(gene diagnosis and therapy)

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• ????
• ?????????
• (gene diagnosis and therapy)

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

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

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

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?. ??????????

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

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(?)???????? ??????
????????(DNA?RNA)??????,??????????????????????
???? ??????????
DNA?DNA?? AT? GC
DNA?RNA?? AU? GC ??
??????,???????????? ??
???????,???????????????
????
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hybridization

• DNADNA 5 A T G C C G A T
• 3 T A C G G C
• DNARNA 5 A T G C G T A
• 3 U A C G C A
  U
• RNARNA 5 A U G C U A C G
• 3 U A C G A U
  G C

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????????????????????,????????????????????,
???????????????,?????????????????? ????
???????????DNA?RNA??,???????????????????,????
????? ????? ?????????????(???????????
?)????
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• 1.?????????
• ????
• ??????????????????????????
• ????
• ????????????????,??????????????????
• ?????????
• ???????????---

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• 2.??????(????)????

??????
????????
???????????DNA??
??????
????????
???
??
??????????????
??????
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• 3. ??????????
• Southern ?????(Southern blotting )
• Northern ?????(Northern blotting )
• ??????????
• (Spot/ Slot blot hybridization)
• ????(colony hybridization)
• ?????(sanwich hybridization)
• ????(nucleic acid hybridization in situ)

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(1) Southern ????? (Southern blotting )

???????? ??????
??DNA
Southern ?????????DNA?????????????????
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• (2)Northern ?????
• (Northern blotting )
• (??????????Southern?)
• ??RNA???RNA????????????????????(??DNA?RNA)??
  ??????????????
• Northern????????????RNA?mRNA

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• (3)??????????
• ????
• ????DNA?RNA?????????????????????????????????
  ??????????
• ??
• ??????????????
• ??
• ?????,??????????

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• (4)????(colony hybridization)

???????????????DNA???????????
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• (5)?????(sanwich hybridization)

??A ???? ????
????,?????????,??????
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• (6)????(nucleic acid hybridization in situ)
• ?????????????????????,???????DNA?RNA???
• ??????
• ????????
• DNA-DNA
• RNA-DNA ????
• RNA-RNA
• ????
• ??????,??????????????,???????????????????

?
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(?)??????(PCR,polymerase chain
reaction)??
PCR???????????????DNA??????DNA?????????????
?DNA?????? 1. PCR????
PCR??????dNTP?Mg2????,????Taq???DNA???,????DNA???
?RNA??,??DNA??????????(??)???3????????(25?30???),?
?DNA???100?????
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• (1)DNA?????
• ????DNA???950C??,???DNA??????(????DNA??????
  ?DNA????? ),?????????????
• (2)????????(?????)
• ???????????(550C??),?????????DNA??(3?)?????
  ????(??????????????,??????????????????????)

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• (3)????
• ???????720C??,Taq?????dNTP??????????DNA??3?
  ,?????,?????????????
• ?????PCR??(????????)
• (????????????????)
• ??????????,PCR??????????DNA ????T2n
  (n ????)?

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PCR???????
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• 2. PCR???????
• (1) ??
• PCR?????DNA?RNA?
• ???DNA????,???,???102?105???
• RNA?????,??
• RNA cDNA PCR, ???RT-PCR.
• (2)??DNA???(Taq DNA???)
• ?????????????DNA???,??95???30???????PCR?94?
  ??? 55????71?????30??????????
• ?PCR?,Taq DNA?????????

???
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• (3)??
• ????????????DNA???????????????????,?????15?30
  ????
• ?????PCR????????????????,???????PCR????????
• ????????

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????????????? ? ????,???15?30??? ?
GC??,???40?60 ? 4????????? ?
???????????? ? ???????????4?????? ?
??3???????? ? ??5?????
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• (4)?????Mg2
• PCR????10?50mmol/L Tris-HCl?Ph8.3?8.88??????
  ???????Mg2??
• (5)dNTP??
• PCR???50?200µmol/L?dNTP??4?dNTP?????

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• (6)??
• ???????
• ???? 940C, 30?
• ???????
• ?????????? ?GC??,
• ???? Tm - 5?
• ???????
• ???? 70? ?75?
• ????
• ???????, ????30?

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

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• 3. PCR????
• (1)???????
• ?????(Agrose)??????(PAGE)??????PCR????????
• ??????DNA????????,?????????,???,????????????
  ??
• (????????????????????,????????????)

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(2)?????? ???2? ? ????????????,??????????,?
????????????? ? ???????????,??????PCR?? ?????
?????????DNA??,??????????????????
(??????????,???????)
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• (?)????
• DNA?????????????????

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

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• ?. ?????????

(?) ???????? (?) ??????? (?) ??????????
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?? ???????? ß-????????????A?T
? ???????????
?????????1?Mst?????? ??? 1.1kb ??
1.3kb
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Mst II????(CCTNATG)

3
5
1.3kb
1.1 kb
1.1kb
????
0.2kb
3
5
1
2
3
1.3kb
1???? 2?????? 3???
????
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??? ???? ??????? ?????????
????????? ??????????
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???????? ???? ?????????????????????????,??
?????????? ???? ???????????????,?????????,
????????????

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

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• (?)????(gene correction)
• ??????????????????,????????
• (?)????(gene replacement)
• ??????????????,???DNA????????
  

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• (?)????(gene augmentation)
• ?????????????,?????????????,?????
  ????? ,?????????
• (?)????(gene inactivation)
• ?????????????,?????????mRNA??,???
  ???????????,?????????
• ??RNA ??mRNA???RNA
• ??DNA ??DNA???DNA.

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

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• (?)??????
• ???????(IL-2?GM-CSF?)??????????,??????????
  
• (?)??????
• ????????, ?????????????????,???????????????
  ?

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• ?? ?????????
• (?)????????
• ???????????????, ????????????????
• ?,?????????,?????(???)????????
• ????,???????????????????????????????
• ????????????
• ?????????????DNA????,?cDNA????,PCR??,??????

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

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

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• ???????,?????????????
• ???
• ?????????(in vivo)
• ?,?????????????????
• ??????????(ex vivo)
• ?,??????????????????,????????????????????????
  ?,???????

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(?)??????
???? DEAE-????
???? ???? ???? ????? ??DNA?????
???? ?????(????)

• ????
• ?????
• ????

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

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

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• (?)???????????
• 1????????????????
• 2??????????????????
• 3??????????,????????
• ???
• 4???????????????????
• 5???????????
  

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• Eggs are coaxed to mature in a culture dish. Each
  has a remnant egg cell called the polar body and
  cumulus cells from the ovary clinging to it.

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• While an egg is held still with a pipette, a
  needle is used to drill through the zona
  pellucida, removing a plug.

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• After ejecting the zona plug, the needle is
  inserted back in the egg through the hole to
  withdraw and discard the polar body and the egg's
  genetic material.

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• A cumulus cell from another egg is taken up
  into the needle. Cells called fibroblasts (or
  their nuclei) can also be used in this step.

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• The cumulus cell is injected deep into the egg
  that has been stripped of its genetic material.

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• The injected egg is exposed to a mixture of
  chemicals and growth factors designed to activate
  it to divide.

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• After roughly 24 hours, the activated egg
  begins dividing. The cells contain genetic
  material only from the injected cumulus cell.

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• By the fourth or fifth day, a hollow ball of
  roughly 100 cells has formed. It holds a clump of
  cells called the inner cell mass that contains
  stem cells.

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• The blastocyst is broken open, and the inner
  cell mass is grown in a culture dish to yield
  stem cells.

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• The stem cells, in turn, can be coaxed to grow
  into a variety of cells that might one day be
  injected into patients.
• --

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• ?????????(????)
• 1. ???????????
• ?????? B. ?????????
• C. ????????????????
• ????????? E. ??????
• ??????????????,????
• Southern ????????????RNA????
• Southern ????????????DNA????
• Northern??????????DNA?RNA
• Northern??????????RNA
• ????????????????DNA?RNA

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• 3. ?????PCR?????,???
• PCR??????DNA??,????????DNA????
• ????DNA??????
• ?Taq ???DNA???
• ?RNA????DNA??
• PCR??DNA????????3????????
• ?????????
• ?????? B. ??????
• ???? D. ????
• E. ?????