??????? New technique

1
????
??????? New technique New progress
2
???

• ??DNA??
• Recombination DNA Technology

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? DNA?????
??????????????????? ?????????
????----???????????? ???????????,
????DNA???? ?? ?
???????????DNA?? ?
????????????????(???)
4

• ????
• DNA??
• ???
• ????
• ????
• ????
• ??????? ??????????
• ?????????? DNA?????
• ?????? ???????

5
?? DNA??
???????, ?????????????(???????????????????????DNA)
???DNA?????????????DNA??( ???replicon),???????????
???,???????????????,????????????DNA???
?????????DNA (recombinant DNA)
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???? 1.??(clone)???????? 2.????DNA???DNA??(DNA
cloning)????? (molecular cloning) 3.??????????????
(gene cloning) 4.????DNA????? ???
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• ??DNA???????
• ?????????
• 1????????
• 2???????????
• 3???????????
• 4???DNA????????
• 5??????????????????
• (???)

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? ? ? ? ? ? ?DNA ? ? ? ?
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?? ???

• ????????
• DNA???I
• ????
• T4DNA ???
• ?????
• ?????
• Taq DNA???

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• ????????
• (restriction endonuclease , RE)

?? ??DNA?????, ??????????
????DNA??????
BamH?

???????
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????????? ????
5
5
?? ?????????
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????
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???????????????????????? ????????
BamH I
G?GATCC Cla I
AT?CGAT EcoR I
G?AATTC
Hind ?
A?AGCTT Hind ?
GTPy?PuAC Kpn I
GGTAC?C
Not I
GC?GGCCGC Pst I
CTGCA?G Sal I
G?TCGAC
Sau3A I
?GATC Sfi I
GGCCNNNN?NGGCC Sma I
CCC?GGG
Xba I
T?CTAGA Xho I
C?TCGAG  
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DNA???????????? ?
? ? ? ?
???????? ??DNA????,??DNA?
DNA???I
????????DNA?? ?????(Klenow)
???cDNA????
?????DNA3¹??

?DNA???? ??DNA
??????(PCR)
(?Taq DNA)??? DNA???
????DNA????? ??????
??????5¹???????,??? ?????
?????
?3¹?????????? ???
????????? SI?????????
????DNA?RNA,??DNA??????? DNA?I

??DNA,???DNA??????? RNA?A
???RNA 
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??????

• cDNA(DNA)
• ???DNA

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(?)????????????

• ????? ????????????????????????
• ???DNA(G-??)
• ???DNA??(genomic DNA library)
• cDNA cDNA??(cDNA library)(C-??)
• ??????(polymerase chain reaction,PCR)

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1.???DNA??
 
???????????DNA,?????(?????????)???(??????????????)
?DNA??????????,?????????????(?????????YAC??)??,???
??????,?????????????????DNA??????????DNA??,???????
?,????????????????DNA???????,??????DNA??(genomic
DNA library),???
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????????DNA
??????
????
????
???DNA?? ??????????????????????DNA????
??DNA??
???
?????????
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2. cDNA??
 

• ?mRNA???,?????????DNA,??DNA???mRNA??,????DNA?cDNA(
  complementary DNA)
• ??????????mRNA,???????cDNA,??????(??????????)?????
  ???,?????????CDNA,??????,?????????mRNA???cDNA?????
  ????????cDNA??(cDNA library)?

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• 3.???????
• (polymerase chain reaction ,PCR)
• PCR???????DNA????????????DNA?RNA?????
• PCR??????????,??????DNA??
• ?????2??????DNA?????106-109?,???????????????????

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(1)PCR????

• ???????DNA??
• TaqDNA???
• ??????????(primers)
• DNA??????4????????(dNTPs)
• ??????????Mg2??????

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(2) PCR???????

• 1.??DNA???(Taq?)??????????????DNA???,????950C,30?
  ??????????
• 2.??DNA???????DNA,???????10kb?
• 3.??????????(primers)????DNA?????????????????,???
  ???,??????????DNA??3-??
• 4.DNA??????4????????(dNTPs)?????dNTPS?
• 5.??????????????Mg2??????

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(3)PCR????
 
1.??????,DNA????? 2.????????,??????? 3.?TaqDNA??
?,Mg2????????????,????????????3????,?????PCR????
,????1??2???????? 4.????????????20-30???,?????????
?????????????????????????
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PCR?????
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(4) PCR?????
PCR???????????????,??????PCR????? ?????????
???????????,?????200800bp?? ?????
???1825bp ????? 3???????? GC???Tm?
???40-60??,????2-3 ?
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(5) PCR?????
??DNA???????95???1min,?DNA ??????
???????????????,????30s?1.5min ????72?
1min ??? ?????????,??25-45???
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(6) PCR?????

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

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

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?? ????
?????????,???????????????????????DNA???
??????DNA????DNA???DNA
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????(cloning vector)-----???????DNA?????????????
????(expression vector)-----???????DNA????????????
???????
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???????

• ?????
• ????????????,???????????
• ?????(??DNA???),???????????,???????
• ????,????????DNA?

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??(plasmid)???

• ????????????????DNA??
• ???????????????
• ??????????????
• ???????????????
• ?????????????
• ????????????????

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????????????
1??????? ?????????????
??????
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(No Transcript)
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2????? ????????????????
????????????
3???????? ?????????,?DNA?????????????????
??,????????
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(No Transcript)
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4.???? ???????????,?????????????,????????

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?????????
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????DNA?????
????? ????? ?????????
?????
?????? (transformation) ??
(transfaction) ?? (infection)
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??????
????? ??????? ?????
???? ????? ????
Southern?? ??????
????? ??????? ?????
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? ? ? ?
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(?)?????????????????

• ????
• ??????
• ??????
• SD????????
• ????
• ????
• ????????
• 5????GC????SD???????
• ?????
• ???????

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????DNA????

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

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???DNA????
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????

• DNA????????????????
• 1.DNA polymerase????????
• 2.????????
• 3.???????????????????
• ??Sanger?????????
• Maxam-Gilbert??????

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(?)Sanger?????????

• ??DNA??????3,5-???????,??DNA??????
  2-????????2,3ddNTP???dNTP??,????????3?????????
  ?DNA???????????????????DNA??,?????3??,??????dNTP?
  ??????,??,?????DNA????????

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????

• DNA??????3,5-???????,??DNA??????
  2-????????2,3ddNTP???dNTP??,????????3?????????
  ?DNA???????????????????DNA??,?????3??,??????dNTP?
  ??????,??,?????DNA????????
• ?DNA????????4???dNTP????????ddNTP,???????
  ??????????????,???????????,???????????????????????
  ???4???????????4????ddNTP,?????4?????,????????????
  A?C?G?T???

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• ????
• 1.??????DNA
• 2.????????(??)
• 3.DNA???????
• 4.??
• 5.?????
• 6.??

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?????????
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??DNA?????
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? ?
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Maxam-Gilbert?????

• ??????????DNA????????,??????????DNA??,???????DNA??
  ?
• ???????DNA?5?,?????????????90C,???DNA???????????
  ???????,????????????????,?????????????,????,??????
  ?????????(??,?????),?????????????????????????

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???DNA?????
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(?)DNA?????

• Sanger?????????,Maxam-Gilbert?????????????????,???
  ?????????????
• 1987?,????????(BD)????????????????DNA??????,??????
  ??????????

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(No Transcript)
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????

• ??????????????ddNTP?????????DNA??????????????????,
  4??????????????,??????????????????????????????????
  ????,????DNA???
• ????????4????????????????

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(No Transcript)
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??? ?????
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?????????????
?
?????? (post genome era)
??????,HapMap,?????
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?????????
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????

• 2000?6???????????????????????????6????,?????????
  ???????,????????????,??????,??????????????
• ???,??????????????????????????????,???????????????
  ??????????????????????????????,???????????????????
  ??????????,?????????,????????????????

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??????
??????????????? ????????????????,
???????????????????????????
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???????

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

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????????????
????????? ????????? ????????? ?????????????
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? Hapmap??

• ???????????SNP????,?????SNP?????,?????????????????
  ????,?????????SNP?????????????????????????????,???
  ??SNP?????????????????

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

1. ???????,?????????????????????????,??????
2. ???????????????????????,??????????????????????????
   ??????
3. ?????????,????DNA?????????????

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(?)????????

1. ????(Proteome)??????????? Wilkins??1994???,???????
   ??geneome?????????????protein??????(proteomics)???
   ?????????????????????
2. ?????????????????????????????????

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(?)??????????

• ???????(???????)???
• ?????????????????????????????????????????????,?
  ???????????????????

• ???????????
• ??????????????????

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????????
70
(?)??????????
?????????--?????????(2 -DE) ?????????????(Iden
tication) ??????????????(database)
???????????????(HTS)
??????(2-DE)???(Mass spectrometry)????????????????
???
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?????
72
(?)?????????-????

• ??????(Image analysis)
• ????(micro-sequencing)
• ???(mass spectrometry)?????
• ???????

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  (1) ??????(Image analysis)

• ?????2-DE?????????????,????????????????????
  ,??????????????,???????????????,??????.
  ????????2-DE????(?????,??????)????,???????????????
  ????????????

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(2) ????(microsequencing)

• ??????????????????????,?????????
• ??????????????????????????????????PVDF????????,???
  ??,??????????,???subpicomole?????????
• ??????????,??????????????????????????,????????????
  
• ??BLAST??,?????????

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(3) ????????

• ??????????????????,??????????????????
• ?????????????????(MALDI-TOF)??????????.
  ???????????,???????????
• ?????(ESI-MS) ?????????,????????,????????????????
  ??????
• ???????????(tandem MS)???????picomole???????????
  ?????????,????picomole??femtomole????
• ????????????????femtomole??????
  ????attomole????.

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  (4) ???????

• 1977???????????????,???????????????????????????,
  ????????????,????????????
• Internet?????????????????,?AACompIdent,ASA,FINDER,
  AAC-PI,PROP-SEARCH?,??,?PROP-SEARCH?,?????????????
  ??????????
• ????????,?????????????????????????,???????????????
  ?.

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(?)?????NMR??

• ?X??????,?????????????????NMR?,?????????,?????????
  ???????????????,??????????????????????
• NMR???????????????????????,NMR??????????,?????????
  ?,??????,?????????,????????????

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?????????

• ?????????????????????
• ??????????????????????????2DEMS??????????,???????
  ????,???LC-MS/MS?????????????????,????????????????
  
• ?????????????????????????,?????????,???????,??????
  ????????????????,???????????????????????
• ????????? N??????,???2DEMS??,?????????????????

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

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

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???????????

• ????????????????,????????,?????????????????
• ??????????????,?????!??????????????????,?????????
• ????????,???????????????,???????????????,?????????
  ??????

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1.???????
????????????????????????????????,?????????????????
??????????????????????
82
2.????????
??????????????????????
?????????????????? ????????????????????
83
?????????????????

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

84
???????????????

• ??????????????????????????????,???????????????????
  ?????????????????????,?2002???????????????????????
  ?????????????????????????

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? ??????
???????????--????
86
????????

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

?????? ????????
87
????????????????

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

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

• ???????????????????, ????????????????????????????
  ?????
• ?????????????????????,???????????????????????????
  ???????????????????GPCR???????
• ???????????

????
??????
?????(?????????????????????????? )
89
?????
90

• ????????

91
???????
92
????????
93
(No Transcript)