基因工程基因工程chapter09

1、1.The polymerase chain reaction in outlinePCR in more detailApplications of PCR2.3.The Polymerase Chain Reaction94,303068 , 1 72 , 13Extension(synthesis)Cycling for 2535 cyclesAnnealing(hybridization)Denaturation9.1PCR in outline1. Denature（变性）94,302. Annealing（退火)( hybridization)3068 , 1 Fig 9.13.

2、Extension（延伸）72 , 13Fig 9.1Fig 9.3PCRPCR reaction: Templates ( DNA molecules) Primers (oligonucleotides) DNA polymerase dNTPs (substrates) Buffer1050 mM Tris.Cl (protect the Taq polymerase) 50 mM KCl (annealing of primer and template ) 1.5mM MgCl2 ( annealing temperature andamplification specificity

3、, 0.5-2.5 mM)The sequence of the primers are critical to a success of PCR.9.2PCR in more detailFig 9.4 The result of PCRs withwell- designed and poorly- designed primers.Lane 1: goodLane 2: nothing Lane 3: wrong sizeLane 4: mixture9.2PCR in more detail1.Designing the oligonucleotide primers for a PC

4、RAGACCAAGGTCTTGTTATGAACTTCTACAAGGATTCTTGTCCTCA GGCTGAAGACATTATCAAGGAGCAAGTTAAGCTACTGTACAAGC GACACAAAAACACTGCTTTTTCTTGGCTGAGAAACATTTTCCAT GACTGTGCTGTCCAGTCATGTGATGCTTCACTGCTGCTGGACTC CACAAGAAGGAGCTTGTCTGAGAAGGAGACAGACAGGAGCTTT GGCCTAAGGAATTTCAGGTACATTGAGACCATCAAAGAAGCTGT AGAAAGGGAATGTCCTGGAGTTGTTTCTT

5、GTGCTGATATCCTTG TTCTGTCTGATATTCCTCTCCAGATGTTGGTGCATCGTTTGTACC CAGAGGTTGACCCTGCCCTCAACCCCGACCATGTTCCACACATA CTCCATAAATGCCCTGATCAGATCCCAGACCCCAAGGCCGTCCA GTATGTGAGAAACGACCGTGGGACACCCATGGTTTTGGATAACA ATTATTACAGGAACATACTGGACAACAAGGGTTTGTTGATTGTGG ATCACCAACTGGCGTACGACAAAAGGACCAGACCTTATGTGAAG AAAATGGCCAAG

6、AGCCAAGACTATTTTTTCAAGGAGTTCTCAAG GGCCATTACTTTCCTTTTTGAGAACAATCCTCTCACTGGTAGCAA GGGTGAGATCAGGAAGCAATGCAATCTTGCAAACAAGCTTCATTAAAATGCCTCGCCAAAGAGAAAGCCGAGTGTGGCCGPrimers for a PCRBased on sites：Based on specifity：Specific primer (专一引物, 特异引物）Degenerated primer (兼并引物) Random primer (随机引物)Forward primerRe

7、verse primerSense primerAntisense primer5-end primer 3-end primerUpstream primerDownstream primerPrimerLength48 = 65536 bp46 000 sitesFig 9.6417=17 179 869 184bponly one site94 1 or97 15 sec72 or 74 35100个核苷酸/秒50-70 Fig 9.72. Working out the correct temperature for PCRFig 9.8Calculating the Tm of a

8、primerin which G + C is the number of G and C nucleotidesin the primer sequence, and and T nucleotides.A + T is the number of AThe annealing temperature is 12C below this figure.30 nt:（G+C）%=(Tm-69.3) x 2.44(30 nt)The principles of primer designing (引物设计的原则)：1、 the length of the primers:1630bp, best

9、 2024bpthe GC content of the primers: 4055%random distribution of four nucleotides avoid the secondary structure of the primers2、3、4、5、the 3-end nucleotide: T is best,A is worst6、 For the degenerated primers(兼并引物)， thedegeneration（兼并性） of 3-end should be smallGGAGACCAAGGTCTTGTTATGAACTTCTACAAGGATTCTT

10、GTCCTC AGGCTGAAGACATTATCAAGGAGCAAGTTAAGCTACTGTACAAG CGACACAAAAACACTGCTTTTTCTTGGCTGAGAAACATTTTCCA TGACTGTGCTGTCCAGTCATGTGATGCTTCACTGCTGCTGGACT CCACAAGAAGGAGCTTGTCTGAGAAGGAGACAGACAGGAGCTT TGGCCTAAGGAATTTCAGGTACATTGAGACCATCAAAGAAGCTG TAGAAAGGGAATGTCCTGGAGTTGTTTCTTGTGCTGATATCCTT GTTCTGTCTGATATTCCTCTCCAG

11、ATGTTGGTGCATCGTTTGTAC CCAGAGGTTGACCCTGCCCTCAACCCCGACCATGTTCCACACAT ACTCCATAAATGCCCTGATCAGATCCCAGACCCCAAGGCCGTCC AGTATGTGAGAAACGACCGTGGGACACCCATGGTTTTGGATAAC AATTATTACAGGAACATACTGGACAACAAGGGTTTGTTGATTGTG GATCACCAACTGGCGTACGACAAAAGGACCAGACCTTATGTGAA GAAAATGGCCAAGAGCCAAGACTATTTTTTCAAGGAGTTCTCAA GGGCCAT

12、TACTTTCCTTTTTGAGAACAATCCTCTCACTGGTAGCA AGGGTGAGATCAGGAAGCAATGCAATCTTGCAAACAAGCTTCATTAAAATGCCTCGCCAAAGAGAAAGCCGAGTGTGGCCG(1) Gel electrophoresis of PCR products(2) Cloning of PCR products(3) Sequencing of PCR products9.3After the PCR:studying PCR products3.After the PCR:studying PCR products (1)Gel e

13、lectrophoresis of PCR productsFig 9.10(2)Cloning PCR productsUsing the property of Taq polymeraseMany Taq polymerase can add a A at the 3end of a DNA strandT cloning vectorvectorBlunt end vector dTTPNo primer polymeraseFig 9.12 (2)Cloning PCR products PCR produce stiky endand PCR products restrictio

14、n enzyme(3)Problem with the error rate of Taq polymerase30 cycles1/9000bp1/300bp高保真 DNA polymerasepfu DNA polymerase9.4.1 Carrying out a quantitative PCR (qPCR ) experimentWTSPI2SPI3SPI7SPI8SPIEF1-a9.4Real-time PCR enables the amount of starting material to be quantifiedReal-time quantitative Polymerase Chain Reaction(Real T