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

55

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5 5

Cycle 3

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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 !