Genetic Recombination, Genetic diagnosis and gene thereapy

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????

• ??????????????
• Genetic Recombination, Genetic diagnosis and
  gene thereapy

? ?
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? ???DNA??? DNA Recombination
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DNA??
???? (homologous recombination)
???? (conjugation)
???? (transformation)
???? (transduction)
???????(site-specific recombination)
? ? (transposition)
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???? ??DNA??
DNA Recombination Technique
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??DNA??????

• ? G.J.Mendel???????
• 1944? O.T.Avery?????????
• 1973? ??????????????????DNA??
• 1977? ???????????????????????????,??????DNA??????
  ???????
• 1980? ???????????DNA??????????
• 1997? ???????????????

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??????

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

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????DNA??????
(?) DNA??
??(clone) ??????????????????
??????????????(cloning),??????
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????????(molecular clone) ?DNA ??
???? ????(?????)
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DNA?? ???????,?????????????(??????????????????????
?DNA)???DNA?????????????DNA?????(replicon),?????
????????,???????????????,?????????????DNA??,??????
???DNA (recombinant DNA) ?
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?????? ????????????????????
????(genetic engineering) ?????????????????????
?,????DNA????
?? ? ?????????????DNA ? ????????????(???)
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(?)???

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

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??DNA?????????
? ? ? ? ?
???????? ??????,??DNA
DNA??? ??DNA????5????3?????????????,?DNA????????DNA???????
DNA???? ????cDNA??????? ??????????? DNA???? ??3??
Klenow?? ??DNA???I???,????DNA???I?5??3????3??5?????,??5??3?????????cDNA?????,??DNA 3??????
???? ??cDNA ??DNA???I????,???DNA????
??????? ???????5???????,?????
????? ?3?????????????
????? ???????
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????????
?? ????????(restriction endonuclease,
RE)???DNA?????, ??????????????DNA???????
Bam H?

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?? ????? (???????????)
?? ??????????????????,????DNA, ????DNA?
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??
Hin d?
Haemophilus influenzae d? ??????d??????
????????????????,??? ???????????????,??? ???????
? ???????????????
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????????? ????(palindrome)
?? ?????????
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Hind?
Bam H?


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

• cDNA (complementary DNA)
• ???DNA (genomic 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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1. ?? (plasmid)
?? ?????????????????????, ??????????????
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? ?
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(No Transcript)
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2. ???(phage)
????DNA???? ?gt??(???,??cDNA??) EMBL??(???,??????
?)
M13???DNA????(?lacZ??) M13mp?? pUC??
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3. ????(cosmid)
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??
??????? (yeast artificial chromosome,
YAC) ??????? (bacterial artificial chromosome,
BAC) ????DNA????? (????,???????,?????)
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????DNA??????
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? ? ? ? ? ? ? DNA ? ? ? ?
? ?
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(?)???????

• 1. ?????
• ???????????????????????? ?
• 2.???DNA??(genomic DNA library)
• 3. cDNA??(cDNA library)
• 4. ??????(polymerase chain reaction, PCR)

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???????????
?????????????DNA??
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????DNA????????
????????DNA
??????
????
????
???DNA?? ??????????????????????DNA???
??DNA??
???
?????????
? ?
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? ?
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? ?
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(?)??????????
(?)??????????
1. ?????? ??(1)?????????? (2)?????????? ?????? ??
?????
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??????????
Bam H?????
T4 DNA??? 15ºC
? ?
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????????(?????)???
Eco R?????
Bg l?????
???????????????????????
? ?
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2. ???? ???????????????? ????????????
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? ?
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(?)??DNA?????
????? ?????
????????? ?????(competent)
???? ?? (transformation) ?? (transfection) ??
(infection)
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(?)?????? 1. ????? (1) ??????? (2)
????(marker rescue) (3) ????? ???? Southern??
2. ????? ???????????????
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(?????) ???????
? ?
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? ?
??DNA????????????
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??DNA?????????
? ?
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?????????
(?)?????????? ???????????????,???????????
(?)????
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??DNA????
? ? ? ?
????????? ??
????VIII ????
????-?????????? ???????
??????? ???????
????(bFGF, EGF) ?????????
??? ?????
??? ?????
???(? 1b, ?2a, ? 2b, ?) ??????????
????? ??????????
??????? ?????
????? ?????????????????????
????(CHO, ??) ????
????B????????? ????
? ?
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(?)????
????(genetic diagnosis)???????????????????,?DNA???
??????????????????????????
????
????????DNA??
?????DNA???
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?? . ???????? . ???????????? .
??????,???DNA??? . ?????????,????????????
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?? . ???????????? . ?????? . ???????(PCR)
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?????????Molecular Hybridization Blotting
Technology
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??????????????
?????? (nucleic acid hybridization )
?DNA?????,?????DNA???????????,??DNA?RNA????,???DN
A?RNA?????????????????,?????????????????(heterodup
lex) ?
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??
RNA
DNA
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(?)???? (?)????
?? (probe) ????????????????????????????????????
,????NC????????,???????????????
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????????????
(?)DNA???? (Southern blotting)
?????DNA?????????????
(?)RNA???? (Northern blotting) ??RNA????????
(?)???????? (Western blotting)
?????????????????
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?? ???? (dot blotting) ???? (in situ
hybridization) DNA?? (DNA array) DNA????
(DNA chip)
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?????????
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? ?
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???????
? ?
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         ? ? ? ? ? ?
Biological Chip Technology
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??????
????(gene chip)

• DNA??(DNA chip)
• cDNA??(cDNA chip)
• ????????DNA???cDNA??????,???????????????????? ?

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? ?
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???????
?????(protein chip)
????????????????????????????,???????????,?????????
?,?????????????????????????
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? ? ? ? ? ?Polymerase Chain Reaction
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????????
Template DNA
Primer 1
5?
5?
5?
Primer 2
5?
? ?
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? ?
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??PCR????????

• ??DNA
• ?????
• ??DNA???
• dNTPs
• Mg2

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??PCR???????
?? 95C
?? Tm-5C
?? 72C
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??PCR?????
(?)??????? (?)??????? (?)DNA?RNA????? (?)DNA???? (
?)??????
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???????PCR????

• (?)???PCR??
• (?)??PCR??
• (?)??PCR??

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??PCR????
? ?
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(?)????
?? ????(gene therapy)???????????????????,?????????
???,??????????
?? ??????? (somatic cell gene therapy) ???????
(germ line gene therapy)
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????????????
1. ???? 2. ???? 3. ???? 4. ????