pcr ( p olymerase c hain r eaction)
DESCRIPTION
PCR ( P olymerase C hain R eaction). 聚合酶 链 式 反应. 生化教研室: 牛永东. What is PCR ?. P CR is to make a huge number of copies of a gene in vitro. HISTORY. lives in a small apt. near the beach. - PowerPoint PPT PresentationTRANSCRIPT
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 !