??DNA??

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

• ??DNA??
• ??????
• PCR?????

3
??DNA
??DNA??(recombination DNA technique)????DNA???????
???,??????????????????????????,??????????????????,
?????????????DNA??(DNA cloning)?????(molecular
hybridization)?
4
??DNA

• ??

• ??

• ??(??)

• ?????

5
foreign DNA
bacterial cell
vector
restriction endonuclease
ligase
??DNA??????
host
6
????????
???????DNA?????????????

• Type?

• Type?
• Type?

?
7
????????
???????DNA?????????????
EcoR?
8
????????
Hae?
Mbo?
BamH?
Pst?
9
????????
??DNA??,????DNA???DNA????????????

• ??????

• ?????????????

• ??????,??????????????

10
DNA???
????DNA??????,??ATP?????????DNA??5
-PO4???DNA??3-OH????????

• E.coli DNA???

• T4 DNA???

11
???(Vector)
??????????DNA??????,?????????????

• ?????

• ??????????

• ????????????????

• ?????????,?????????

12
???(Vector)
??????????DNA??????,?????????????

• ??? ???

• ???? ????

13
??(plasmid)
???????????,??????????????DNA???
14
??(plasmid)
pUC19 2686 bp
15
????(Lambda phage)
??????????????????,????????????????????DNA??????

• Cos site

• ?? ??

• ????????

• ????????

16
??(cosmid)
?????????????????????
Cosmid????????????,????(????)??cos????,???????????
????
17
??(cosmid)
?????????????????????

• ???,????????(?phage)
• ?????(plasmid)
• 45 kb??DNA
• ??????????

18
??(cosmid)
19
???DNA????

• P1??? PAC

• BAC, ???????
• YAC, ???????

• ???
• ??
• ??????,ARS

20
???(Vector)
?? ??????
Plasmid 010 kb
Insertion ? vector 010 kb
Replacement ? vector 923 kb
Cosmid 3344 kb
P1 phage 70100 kb
PAC 130150 kb
BAC 300 kb
YAC 0.22.0 Mb
21
????(Cell clone)
22
??DNA?????
????
23
??DNA?????
24
??DNA?????
????
25
????

• ?????(genomic library)

• ??????????
• cDNA??(cDNA library)

26
????

• ????(homoduplex)

• ????(heteroduplex)

27
????????
??(probe)??????????????DNA?RNA??,????DNA?RNA?????
??????????????

• ??????(oligonucleotide probe)

• ????(degenerate probe)

28
????????

• ????(degenerate probe)

29
????????
??(probe)??????????????DNA?RNA??,????DNA?RNA?????
??????????????

• ??????(oligonucleotide probe)

• ????(degenerate probe)

• ??EST??(unique EST-base probe)

30
????????

• ?????

• 32P,35S,3H

• ??????

• ???
• ???

31
????????

• ?????(nick translation)

• ???????(random-primed labeling)

• ?????(end labelling)

• PCR???(PCR amplification)

32
????

• ??? (dot-blot)?

• dot and slot hybridization

• allele-specific oligonucleotide, ASO

33
ASO??
CTG ACT CCT GAG GAG AAG TCT
CAT CTG ACT CCT GAG GAG AAG TCT GCC
His Leu Thr Pro Glu Glu Lys Ser Ala
His Leu Thr Pro Val Glu Lys Ser Ala
CAT CTG ACT CCT GTG GAG AAG TCT GCC
CTG ACT CCT GTG GAG AAG TCT
34
ASO??
CTG ACT CCT GAG GAG AAG TCT
CTG ACT CCT GTG GAG AAG TCT
35
????

• ??? (dot-blot)?

• Southern??(Southern blot)

36
Southern Blot
37
Southern Blot
??????(HbS) ?57????
Mst? CCTNAGG
?A CCTGAGGAG ?S CCTGTGGAG
38
????

• ??? (dot-blot)?

• Southern??(Southern blot)

• Northern??(Northern blot)

• Western??(Western blot)

• ????(immunoblotting)

39
???????(PCR)
???????(Polymerase Chain Reaction,PCR),??????(in
vitro)??????????????????DNA??????????

• 1971, Dr. Kjell Kleppe

• 1983, Dr. Mullis

40
???????(PCR)
"for his invention of the polymerase chain
reaction (PCR) method"
The Nobel Prize in Chemistry 1993
41
???????(PCR)

• Denaturing step 90?95?

• Denatures the double stranded DNA into single
  strands

42
???????(PCR)

• Denaturing step 90?95?

• Annealing step 50?68?

• Allows the primers to attach to complementary
  strand of DNA

43
???????(PCR)

• Denaturing step 90?95?

• Annealing step 50?68?

• Extension step 70?72?

• Optimal temperature for Taq polymerase to attach
  to and extend the new strand of DNA

44
???????(PCR)
45
???????(PCR)

• ??

• ??
• ??
• ??

46
???????(PCR)

• ??

• ????????
• PCR??????
• DNA???????

47
?????

• ??DNA??
• ??????
• PCR?????