??DNA?????

1
?????????

• ??DNA?????

2

• ??????????
• ????
• DGGE????????(denaturing gradient gel
  electrophoresis
• PCR??????????
• ???????????????????????
• ??????????????????
• ????????????,?DNA ????????,?????????????

3

• DNA??????????,??????????
• DNA?????????DNA??????????
• ????CG-rich??????
• DGGE?????
• ???DNA????????,????????????
• ????????,??????????????? ?
• DNA?????????????????????,?????????
• DNA?????????????????????,???????? ??
• ?????????,????????,?????????????????

4

• ??????????????,??????????
• ???????DNA?????????????,??????
• ??????DNA?????????????,????????????????
• ??????????????DNA????
• ?????????? ?????????
• ??PCR???,?????????????
• ???5???2040 bp???CG,?CG clamp,??DNA?????
• ?????????????????
• ?PCR??????????,????,?????????????,?????????????
• ????????????????????,?????????????

5
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6

• ??DGGE??polyacrylamide gel(???????????
• ????????,??TGGE
• ????????CDGE (constant denaturant gel
  electrophoresis)
• DGGE???
• ??????
• ???????melting domain
• ????????

7

• PCR????????
• ARMS (Amplified refractory mutation system)
• ????Primer??????????????????
• Allele-Specific PCR??????????????????
• ????????????????, ??????????
• 1993 Rust et al.??MS-PCR (Mutagenically
  separated-PCR)
• ???(???)???5'????20 bp
• ?Primer??????????????
• ??3???????mismatch???,?????????
• ???????????????????????????,????????????

8

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

9

• ACRS (Amplified Created restriction site)
• ????????????????
• ?????????????????mismatch primer
  ?primer??????????
• PCR?,????primer??mismatch????????????????????????
• ????mismatch?primer?????????PCR
  ??,?????????????,???????
• Mismatch base?????????,????????,??????bands?,?????
  
• ??ACRS?, Primer??????????base?????????????

10
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11
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12

• ??PCR (multiplex PCR)
• ?DNA??????,?????????????,??????????
• ?????????PCR?????????,??????PCR??????
• ??????????,????????????
• ??????????
• ???? Primer ,?PCR??,????????????????????,?????
• ???primer pairs???????????????,?????????????primer
  -dimer???

13

• ???????????,??? ?????????( positive
  control)???????????

14

• SSCP???????single strand conformation
  polymorphism
• ???DNA???????????????????,???????????
• ???DNA??,????????????????? ?????
• ????????,???????????
• ?????????????????????,???????????????
• ???????????,???????????,??????????
• ??SSCP?????DNA???????,???????????,???DNA?????(???
  l50300 bp??)

15

• SSCP??????????????
• ????????????????
• ??????????????band,???????????????????
• ????????,??????,????????

16

• SSCP?????????????????
• ????????250 bp??
• 1992?Sarkar???Sanger?????,????????( dideoxyl
  fingerprinting, ddF)
• ddF?????????SSCP???????,
• ?????,?PCR???????????????,?ddCTP??????,
  ??????????????DNA,?????SSCP????
• ? SSCP??????????????????ddF????

17
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18
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19
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20
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21

• ????????Heteroduplex Analysis,HA
• HA ????????,?????????????????,???????
• ??????????PCR??,????????????????????

22

• ?????????,?????PCR????????PCR?? ?11??
• ?????,???????????DNA??,????
• HA?????1 kb???,?????????????
• ??????????,??????SSCP??,??????comformation-sensiti
  ve gel electrophoresis (CSGE)
• ??200 800 bp??DNA??????

23

• Carbodiimide??Carbodiimide Modification
• ???????,?C?T??????,??Carbodiimide????????
• ????????????,????????????(????DNA??????)
• ???? ?????????????
• ???primer?????,???????mismatch????????????,???????
  ????????????

24

• DHPLC????????Denaturing high performance liquid
  chromatography
• 1995???????Peter Oefner?Umderhill??
  HPLC??DNA?????
• ??150 1000 bp?????
• HPLC???????????????? ????????,?????????????,?????
  ???,??????,??????????( retention time) ??????
• DHPLC???TMHA (temperature-modulated heteroduplex
  analysis)
• ????????,????????,?????,??????????????????,???????
  DNA???????????,?????????????

25
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26

• ?????????????????,DNA?????????????DNA???
• ????????????????
• ???????????
• ?PCR ???????????1 1??
• 95C??5 ?, ??????
• ???cycle?1.5C,?45?
• ???????????8 ml??????
• PCR??????
• DNA???????????
• ??????????????????
• ??????DNA???????(?????,????????),?????????
• ??????????????????
• ???????????,??????????????

27

• ????peak???,???????????
• DHPLC??????????,???????,?????????????????????????
  

28

• ????
• ?????????Chemical cleavage of mismatch, CCM
• ????PCR????,????????
• ?????DNA??C?T?????hydroxylamine?????osmium
  tetroxide??
• ?peperidine??DNA??????
• ??????????,?????????????????
• ??1 kb????DNA?????,?? ?????
• ??????????????????

29
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30

• ??????????Enzyme cleavage of mismatch
• T4???VII,??????????
• ????????????? ( small loop )?????
• ???????1 kb??,?????????? ???,????????
• ??????CELI????, ???????????
• ???????ligase chain reaction, LCR
• 1991 Barany??????????????,????????????????
• ??????????????????,??????
• ?????????????????????? ??,??30???,???????????????
  

31
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32
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33
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34
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35

• RFLP (restriction fragment length
  polymophisms)?PCR-RFLP
• ????????20004000 bp????????,?????1000
  bp?????????
• ????????(???)???(SNP)???
• ???????????????????,??????DNA blotting?PCR-RFLP???
  ?
• SNP (Single nucleotide polymorphism)
• ????????????????,??????????
• ???????????,??????????
• ???? ??Allele????,???????????????
• ??????SNP???,??????????????????,???????????

36

• CFLP (cleavage fragment length polymorphism )
• ???DNA????????????????,??????????????????????
• ??structure-specific???? cleavase
  I??,??????????????????( band pattern)
• Muts
• ?E. coli???, Muts????????DNA??
• ????DNA??,?????DNA ??????

37

• ????Automated sequencing
• ?? Sanger??????????????
• ??DNA???,???????dNTP???ddNTP???DNA ??,??PCR??,
  DNA?????5?3???
• ?ddNTP??3-OH,???????? dNTP?????,???????,?????????
  ?
• ?????????ddNTP?dNTP???,????????????ddNTP???,??????
  ??????
• ??????????????DNA
• ??4???????4????ddNTPs
• ???????????,???????????, ???????

38
Fluorescence dideoxy Method I
????http//www.cbs.dtu.dk/staff/dave/roanoke/gene
tics15.htm
39
Fluorescence dideoxy Method II
40

• ??????????PCR-RFLP???PCR-SSCP??,????? ?????
• ???????????,????PCR??????
• ?????????,??????????
• ?????????,??3??5??????????
• ??????????1000 bp ??

41

• Minisequencing
• PCR?,??????????????dNTP?????
• ??????ddNTP????????3????????????????
• ?ddNTP??????????
• ?????????
• Minisequencing??????peak??homozygote (????,?? ?)
  , 2?peak??heterozygote (????,???)

42
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43

• ????????????Sequencing , SSCP?DHPLC
• ??????????
• ?????????,????????????????? ??????
• ?????Microarray Sequencing
• ???????DNA???????????????????
• ????????,???????????????????????