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 ， 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 Mullis In 1993 ， 7 years after the invention of PCR, he won the Noble prize.

5 Primer 1 5 Primer 2 Cycle 2 Cycle Template DNA I. The principle of PCR

Cycle 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 elongation 72 ˚ C annealing Tm-5 ˚ C

PCR cycle 72 ℃ ℃ 55 ℃ 1 PCR cycle denaturationannealingelongation

Template DNA Specific primers dNTPs Mg 2+ Hot-resistance DNA polymerase II. Basic components of PCR system

PCR reaction system Taq P1 P2 dATP dTTP dGTP dCTP Mg 2+ Co-factor ： Mg 2+ 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 sequenceNew

Mg 2+ Proper concentration is mmol/L Mg 2+ is an activator of DNA polymerase Mg 2+ in a low concentration: PCR product decrease Mg 2+ in a high concentration: non-specific amplification 返回

① 5’→3’polymer activity ② 3’→5’nucleic excision activity ： mistake correction ③ 5’→3’excision activity ： DNA damage repair the Klenow fragment of DNA polymerase Ⅰ of E.coli: 5’→3 polymerase activity 3’→5’exision activity suitable for filling the blank of DNA single strand. DNA polymerase

Temperature ( ℃ ) Enzymeactivity(%) 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 ℃ 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 buffer125 dNTP Primer F Primer R Template r-Taq ddH 2 O Total volume10µl20µl50µl

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 dNTP1.648 Primer F Primer R Template LA-Taq ddH2O Total volume10µl25µl50µl

Pyrobest DNA Polymerase bp/min Amplification condition is hard to verify HI-FI PCR REACTION Enzyme activity: 5’-3’polymerase 3'-5’ excision activity Amplification rate ：

10*pyrobest buffer12.55 dNTP0.824 Primer F Primer R Template LA-Taq ddH2O Total volume10µl25µl50µl 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 buffer51025 Primer F Primer R Template ddH2O Total volume10µl20µl50µl Amplification system:

III. PCR amplification condition 2 steps: 3 steps: Suitable for short fragment amplification 95 ℃ 68 ℃ 5min 95 ℃ 30sec 1min 95 ℃ ℃ 5min 95 ℃ 30sec 72 ℃ 30sec 72 ℃ 10mi n 8℃8℃ 3hour 35cycle s 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 ） ～ ～ ～ ～ ～ ～ 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 resultTaqP1 P2 dATP dTTP dGTP dCTP Mg 2+ 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-R P2-F P1-R P1-F

Low concentration target fragment primer ： DNA decay Order the primer again or Increase the concentration template ： DNA decay Do extraction again or increase the con. Secondary amplification Do a PCR again by using the PCR product of the first time

target High Specific target DNA fragment !