(?)???????(PCR:Polymerase Chain Reaction)

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(?)???????(PCRPolymerase Chain Reaction)
PCR???????Kary B. Mullis??????????????DNA?
???????????,1993??????????
Kary B. Mullis
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Kary B. Mullis(1944-)
http//www.karymullis.com
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??DNA?????
????? ???? SSB
DNA???(I II III)
?? dNTP Mg2
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Mullis?PCR??
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• 1?????
• PCR??????DNA???(????)?Mg2?4????????(dNTP)
  ??????,??DNA???(Taq?)????,??DNA??????????(??)?????
  ???,????????DNA???
• (1)???????
• ?????????DNA???????????????????????????????
  ???????????,?????????????????????????

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PCR??? ??DNA??? ?????????????????
??
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(2)DNA????? ????????????? DNA????????,????,????DNA
,??? DNA???? (3)????????(?????) ????????,
?????????????,????,?????????????,??DNA??, ?????
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(4)???? ?????????72???,?Mg2??????,Taq?????d
NTP??????????DNA??3?(5?3????),?????,???????????
?? ?????PCR??(????????),????????????????
PCR??Flash ??
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PCR?????????
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PCR????
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2?PCR????????? (1) PCR????
dTTP
Taq
dCTP
P2
P1
??DNA ??P1 P2 DNA???Taq ??dNTP ?????Buffer ?
???Mg2
dGTP
dATP
Mg2
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• ??
• ????DNA???
• ????????????DNA???????DNA??????
• DNA????100ng /100?L?
• ???????????????????
• ????
• 0.1-0.5 ?mol/L
• ??????????????,????????

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• Taq DNA???
• 0.5-2.5 U/50 ?l
• ???????????????????????
• dNTP
• dNTP????????????
• ??dNTP?????
• ??????????????,???????????,dNTP??Mg2??,??
  ??Mg2????,??DNA???????

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• Mg2
• Mg2?DNA?????????????0.5-2.5mmol/L?????
• Mg2??????Taq??????PCR???? Mg2??????????
• Mg2???????,???????dNTP?EDTA????????????Mg2???

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(2)????
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• 3??????(??)??
• ??????????????????GC??????
• ????1525bp?,?????(??)??????Tm4(GC)2(A
  T)?????
• ?Tm?????,Tm??,PCR??????

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• 4?PCR?????????
• (1)??????????,???????????
• (2)??????,???15-30 bp
• (3)???GC?40-60
• (4)?????????,??3????3???G??3???C?????,???????G?C?
  ??????
• (5)????????????(???????)
• (6)?????????????(???????4?)
• (7)??3??????5??????
• (8)??3??????????????,????????????????

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??????
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• Cytb????????(??)
• L14724 5 GACTTGAAAAACCACCGTTG 3
• H15915 5 CTCCGATCTCCGGATTACAAGAC 3

???????cytb??
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PCR???
Real time PCR???
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5????5 ???

• PCR?????????3???,??PCR????????5?????PCR??
  ???????5?????????????PCR,??????PCR?????????????

??????(?????????????????
???????????? ) ?????? (?????????????
)
????
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6????????

• (1)??PCR(anchored PCR, A-PCR)
• ??PCR?????????????DNA??????????????dG(Poly
  G)???,???????dC(Poly C)????????????PCR?????PCR????
  ??????????????????????????????,???????????????????
  ????,???????????????????,?????????

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cDNA
???????
5
3GGGG
CCCC ????
3GGGG
5
CCCC
3GGGG
CCCC
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• (2)????PCR (degenerate primer PCR,DP-PCR)
• ??????????????,???????????????????????????
  ??
• ??????,??????????????,?????????????????????
  
• ????PCR???????????????????????????????????
  ????????

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(3)??PCR (reverse PCR)
??????????????????DNA???????DNA??????????????
?????????????,???????DNA????????????????cDNA???,?
????????DNA??????????
????
????
????
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???
???
????
????
????
???
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• (4)??????PCR(single specific primer PCR,SSP-PCR)
• ?????????????????DNA,?????????????????????
  (???????????)?????(???????)??????????

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• (5)????PCR(arbitrary primer PCR,AP-PCR)
• ?????????????,????????????????????DNA??????????
  ??????????????????DNA??????????,????PCR,??????????
  DNA?????????????????????????,?????????????????????

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7??????PCR??

• (1)???PCR(reverse transcribed PCR,RT-PCR)
• ??RNA?mRNA????????,?????PCR????????????????
  ?

?RNA
cDNA
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(2)???PCR(asyrmnetric PCR ) ?????????????DNA?
??????????????????????????????,????????150-1
00,?????????????????????????????????,?????????PCR?
?????????DNA ???????????????????
???DNA???????
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????? ??????
????? ?????
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• (3)??PCR (multiplex PCR)
• ????????????,??????DNA??????
• ?????????????????????????

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??
1
2
3
4
1
2
3
4
??
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• (4)??PCR (nested PCR)
• ???????????????,?????????????DNA,?????????
  ???????????????????PCR??,?????????????DNA?
• ?????????????,??????PCR????????????????????
  ?????

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??
??
??
????
??
???
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• 8.PCR??????????????????
• (1)????????????????
• (2)??????????????
• (3)????????????
• (4)???????,????????????????????
• (5)?????????????????TaqDNA?????,??????95???10min
  
• (6)????????????,????
• (7)????????????,?????????