????????(Q-PCR)???? ??????

1
????????(Q-PCR)??????????

• ????????? ???

2
PCR?

• PCR????????????
• ?????(Kary Mullis)
• PCR??????,???????PCR?????????????,????????
  ??,????????????
• ?????????????????
• ??Making PCR A Story of
  Biotechnology by Paul Rabinow

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(Kary Mullis)
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???PCR?

• ????PCR???????(?????????)???????????
  ?????????????PCR,???????????????????????,?????????
  ?????????????PCR????????????
• ?????(Kary Mullis)

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(No Transcript)
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PCR??????????(1)

• 1983 Cetus ??? K. Mullis????(12?16????????
  )???PCR?
• ?????????,????????DNA???????????????,?????????
  ?????????????????
• --KB Mullis, Scientific American,
  april 1990

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PCR?????????
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(No Transcript)
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(No Transcript)
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(No Transcript)
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(No Transcript)
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PCR??????????(2)

• ??PCR ??????Cetus??Saiki??? Science ? ? ? (R.
  Saiki, S. Scharf, F. Faloona, K. Mullis, G.
  Horn, H. Erlich and N. Arnheim)
• 1987 KB Mullis et al. Specific synthesis of DNA
  in vitro via a polymerase-catalyzed chain
  reaction. Methods in Enzymology 155335

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PCR??????????(3)

• 1987 ??7?Cetus ???????PCR?????????
• 1985?????Perkin-Elmer Cetus???? (
  PECI ) ?????11?????? ?PCR?????????????
• 1989 Science ??????DNA??? Taq ????,???????????12?
  Science???PCR????????????????????
• Hoffmann-La Roche ???Cetus ?
  ????PCR??????? ?
• RK Saiki, KB Mullis et al. (1988)
  Primer-directed enzymatic amplification of DNA
  with a thermo-stable DNA polymerase, Science
  239487

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Taq DNA polymerase
Thermus aquaticus
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Taq DNA polymerase A thermostable DNA
polymerase from Thermus aquaticus.
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PCR??????????(4)

• DuPont???,1971?PCR?????Khorana???????,?????,?????
  ????????Kornberg(??DNA????????)???,???????????????
  ?Khorana???????????PCR?

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PCR??????????(5)
Study on polynucleotides repair replication of
short synthetic DNAs as catalyzed by DNA
polymerases. J Mol Biol, 1971, 56341
??DNA??,???????,?DNA???????,????????????tRNA???
????????????????? ?????????????????
??(1970)Smith???????,??????????  
H.G. Khorana ??MIT???1968?????????
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The article from 1971 in which Kleppe et al.
describe Repair replication basically the same
principle as PCR! J. Mol. Biol. (1971) 56
341-361
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PCR??????????(6)

• 1990 Cetus??? D.Gelfand?S.Stoffel???? ?Taq DNA
  ???????
• Cetus ???H. Erlich ?K. Mullis??????????????
  ???
• 1991 TaqMan ? ? ? ??
• ??11?Hoffmann-La Roche???Cetus???? ?PCR????
• Roche Molecular Systems ??????????rTth
  ??RT-PCR??,?????RNA?????
• ?????????Cetus?????????

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????PCR??
1986?? Russ Higuchi??PCR????Cetus????
????PCR??? ??????? ???????
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PCR??????????(7)

• 1992 ??3?Cetus ????Taq???????????
  
• 1993 AMPLICOR HCV??????RNA PCR ? ?????
• Kary Mullis ?PCR????????????

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PCR??????????(8)

• ??????PCR???????????
• 1998? ????PCR??????????????????
• 1999? ?????????PCR???????

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PCR ?? ??? ????
???
???
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PCR ??- ??? ????????
???
3
5
5
3
Biotin
Primer 1
Primer 2
Biotin
3
5
5
3
???
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PCR ?? - ??? - ????
???
3
5
5
3
Biotin
Primer 1
Taq DNA Polymerase
Primer 2
Biotin
3
5
5
3
???
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?1? PCR ????? ??????????
???
Biotin
Biotin
???
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30????????????
No. of No. Amplicon Cycles Copies of
Target 1 2 2 4 3 8 4 16 5 32 6 64 20 1,048,576 30
1,073,741,824
1 cycle 2 Amplicon
2 cycle 4 Amplicon
3 cycle 8 Amplicon
4 cycle 16 Amplicon
5 cycle 32 Amplicon
6 cycle 64 Amplicon
7 cycle 128 Amplicon
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(No Transcript)
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PCR??????
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PCR???????

• PCR???????,?????????????????
• YnYn-1(1E) Yn-2(1E)2 X(1E)n
  
• 0E1
• ??E??????,Yn???n????PCR???????,Yn-1?n-1?
  ???PCR????????
• ????????????(???20?30)??????????,????????
  ????????????,??????,????.

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??PCR??????? PCR??????

• ??/????
• ????????
• ?????????????
• ?????DNA?????????
• PCR??????
• ??????????????E??

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PCR ?????

• ???/???
• ???/???
• ???/???
• N ???????
• n ???????

log-phase analysis
end-point analysis
N
n
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??PCR???PCR?????????

• ??PCR???????PCR???????????PCR?????????P
  CR???????

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??PCR????

• ??PCR( Taq Man??????)
• PCR-????
• PCR-ELISA

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??PCR???

• ??PCR????????,???????????????????
• ??????????(???X?????)?????(????????X???)

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???????PCR??
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???????PCR??????

• ??(1)????,????(2)????????,??????????????,???????
  ???,???????????
• ??(1)PCR???????????????????????????????????,?????
  ?????????????
• ??,????????????PCR????,???????????(????)????(????)
  ??,???????????

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TaqMan??????PCR
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(No Transcript)
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??????????PCR
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?????PCR??

• ?????PCR?????
• ??????????????????

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???????(1)

• ???PCR???????????????,?????PCR????????????????????
  ?????,????????????Yn?????E???????2??????,?????????
  ????????????????,???PCR??????????????????????????
  ?,?????(1)??E?

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???????
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???????(2)

• ?????????,?????????(2)?????????????,???j(?????????
  )??n,Yj(??????????????)??Yn,Yi-j(??????????????)??
  X
• E-1(Yi/Yi-j)1/j (3)

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X(?????)???

• ???????,????(4)??????????????????X???(4)??
  ???(2)?????
• XYn /(1E)n (4)

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X????????(1)

• ??????,????(2)?????,?????Yn???????n????X?
  
• logYn logX nlog(1E) (5)
• ?n0?,??????X?,??????(5)???????X?(?2)?

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(No Transcript)
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????????

• ??????? (1)??????(2)?????PCR?????????,???????
  ?????????(Ee)?

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????????????PCR??

• ???????(1)?????????????(2)????????????(3)??????
  ??????????????
• ??DNA???,?????????????????,?????????????????ß?????
  ???
• ??RNA???,???????????????mRNA?????????????????????,
  ??,??????????RNA,????????????,????????????????????
  mRNA???????????,?HLA?ß?????GPDH????H3.3?mRNA?14S
  rRNA??

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??????????????

• ???????
• ???????????,????????,??????????

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????????????

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

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???????????PCR??

• ??????????????????????????????????,????,???????
  ??????????????????????,???????????????????????????
  ???????????????
• ???????????????????????????????????,??????????????
  ???????

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???????????PCR??

• ????????????????????????
• ??????????????????????,???????????????????????????
  ????????????????????????????????

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???????????PCR??

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

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?????PCR??????????????

• ??????????
• ????????????????

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??????????

• ?????????????????????????????????
• ???????????,????mRNA??????

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??????????

• ?????????
• ??????
• ????

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????????????

• ?????????????????????????????,???????????
  ?
• ???????????????????????????,????????????,????gt??,
  ?????????????????,???lt??,?lt103???/ml,??????0???/m
  l????
• ???????????????, ??????, ??????

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???????????????

• ?????
• ??
• ??
• ????????
• ???????????
• ????
• ????
• ??????

60
???????????????????

• a??
• ß??
• ???
• ?????

61
?????????

• ??????????????,?????????????????????????,?????????
  ???,????????????????,??????????????????
• ?????Y??????,?????????????,???????????????????????
  ??DNA,?????????????,?????????????????

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?????????

• ??????DNA??,???????????????????DNA????????????????
  A?C?G?T??????????????30??????,????????????????????
  ???,????????????????????????????????????,?????????
  ??????????,????????(??????)??????????????????

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???????????

• ????
• ??????
• ?????

64
?????????

• HCV?HIV?????
• HBV DNA PCR?????????

65
? ?

• Immuno-PCR

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??PCR?????????

• ??????????????
• ???????????????????????????????????
• ?????????????????
• ??????????????????????
• ??PCR?????????????

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? ?

• 1.????????,?????????????????????????????
• 2.????????????????????
• 3.????????????????????????????????????????????????
  ??????????,????????????????????
• 4.??????????????,??,????????????????????????,?????
  ???,??????? ????????????,????????????

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? ?

• 5.?????(????)???????????????????????
• 6.??????,??????????(????????)?????,???????????????
  ???????????????????,?????????

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??!