??AFLP?????????

1
?????????
??AFLP?????????
2
???? 1????????DNA????? 2???AFLP??????????????
3
???? 1 ?????DNA??? 2 ?????DNA?AFLP???????
4
????
?????????????,????????,????,????!??????????,??????
????
5
???????????? !!??

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

6
a.???? b.?????,?????????????????? c.?????????,??
?????????? d.???? e.???? f.????,?????? g.????
h.????????????????

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

7
???????

• ????????? RFLP (Restriction Fragment Length
  Polymorphism,??????????)
• ????????? RAPD(Random Amplified Polymorphic
  DNA,???????DNA )
• AFLP (Amplified Fragment Length
  Polymorphism,?????????)
• ???,???????????SSR?ISSR?SRAP(?????????)??,????????
  ???????SNP( ???????) ?

8
AFLP(Amplidied Fragment Length Polymorphism)?1993?
?????Zbaeau?Vos?????????DNA???????? ????????DNA??
?????????,???????????????????,????????????????????
????,????????????,???????PCR??3?????????,????????
?????????????????????????????????????????????????
?????????????????????,??????????
9
????????????????????????????????????3????????(1
10 bp)??????????????????????????
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AFLP?????

• ????
• DNA???(SDS??CTAB?)
• ??
• ??
• PCR??(PCR AMPLIFIED )
• ???(Pre-amplified)
• ?????(Elective amplified)

11

• ????????

??????????????????,??????????????4?10??????

• ????

???????????????????????,??Gene-Scan,
Gel-Compare??????????
12
DNA???(SDS??CTAB?)

• SDS?Sodium dodecyl sulfate????????????,
• CTAB?Cetyl trimethyl ammonium
  bromide?????????????,
• ???????,?????????????????,?????????,??,?????DNA???
  ????????

13
??

• ?????????????,????????????,???6??????????????(??Ec
  oRI,?PstI?SacI ),????4??????????????(??MseI
  ?TaqI)????????????(1)?????????DNA??,?????????????
  ??????,??????????????????????????????,????????????
  ??????????(2)???????????,??????????????(3)????????
  ??????????(4)??????????????????,??????????AFLP????
  ?,??????????????????,?EcoRI/MseI????EcoRI?EcoRI???
  EcoRI?MseI???Mse??MseI??,??AFLP???????????DNA?????
  ,???????????,???????DNA??????????

14
??

• ????DNA???T4 DNA??????????????????????,???????????
  ??????,??????,????????,?????????(??????????),?????
  ???????????????????,????????????????????????????
  ?????????????

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PCR??(PCR AMPLIFIED )

• ????????????????AFLP????????(1)5'????????????????
  (core sequence)(2)????????????(enzyme-specific
  sequence) (3)3'??????????????(selective
  extension),??AFLP?????(????????????)?????,?????Eco
  RI?MseI??,??????????????????????????,???????????94
  ???,37???,4??????????????????,??????????1/4,?????
  ???????????????,????1/4096???????,???????????????
  ??????????????????????????????????????????????

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• ???(Pre-amplified)
• ??????3'????????,????????????????,????????????????
  ????????,???????????????3'?3???????????????
• ?????(Elective amplified)
• ????????????????,???????????????3'??3????????,??3?
  ????????????DNA???

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Mse I
EcoR I
(??????)
(??)
EcoR I??
Mse I??
EcoR I??A
Mse I??C
(???)
EcoR I??AAC
Mse I??CAA
(????)
(????)
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???? 1 ?????DNA??? 2 DNA????????? 3
?AFLP??????(adapter)?? 4 ??PCR??,????????????,??
???????????,??????,?????????0.51µg 5
????????????????DNA?? 6 ???
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???????????????DNA??????,????????????????????AFLP?
????????????DNA??,???????????????,??,?????????DNA?
?????DNA????????????????AFLP?????????????DNA?????

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???? ?????? ?????? PCR? ????? ??? DNA?????
?? ???????
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???????
1 ?????(adapter)??    ????????AFLP????????,??????
???AFLP?RAPD?PCR??????? ?????DNA?????????,AFLP??
?????????????,AFLP?????????????????5'??,5'????????
???? ?????3?????????(CORE)??????(ENZ)????????
(EXT)?
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?EcoRI???MseI??(EXT?NNN??,N??????)
CORE ENZ EXT
EcoRI 5-GACTGCGTACC AATTC NNN-3
MseI 5-GATGAGTCCTGAG TAA NNN-3
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????????????????????, ?EcoRI???MseI??
EcoRI-adapter 5-CTCGTAGACTGCGTACC
CATCTGACGCATGGTTAA-
5 MseI-adapter5-GACGATGAGTCCTGAG
TACTCAGGACTCAT-5
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2 ??DNA?? DNA??,???,???? ????????DNA??????DNA?
???cDNA? ??,??CTAB????????DNA,?????? ?DNA,?EcoRI/
MseI???????T4 DNA???,?EcoRI?MseI???????????,???,??
10?,-20???????DNA???????? AFLP?????DNA???????,?
25ng??25pg?,??????????,?????????,DNA??????,????DNA
?????,???????
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3 DNA???? AFLP??????????,????????AFLP???????????
?,????????????? ???????????????,AFLP??20???,??????
?????94?,DNA????30?,56???1??,72???1??? ????2?3??
??????,AFLP???36?????1????94?DNA??30?,65???30?,72
???1???2?13???,DNA????????0.7?,???????????1436?
?,?????56?,?????????? ?
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???????DNA,AFLP??????????,??????,????????,???AFLP?
?,AFLP????????????,???,???????10?,DNA????,???????
???????????3???????????????????,?????Smear????
????AFLP?????????DNA?
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4 ??
???????? ???????????????????????????,??????????,
???????????,????????,?????????????????????????????
???????????????????????????????
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(1)??????? a) ?????????(1ml??)??????????????????
?????????????????????b) ??????????95???????3?,?
????Sigmacote??? (2)??????? a)
????,??????????b) ?????????(1.5ml)??????????????
??????????c) ????,???????????,?????????????????,
??????????,??????????d) ????????????????????????
??????????????????????
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• (3)??????????
• ??????????????,???????????,??????????
• b) ???????,????????,?????????
• c) ???????????????????????????????????????????10
  NaOH??3060min???
• d) ???????, ?????????????????????????, ????????,
  ?????????????????

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????? (1) ??????0.4mm??????????????,???????????
???????????????????,??????? (2)??????(6)???????
????,???AcrBis?10TBE???,??????????99.2ml,??0.45m
m?????,?????????TEMED?
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???????(PAG)?????(Acr)?????????(Bis)???????????,??
?????????????,?????????????,??????????????????????
???????,???????????? ??????????????????????(APS)
???????????????????? (TEMED)?
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(3)???? ????????????????????,???,???????????????
???4-6??,?????,??????,????????TEMED??????
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4)???????????? a) ??????????,???????????,???????
??????????????? b) ??????????????????????????,???
?TEMED??????c) ???????????????,??????????
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5 ???? ???????,???,????,??DNA???2µl,?5?????????
???,????110W?,??2??
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???(1)????????,??????,???????,???????????,??????(
???????????,????) (2)??????????????,?????????????
??,????????????,????TBE???? (3)??10TBE????1TBE,
???????????????,???????,?????????? (4)???????55??
?????? (5)?30V/cm??????20-30??(???80-90W,?30min)?
????????????????,?????????????????????GC??????????
?,????????(6)???????? a) ???????????,??????????0
.5mm??,????????????,??????????b)
???????????????????,??????????????c)
????0.4mm???????????
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?????(1)?????,?????????????,??????????????? (2)P
CR??????????(98???,10mmEDTA,0.25???,????)83??,
95???8min,????????????????,??????????????????????,
?????????????????AFLP???????95???8min,??????? (3)
?????????????,????????????,?????????30V/cm????,5??
?????40-60V/cm,????????????,??55cm?,0.4mm?????,?25
00V???????2????????,????????,???????28mA??25mA?(4
)???? ????????????????,????.
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6 ???????? ??????DNA??,???????????? DNA??????
????? ??????(Ag)??????DNA???????????? ????????,
??????????????,???? ????????DNA?????
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?????????????????????????,????????????????????????
???????? (1)???????????????????,????????????????
(2)???????(????)?????,?????(????)??,????20?????
????????,????????????(???)???????,????????? ?????
??????,?????DNA?? ?????,?????????????,????????,???
?? ? (3)??????????2?,??8????????,???????????????
??10-20?,????,?????????
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• (4)?????????????????30???
• (5) ??
• ???????????10???????(?NaOH)?,??37??(3ml)?10mg/ml
  ????????(400µl)??????????????1?,????,????????????
• b) ????????,????,?????????,??????????
• c) ??????????????????????5-10?????????????????????
  ??5-10????????????????????????????,????????????
• d) ????????1?(?????)?????????????????????????????,
  ????????????????2-3??,??????????
• e) ???????????,??1?????,????2-3??,??????,????

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????? DNA???
????
????
???
??
?????????
????
???????????
???
??
??
?????
41
????
1. DNA???????????,??????? 2. ???DNA????,ODgt1.7, 3.
????,??????????buffer, 4.?????????????, 5.????????
????,????,?????? 6.???????????,???????,??????,???
? 7.??????????,??????
42
AFLP?????

• ??????
• ?????????EcoRI?PstI? SacI? HindIII?Apal?MseI?
  TaqI??AFLP??
• ????????????(???)
• ????????????(???)TE-AFLP
• ??????????
• ?????????????????????,????????????????,???????????
  ??,???????,???AFLP?????????,?????????????????????,
  ?????,???????????????????????????????,????????????
  ?
• ????
• AFLP?????????DNA???????,????cDNA-AFLP,???????-AFLP
  ?

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AFLP????? ???? ??AFLP??????????????????
AFLP????????? ??AFLP??????????? ??????? DNA??
???
44
?? ???? CTAB????????DNA??? AFLP??????
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???? 1 ???? 2 ???? 3 ??????
46
?????? 1 ?????????????????(08.12.10)??????????
2 ???? 3 ???? 4 ????????? 5 ???? 6
????????????? 7 ?????????? 8 ???????????????? 9
????? ???????????????????????