The Polymerase Chain Reaction 1. The polymerase chain reaction in outline outline 2. PCR in more detail 3. Applications of PCR

Denaturation Annealing (hybridization) Extension (synthesis) 94 ℃, 30″ 30~68 ℃, 1 ′ 72 ℃, 1~3′ Cycling for 25~35 cycles 9.1 PCR in outline

1.Denature （变性） （变性） 2.Annealing （退火 ) ( hybridization) 94 ℃, 30″ 30~68 ℃, 1 ′ Fig 9.1

3.Extension （延伸） （延伸） 72 ℃, 1~3′ Fig 9.1

Fig 9.3

PCR

9.2 PCR in more detail The sequence of the primers are critical to a success of PCR. The sequence of the primers are critical to a success of PCR. PCR reaction: ● Templates ( DNA molecules) ● Primers (oligonucleotides) ● DNA polymerase ● dNTPs (substrates) ● Buffer 10~50 mM Tris.Cl (protect the Taq polymerase) 10~50 mM Tris.Cl (protect the Taq polymerase) 50 mM KCl (annealing of primer and template ) 50 mM KCl (annealing of primer and template ) 1.5mM MgCl 2 ( annealing temperature and amplification specificity, mM) 1.5mM MgCl 2 ( annealing temperature and amplification specificity, mM)

9.2 PCR in more detail Fig 9.4 The result of PCRs with well- designed and poorly- designed primers. Lane 1: good Lane 2: nothing Lane 3: wrong size Lane 4: mixture

1. Designing the oligonucleotide primers for a PCR

Primers for a PCR 5’-end primer 3’-end primer Sense primer Antisense primer Forward primer Reverse primer Upstream primer Downstream primer Based on sites Based on sites ： Based on specifity 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 )

PrimerLength 4 8 = bp sites 4 17 = bp only one site Fig 9.6

2. Working out the correct temperature for PCR ℃ 1’ or 94 ℃ 1’ or 97 ℃ 15 sec 97 ℃ 15 sec 72 ℃ or 74 ℃ 35 ～ 100 个核苷酸 / 秒 Fig 9.7 ℃ ℃

Fig 9.8

Calculating the Tm of a primer in which [G + C] is the number of G and C nucleotides in the primer sequence, and [A + T] is the number of A and T nucleotides. The annealing temperature is 1–2°C below this figure. (<30 nt) >30 nt : >30 nt : （ G+C ） %=(Tm-69.3) x 2.44

The principles of primer designing ( 引物设计的原则 ) ： 1 、 the length of the primers: 16~30bp, best 20 ～ 24bp 2 、 the GC content of the primers: 40~55% 3 、 random distribution of four nucleotides 4 、 avoid the secondary structure of the primers 5 、 the 3’-end nucleotide: T is best, A is worst 6 、 For the degenerated primers( 兼并引物 ) ， the degeneration （ 兼并性 ） of 3’-end should be small degeneration （ 兼并性 ） of 3’-end should be small

(1)Gel electrophoresis of PCR products (2)Cloning of PCR products (3)Sequencing of PCR products 9.3 After the PCR: studying PCR products

3. After the PCR: studying PCR products (1) Gel electrophoresis of PCR products

(2) Cloning PCR products T cloning vector ● Using the property of Taq polymerase Many Taq polymerase can add a A at the 3’end of a DNA strand vector Blunt end vector dTTP No primer polymerase

Fig 9.12

● PCR produce stiky end and PCR products restriction enzyme (2) Cloning PCR products

(3) Problem with the error rate of Taq polymerase 高保真 DNA polymerase pfu DNA polymerase pfu DNA polymerase 1/9000bp 1/300bp 30 cycles

9.4 Real-time PCR enables the amount of starting material to be quantified Carrying out a quantitative PCR (qPCR ) experiment SPI EF1-a WT SPI2 SPI3 SPI7 SPI8

荧光定量 PCR ) Real-time quantitative Polymerase Chain Reaction (Real Time PCR )

9.4.2 Real-time PCR can also quantify RNA

Application of PCR ● RFLP, RAPD ● 定量 PCR ● isolaton of homologous gene ● Insertion 、 deletion or point mutation ● Identify a desired gene from a cDNA library

The end of chapter 9