PCR (Polymerase Chain Reaction)

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PCR (Polymerase Chain Reaction)
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? ?

• ??PCR??????????

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1983.4 Kary Mullis??????????????Redwood
country???????????????PCR ?????Roche??
??10.000 1993 Nobel prize
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PCR?????
???????????
PCR???
?? 9097?
?? 4555?
?? 72???
PCR??
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PCR????

1. ????,???5 '?3'???
2. ??1530????
3. ??????????????
4. ?????????????

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PCR???????? ?????dNTP????Mg2 Taq DNA???
??????? Thermusaquaticus ??????
Mg2??? ?????
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PCR???????
Taq polymerase (Thermus aquaticus )
nucleotides
primers
salts (ions)
DNA fragment
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Step 1 Denaturation ??
94C
TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTG
ATCTCGACGATGCATCATGACATCGATTTGATCGAT
ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCAC
TAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA
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Step 2 Annealing ??
T primer-dependent
TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTG
ATCTCGACGATGCATCATGACATCGATTTGATCGAT
GCTGCTACGTAG
CGTAGTAGCA
ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCAC
TAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA
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Step3 Extension ??
72C
TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTG
ATCTCGACGATGCATCATGACATCGATTTGATCGAT
GCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGA
GCTGCTACGTAG
TAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCA
TC
CGTAGTAGCA
ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCAC
TAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA
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Next step Cooling
You get two copies of your DNA
TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTG
ATCTCGACGATGCATCATGACATCGATTTGATCGAT
ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCAC
TAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA
TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTG
ATCTCGACGATGCATCATGACATCGATTTGATCGAT
ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCAC
TAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA
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PCR Process
Process
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??

??
1 cycle
2 cycle
3 cycle
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??????
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(No Transcript)
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?????

• DNA
• ??????DNA??????????????????????
• ????????????????K??
• RNA
• ???????
• ???????????????DEPC??

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• (?) ????
• ????????????????????,?????2ml????,??????

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PCR??

• (?) ??
• 5 TTTTCGTAGTAACGGAAGCC-3
• 5 TAAGGATTCTCAGATGCAAATG-3
• ?????????
• ???StepByStep????????
• Primer premier5.0/oligo??
• www.dxy.cn
• ?????????/????????/ PCR?????

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(?) ??eppendorf??? ?/??? 9 ?l
10??? 2 ?l dNTP???
3 ?L ?? 2 ?l ??DNA
2 ?l 25mmol/L Mg2 2.5 ?l
???? 10 ?l
????5sec, 94???,2min
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• Taq DNA??? 0.55 ?l
• ????
• 94? 30s
• 55? 30s
• 72? 30s 35???
• (???72???????????????35100????/?)
  ?????????10 ?l
• (?)????220-242 bp
• ??100300bp????????,??????????????????
  ???PCR(two-temperature PCR)???????????????????????
  ?????94-95??46-47?

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PCR??????

• PCR??????????
• ???,???????
• ???
• ????????

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(?)???? ?????? ???EP?????? ??????????,???? (?)????
???? ???? ???? ???? ???? ?????????
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PCR?????

• ????
• ????PCR???????????????,??pg10-
  12?????????????(?g 10 -6)??
• ?????PCR?????24 ????????
• ?????????
• DNA ???????????????????????????????????????
  ?

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??????
1????? ?0.5TBE??1?????? ? ?1g????????,?100ml
0.5TBE ? ???????1??,????? ? ???????????6070??,
??EB 0.5µl,?????
2????? ????????,?????????????????,???58mm???
????????????????,????????1mm??? ??????????????
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3???????60??????????? ??,????35mm
4??????1????,????????? ??????,?????????????, ?????
??????
5????????0.5TBE?????,? ???????
6??????????????? ????(???)???????,??? ???,????
?
7??????????????????? 1/6??????,????????
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8???????????,??????? ????????????10µl
9??????,????,???? ?????,????????????????? ??????
50v??0.5????
10??????,????,?????
11??????? ?????????????? ?????
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????

• 1???????????DNA??????,
• ???????DNA??????????
• 2???????????,?????,??
• ????????
• 3???????????,????????,???????
• 4??????60???,????,????
• 5???????????????,?????
• 6????,???????????
• 7??EB??,????????????

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????????DNA?????????
???() ??DNA????(kb)
0.3 560 0.6
120 0.7 0.810 0.9
0.57 1.2 0.46 1.5
0.24 2.0
0.13
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