Genetic Recombination and Genetic Engineering

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? ? ? ?

• ?????????
• Genetic Recombination and Genetic Engineering

? ?
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? ? ?DNA??? DNA Recombination
???????????????!!! ???????!!!
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DNA?? DNA recombination
???? (homologous recombination)
???? (conjugation)
???? (transformation)
???? (transduction)
???????(site-specific recombination)
? ? (transposition)
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??????
?????????????????(homologous recombination),??????
??????DNA????,??????????,???DNA???????????????????
?
?E.coli???????,?????????Holliday???
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Holliday???,????4?????

• ????????DNA????
• ???DNA????????????DNA??????,??Holliday???
• ?????????????DNA
• ?Holliday????????,?????????DNA.

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Holiday???
? ?
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Holliday intermediate?????
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????????????
(?)????
???????????????????,??DNA?????(??)???????(??)?DNA?
???????(conjugation)?
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?? ?????????????DNA??
?????? F ??(F factor)
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(?)????(transformation)
??????????????DNA,???????????????????,??????
(transformation)?
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????,???DNA??????????
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? ?
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(?)????
????????(??)?????????????(??)???,???????????????DN
A?????????????(transduction)?
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?
?????? (lysis pathway) ??????? (lysogenic pathway)
????????
? ?
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? ?
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???????? ??????(site-specific recombination)
???????,???DNA??????????????
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??????
???????????????????????(transposition)?
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(?)??????
????(insertion sequences, IS)?? ?????????(inverte
d repeats, IR)?? ?????????
?????(transposase)????
???? ?????(conservative transposition) ?????(dupl
icative transposition)
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??????????
? ?
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(?)?????
???(transposons) ????????????????????????
??????????? ??????? ???????????
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(No Transcript)
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?????????
? ?
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? ? ? ??DNA??
DNA Recombination Technique
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Gene engineering includes a range of
techniques and methods used by scientists to
control or modify genes, switch them off or move
them between two unrelated species.
The terms 'gene technology', genetic
manipulation?'genetic engineering' and 'genetic
modification' mean the same thing, and refer to
one type of modern biotechnology.
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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??
???????,?????????????(???????????????????????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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????????
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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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????? ????????,???????????,??????????
Bam H?

Bst?

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??? ???????????????????,???DNA?,?????????,????????
??????????????(compatible end)?
Bam H?
Bg l?


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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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???????

1. ?????
2. ????????????,???????????
3. ?????(??DNA???),???????????,???????
4. ????,????????DNA?

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1. ?? (plasmid)
?? ?????????????????????, ??????????????
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? ?
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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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(?)??????????
(?)??????????
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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3. ??????? ??????(terminal transferase)????,?DNA?
??????????????????,????????
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??DNA
????
????????
???
? ?
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4. ????(linker)?? ???????????,?????????????,?????
???
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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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a??
? ?
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a ?????
? ?
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????
? ?
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Southern??
? ?
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??ß??????????
? ?
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? ?
??DNA??????????
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??DNA?????????
? ?
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(?)???????
??????? ??????? ??????? ?????????
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1. ?????? (E.coli????????) ?????? ????
?????? ??????? E.coli???????
?????????DNA ???????????? ???????????????
(inclusion body) ???????????
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??????cDNA ??????
? ?
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(No Transcript)
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2. ?????? ????????????
????????cDNA??????DNA ??????????? ????????? ????
?????????
?? ??????????????
??????? DEAE??????? ??? ????? ????
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????pFASTBACI ?????
? ?
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? ?
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????????????
(?)?????????? ???????????????,???????????
(?)????
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(D) MEDICINE
New drugs
To design more effective therapies for diseases
like cancer, diabetes(???), influenza and
hepatitis C(??)
Gene therapy

• To replace an abnormal gene with a normal gene.
• To insert a missing gene.
• To switch off rogue genes that may cause
  cancers.
• To stop viruses replicating within cells.

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Insulin
To replace production of insulin from pigs
livers. This has increased efficiency of insulin
manufacture while reducing cost.
Therapeutic cloning
Embryos are cloned for their cells, which can
be used in gene therapies. Like all cloning, this
technique has significant moral and ethical
implications and remains the subject of research.
Antibody treatments
To develop antibodies that will help diagnose
and treat diseases like cancer and diabetes.
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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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(?)????
?? ????(gene therapy)???????????????????,?????????
???,??????????
?? ??????? (somatic cell gene therapy) ???????
(germ line gene therapy)
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(?)???????
1. ???? 2. ????? 3. ????? 4. ??????
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???????????????

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

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???????????
?????????????????,????(Andi),?????????????????????
?????????????,????????????????????????????????????
?????????,????????????????????????????????????????
??????? ???????????1???,??????,?????????????,????
????????
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?????????????

• ????????,?????????????????-GT?????
  ,?????????????????????? GT?????????,????????????
  ????????????????,??????????????,????????????
• ??????????,????????????????????????,??????????????
  ???
• ??????,???????????????,???????,????,??????????,???
  ?????????????????

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???????????
????????????
??
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????????transgenic anti-chilling tomatoes
???????????????????????????,???????(?????????2?)??
?(????18??)???(?????C????15.5)???????
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?????????????(transgenic anti-ageing tomatoes)
??????,???????(ACC)?????,??????.
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?????Bt?????????
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Golden rice
vitamin A retinol - produced by animals in
liver, milk and eggs plants do not produce vitA -
but some produce ß-carotene, a precursor for vitA
including yellow vegetables, yellow endosperm
corn and sorghum in many developing countries,
vitA deficiency is a major problem
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???

1. ??????,???????holliday???????
2. ???????(conjugation)?????(transformation)???(trans
   duction)
3. ????? ?????????????????????
4. ???????(genetic engineering),?????????????
5. ????????-???????????????????????
6. ?????????????
7. ?????????????????

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8. ??????????????????? 9. ????????????????????? 10
. ?????????????????????