Chapter 11 ??????? PCR

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Chapter 11??????? PCR

• ?????

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????

• PCR?????
• ????
• ??PCR???????
• PCR????????
• ??PCR?????
• PCR???????DNA
• ???????PCR
• ?PCR???????-????
• ????????(RCAT)

• ????????????
• PCR???
• ?????
• ?PCR
• ????????
• PCR?TA??
• ????????? (RAPD)
• ????PCR
• ????PCR
• cDNA????????(RACE)

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• PCR????,??DNA???DNA??????????DNA?????????????DNA??
  ???????????DNA,??????????PCR?????????DNA?????????,
  ??????(Target sequence),??????DNA??(?11.01)

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

• PCR???????
• 1.???????DNA??
• 2.??DNA???????PCR ??(PCR primers)
• 3.??DNA?????DNA???
• 4.???????????
• 5.?????PCR ??(PCR machine)(?11.02)

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

• PCR???????90C????DNA?????????DNA????????PCR??????
  ,????90C???DNA??
• ??????70C?????
• ????????DNA
• ??????????????????DNA

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

• ?????(Degenerate primer)????????????,?????????
• ?????????DNA??,???????????????????????????????????
  ?????(?11.08)

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?PCR

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

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?PCR
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????????

• ????PCR????????????????????????????,??????????????
  (?11.10)????????????????????,????????????????PCR??
  

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????????
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PCR?TA??

• TA??(TA cloning)????Taq ??????????(thermophilic)?D
  NA?????????????????Taq??????DNA??????3A???(?11.11
  )????TA????(TA Cloning vector)??????????DNA??

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?????????

• RAPDs???????????????????
• RAPD?????????PCR??????????????,?PCR??????????DNA??
  ???????????????DNA??????????(?11.12)

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?????????

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

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

• ????????????,??????????????????????????????(Revers
  e transcriptase)?RNA???DNA??,???DNA(Complementary
  DNA,??cDNA)
• ?????????????????????cDNA,??????????????????,PCR??
  ????cDNA???????????????????????????(Reverse
  transcriptase PCR,??RT-PCR)

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????PCR

• RT-PCR????????????mRNA??????????????????mRNA?????
  ?,???????????????????mRNA???
• ??????????,????PCR??
• ????,????mRNA,????????(?11.15)

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????PCR
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????PCR

• ????PCR??RAPD?RT-PCR,????????????????
• ?RT-PCR?RNA????????,????cDNA????????????????????
• 1.???????????mRNA??cDNA???3'??
• 2.mRNA????????????,?????????RAPDs?????,?????????

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cDNA????????

• cDNA????????(Rapid amplification of cDNA
  ends,??RACE)?????????cDNA??????????,??????mRNA?cDN
  A?????
• RACE?????????RT-PCR,??cDNA???????????????(Anchor
  sequence),???PCR??

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cDNA????????
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PCR?????

• ???PCR????????????????
• a)????DNA??
• b)??????DNA?????,??????? (directed mutagenesis)
• ??????????,?????????????PCR??,???????????????(Over
  lap primer)????????????

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PCR?????
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????

• ?????(Directed mutagenesis)???????????????????????
  ?????PCR
• ????????PCR??????????????Taq??????????????????????
  ?????DNA,?????????

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??PCR???????

• PCR??????DNA??,???????????????????????????????????
  ??????,?????????
• Npt????????????,??????????????,???????????????????
  ???????

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PCR????????

• PCR??????????????????
• ????DNA???????????,????PCR?????(?11.20????No.2)
• ????DNA???????????,???????(?11.20????No.1)
• PCR????????????,???

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

• ??PCR??????????????,??????????????????????????rRNA
  ??,????????????????????,????????????
• PCR?????(community profiling)??????????????????
• ?????PCR-??????(eco-trawling)??????????????

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PCR???????DNA

• ??????DNA???????????????????????????(Siberian
  permafrost)?????????DNA
• ??????????????????????,???????????????????????

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PCR???????DNA
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???????PCR

• DNA??????????????DNA???,????PCR?????????????DNA???
  ?,???????DNA,????????
• ????DNA????????????????SYBR Green I??
• ??????????????,?????????????,??TaqMan??

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???????PCR

• ????????(Fluorescence resonance energy
  transfer,??FRET)??????????????????????????????????
  (quench)????(?11.23)
• TaqMan???PCR??????????????????????Taq????????PCR??
  ????,????????????TaqMan ??

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?PCR???????-????

• ????(Molecular beacon)????PCR??,???????????????
• ????(Scorpion primer)???????????????(?hexethylene
  glycol, HEG)??????DNA?????
• PCR??????????DNA?,Taq????????

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????????

• ??PCR?,??????DNA????????????DNA?????DNA???????????
  ?????(Rolling circle amplification
  technology,??RCAT)
• ???RCAT,??DNA???????DNA,???????????????DNA,???????
  ??????????(?11.26)

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????????

• ???RCAT(exponential RCAT,E-RCAT)????????????,?????
  ?????????????????????????????????DNA????????????,?
  ??????????????DNA

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????????
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