a story about section 2. what is pcr? polymerase chain reaction a method to synthesis specific dna...
TRANSCRIPT
A story about
Section 2
What is PCR?
Polymerase Chain Reaction
A method to synthesis specific DNA fragment in vitro. It is composed of many cycles including high temperature denaturation, low temperature annealing and proper temperature elongation to amplify the purpose DNA specifically.
The history of PCR
1971, Khorana : DNA denaturation , hybridization with proper primer , elongation with DNA polymerase primer , repeat the cycle to gain a clone of gene 。
1983, Mullis wanted to synthesis DNA primer to do sequencing, but he is in trouble in lacking of template DNA.
On a Friday night in April 1983, he got the idea about PCR reaction while he was driving.
1985, the first article about PCR technique was published on Science.
1986 , Mullis gave a lecture about PCR technique, people from all over the world began to study PCR.
In December 1983 , Mullis saw the first PCR product in the world, a 49bp DNA fragment after 10 cycles.
Kary MullisIn 1993 , 7 years after the invention of PCR, he won the Noble prize.
5 Primer 15 Primer 2
Cycle 2
Cycle 1
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5 5
Template DNA
I. The principle of PCR
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5 5
Cycle 3
5
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5 5 5
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After 25 ~ 30 cycle , template DNA will be amplified over 1 million times 。
PCR principle
PCR 的基本反应步骤
denaturation
95˚C
elongation72˚C
annealingTm-5˚C
PCR cycle
72℃72℃9494℃ 55℃55℃
1 PCR cycle
denaturation annealing elongation
•Template DNA
•Specific primers
•dNTPs
•Mg2+
•Hot-resistance DNA polymerase
II. Basic components of PCR system
PCR reaction system
TaqTaq
P1P1
P2P2
dATP
dTTP
dGTPdCTP
Mg2+
Co-factor : Mg2+
template : DNA
primer : P1 P2
DNA polymerase : Taq
Synthesis material : dNTP
PCR buffer
template
• Genomic DNA• cDNA
No proteinase, nulease, DNA binding protein or DNA polymerase inhibitors were permitted.
Template in extremely high concentration would lead to non-specific amplify.
primer
PRIMER PREMIER 5.0
File DNA sequence New
Mg2+
Proper concentration is 0.5-2.5mmol/L Mg2+ is an activator of DNA polymerase
Mg2+ in a low concentration: PCR product decreaseMg2+ in a high concentration: non-specific amplification
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①5’→3’polymer activity②3’→5’nucleic excision activity : mistake correction ③5’→3’excision activity : DNA damage repair
the Klenow fragment of DNA polymeraseⅠof E.coli:5’→3 polymerase activity 3’→5’exision activity suitable for filling the blank of DNA single strand.
DNA polymerase
TemperatureTemperature
(℃)
En
zy
En
zy
me
me
activ
itactiv
ity(%
)y(%
)
40 50 60 70 80 90 100
100
80
60
40
20
• hot-resistance DNA polymerase—Taq polymerase
Taq DNA polymerase is a kind of polymerase extracted from yT1.yT is a kind of fungi, which can live in 70-75 hot spring.℃ It can duplicate DNA in 74 , ℃and still keep the enzyme activity in 95 . ℃
Taq DNA polymerase
Some commercial production of PCR polymerase
TaKaRa rTaq™
5'-3’exision activity
“ A” base will be added to the 3′end of The PCR product.
Amplification rate :
1kb/min
Routine PCR and identification
Activity: 5’-3’ polymerase
Amplification system:
10*PCR buffer 1 2 5dNTP 0.8 1.6 4Primer F 0.5 1 2.5Primer R 0.5 1 2.5Template 0.5 1 2.5r-Taq 0.05 0.1 0.25ddH2O 6.65 13.3 33.25
Total volume 10µl 20µl 50µl
TaKaRa LA Taq
Enzyme activity:
5’-3’polymerase
3'-5’ excision activity
Suitable for fragment with high GC content
Amplification rate:
1kb/min
“ A” base will be added to the 3′end of The PCR product.
5’-3’polymerase
3'-5’ excision activity
“ A” base will be added to the 3′end of The PCR product.
5’-3’polymerase
3'-5’ excision activity
Amplification system:
2*GC buffer 5 12.5 25dNTP 1.6 4 8Primer F 0.5 1.25 2.5Primer R 0.5 1.25 2.5Template 0.5 1.25 2.5LA-Taq 0.1 0.25 0.5ddH2O 1.8 4.5 9Total volume 10µl 25µl 50µl
Pyrobest DNA Polymerase
600-800bp/min
Amplification condition is hard to verify
HI-FI PCR REACTION
Enzyme activity: 5’-3’polymerase
3'-5’ excision activity
Amplification rate:
10*pyrobest buffer 1 2.5 5dNTP 0.8 2 4
Primer F 0.5 1.25 2.5Primer R 0.5 1.25 2.5Template 0.5 1.25 2.5LA-Taq 0.05 0.125 0.25
ddH2O 6.65 16.625 33.25Total volume 10µl 25µl 50µl
Amplification system:
2*Power Taq DNA polymerase
The mixture of DNA Polymerase 、 buffer 、 dNTP in PCR reaction.
1-2kb/min
The easiest PCR
Enzyme activity:
“ A” base will be added to the 3′end of The PCR product.
5’-3’polymerase
3'-5’ excision activity
Amplification rate:
2*Power taq buffer 5 10 25Primer F 0.5 1 2.5Primer R 0.5 1 2.5Template 0.5 1 2.5ddH2O 3.5 7 17.5Total volume 10µl 20µl 50µl
Amplification system:
III. PCR amplification condition 2
steps:
3 steps:
Suitable for short fragment amplification95℃68℃
5min95
℃30sec
1min
95℃
55-60℃
5min95
℃30sec
30sec
72℃
30sec
72℃
10min8
℃3hour
35cycles
Common PCR
25cycles
Ⅳ. PCR PRODUCT DETECTION
2 MEDIA ELECTROPHORESIS :
Agrose gel +EB PAGE + Ag
Concentration of agrose and suitable DNA separation range:
Agrose ( % ) DNA length ( bp ) 0.5 1000 ~ 30000 0.7 800 ~ 12000 1.0 500 ~ 10000 1.2 400 ~ 7000 1.5 200 ~ 3000 2.0 50 ~ 2000
Polyacrylamide del application :•The detection of protein molecules•The determination of protein molecular weight•The analysis of nucleic acid
Polyacrylamide gel Suitable for small ( 5 ~ 500bp ) DNA molecule
Ⅴ. The optimize of PCR conditions
Negative result
TaqTaq
P1P1
P2P2
dATP dTTPdGTPdCTP
Mg2+
primer : Decrease the annealing temperature
Regulate the amount of primer
template:
Increase or decrease the template concentration
enzyme :To use proper enzyme and buffer
Non-specific result
primer:
Specificity is not good
Increase the annealing temperature
DMSO decrease DNA secondary structure
template :To check if there has secondary structure of template
Change buffer
touch down PCR
touch down PCR : the annealing temperature will be decrease 1 °C every cycle or every n cycle until to a low annealing temperature , which is called ‘touchdown’ temperature , then go on 10 cycles at this temperature.
nest PCR
目的片段
P2-RP2-F
P1-RP1-F
Low concentration target fragment
primer:
DNA decay Order the primer again or Increase the concentrationtemplate
:DNA decay Do extraction
again or increase the con.
Secondary amplification
Do a PCR again by using the PCR product of the first time
targetHigh Specific target DNA fragment !