A ????? ATG

1
??? ???????
2
??

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

3
1. ????

• 1.1 ???????????
• A ????? ATG
• ???ATG???(??Kozak??)
• Kozak??????????????.
• ??Kozak??,????ATG?????????????????.

4

• Kozak??
• ?????ATG????A,T,G????1,2,3?,?Kozak????
  ???
• (1)?4???????G
• (2)ATG?5??15bp????????????T
• (3)?-3,-6?-9??,G?????
• (4)?-3,-6?-9?,????????,C??????

5

• B ?????
• ???????(SignalP http//www.cbs.dtu.dk/services/
  signalP )
• ???????????mRNA 5???????????
• ???SignalP????50??????(????ATG???????Met?
  ?)????,??SignalP????????,????????????

6

• C ?????
• ????? TAA, TAG,TGA
• GC 50 ?????? 64 bp????
• GC gt 50 ??????100-200 bp ????
• ?????? ORF ???50????,??????????? ORF ???100
  ?????

7

• D 3????
• 3????????Poly(A)???,???DNA????Poly(A)??,?
  ????????AATAAA?BLAST????????????

8

• E ?????????
• ???????????????100 ????,????50????????

9

• F ??????
• ???????????????????,??????????3??????
• ???????????????????,??????,???(Ale)?????GC
  A,GCC?GCT,?GCG?????

10

• G ???-?????
• ??????????????????,
• ?????5??????(donor site)??????
  5-AG?GTTAAGT-3
• 3??????(acceptor site),
  ??5PyPyPyPyPyPyCAG-3(Py?????,T?C)

11

• H ??????
• ??????(????)????????,????DNA??????,???????
  
• ??????????mRNA?5?,????????????????????
  ??????(The TRADAT Project , Eukaryotic Promoter
  Database, EPD. http//www.epd.unil.ch/ )?
• ??????????????????????,?????????????????C
  pG??

12

• I ????
• ??NCBI?ORF???? ( ORF finder
  http//www.ncbi.nlm.nih.gov/gorf/orfig.cgi
  )??ORF??????

13

• 1.2 ??????
• ???????????????????????????,??????????????
  ????,????????????????

14

• ?????????
• A DNA??????????
• B ?????(ORF)????,??????
• C ?????????????????
• D ??????????

15

• 1.3 ????
• Northern ????DNA???????.
• ????
• a ?????????????????,??????????,??????????????
• ???????????????????????
• b ???????????????

16
(No Transcript)
17
(No Transcript)
18

• C ???????????
• ??????,??Northern ???????(Zoo-blotting)???
• ?Northern ?? ?????DNA??????,?mRNA?????????,??
  DNA????????

19

• ????? ???????????,????????????,?????????????????
  ?????DNA ????????????DNA??????????,???????1??????,
  ?????????????

20
(No Transcript)
21
2 ??????cDNA??

• A ??cDNA??,?????DNA???????

22
cDNA????(CLONTECH)
23
cDNA????
24

• B ??????????,RACE ?????????cDNA???

25
5RACE (CLONTECH)
26
3RACE (CLONTECH)
27
(No Transcript)
28
(No Transcript)
29
3.??DNA????????

• A ?????cDNA????????,??????DNA???,?????????
• B ??????????????????????

30
4.????????

• 4.1 ????(knock-out)
• ???????????.
• ????
• ?????DNA ???????????????????,???????????,
  ???????????,???????????,???????.??????,???????DNA?
  ??????.

31
tk ???????? ? gangcyclovir neor ????????G418
32
(No Transcript)
33

• 4.2 ??????
• ????????????????????????,???????????????,?
  ???????.

34
4.3??RNA

• ??RNA?????????,????RNA (sense RNA)?DNA
  ??????,??mRNA ???,?????,???????.

35

• ????RNA ????
• ????????????????????? ? ?????? ??????????? ?
  ?????????????????????? ? ?????????

36
??????
??????
37

• ??RNA ????
• A ??????????,???????????????????RNA,????????
• B ?mRNA ???????,????????,????????
• C ??RNA?mRNA???????,?RNA???????,?????

38
4.4 RNAi??

• RNAi???????RNA???,???????????mRNA,???????
  ????????????????.

39
RNAi ????

• A dsRNA?????Dicer???,??dsRNA???2125 ??????RNA?
• B ?????RNA(siRNA)????????????RISC(RNA-induce
  silencing complex,RNA???????)????,???RISC?,???????
  mRNA,???????RNA???????

40
(No Transcript)
41

• RNAi???????
• A Fraser ??????????????RNA?????????????RNA????????
  ??????????
• B Chuang ????????? ? ????????????mRNA??????????

42
HbF???RNAi????
43
RNAi??????

• RNAi??????????
• RNAi?????????,????RNA????????,???????????????,????
  ???????
• RNAi????????????RNAi??????,???????????????,
  RNAi?????????.

44
4.5 ?????(yeast two-hybridization)

• ??
• ???????????????????
• ?????
• ???? (????????)? ??????????????? ?
  ???????RNA??? ? ??????mRNA

45

• ???????
• ??????1
  ??????2

????? ????cDNA
DNA????? ????
???????
??????1 ????
??????2
?????,?????????
46
(No Transcript)
47
4.6 ????

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

48
???????

• ??????
• RACE??
• RNAi??
• ?????

49
5.????6.????
???
????????!