -2年，液体状态于-20℃可保存 6个月。不用时应 20℃保存。生命经纬 www. biox. cn

buffer 10 -50 mmol/L Tris-HCl(p. H 8. 3 -8. 8, 20℃)。 Mg 2+：for Taq polymerase activity conc. : Low: low activity high: non-specific amplification d. NTPs：贮备d. NTP液： 1 mol/L Na. OH 调p. H至中性。贮备浓度为 5 -10 mmol/L，终浓度为 20 -200 umol/L，分装-20℃保存。 4种d. NTP浓度应相同。生命经纬 www. biox. cn

Taq DNA polymerase： from thermophilic bacteria. Taq polymerase from Thermus aquaticus. othermostable DNA polymerase with greater accuracy used for certain applications. mineral oil 生命经纬 www. biox. cn

thermostable DNA polymerases ①Taq DNA polymerase ②Tth DNA polymerase—from T. thermophilus ③VENT DNA polymerase—from T. litoralis ④Sac polymerase ⑤pfu polymerase 生命经纬 www. biox. cn

Molecular marker

RAPD(Random Amplified Polymorphism DNA) 1990: Williams(Du Pont)：RAPD; Welsh(Cal. Biology Inst. )：AP-PCR(Arbitrary Primer-PCR) =Random Primer-PCR =Oligo Primer-PCR =SODA(Small Oligo DNA Analysis） Rapid and simple Random primer: 9 -10 bp 生命经纬 www. biox. cn

其他 • 简单序列重复区间（Inter-simple Sequence Repeat, ISSR) • 序列标记位点（Sequence-Tagged Sites, STS) • 小卫星DNA（Minisatellite DNA) • 单链构型多态性（Single-strand Comformation Polymorphism, SSCP) • 变性梯度凝胶电泳（Denaturing Gradient Gel Electrophoresis, DGGE) 生命经纬 www. biox. cn