PCR ( P olymerase C hain R eaction)

PCR (Polymerase Chain Reaction)

聚合酶 链式 反应

生化教研室： 牛永东

PCR is to make a huge number of copies of a gene in vitro

What is PCR ?

1.In April, 1983, Kary Mullis took a drive on a moonlit California mountain road. During that drive, he conceived the Polymerase Chain Reaction (PCR)

HISTORYHISTORY

lives in a small apt. near the beach

2. He received a $10K bonus from Cetus, Cetus later sold the technolgy to La Roche for $300 million

“I do my best thinking while driving”

1993 Nobel prize

OUTLINE•3 concepts•5 Essential Components

•3 steps•One sentence

3 Concepts of the PCR

1. Denaturation : 变性

2. Renaturation ：复性

3. Semiconservative replication ：半保留复制

the double strand DNA open to single stranded DNA

two single stranded DNA form double strand DNA

replication, one make two

Semi-conservative replication

Old

new

5 Essential Components of PCR Reaction

salts (ions)

dNTPsdNTPs

PrimersPrimers

DNA DNA PolymPolymeraseerase

Template Template DNADNA

Taq DNA Polymerase1. Taq : Thermus aquaticus

2. 95℃ ,T1/2(half life) 40min

3. Polymerization

Polymerization 聚合反应

Taq PolymerasedT

dAdG

dC

dT

5 ’ 3 ’

3 ’ 5 ’A

Taq polymerase: 72℃, 2,000 ～ 4,000 bases/min

•three steps:

How PCR works

Denaturation ( 变性 ): 90 ～ 97℃Annealing ( 退火 ): 45 ～

55℃Extension ( 延伸 ): around 72℃

TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATGACATCGATTTGATCGATACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA

Step 1 : Denaturation 变性 94 ℃

TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATGACATCGATTTGATCGAT

ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA

Step 2 : Annealing 退火T ℃ primer-dependent

CGTAGTAGCA

GCTGCTACGTAG

TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATGACATCGATTTGATCGAT

ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA

Step 3 : Extension 延伸72 ℃

CGTAGTAGCA

GCTGCTACGTAG

Taq Polymerase

Taq Polymerase

TAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATCGCGATAT

TACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGA

聚合酶链式反应

1. Denaturation

2. Annealing

3. Extension

PCR.EXE

Last Step : 72 ℃ 2 min

After 25~30 cycles

?

One sentence

One makes two, two make four, four make anything

一生二，二生四，四生万物

Thank you for your tolerance ！