???????(polymerase chain reaction)??PCR??,?80?????????????????DNA????????????, ?????????????????????DNA?????,PCR???????????, ??????????????????,??????????????,????????, ??????,????????,???,???????????????

1
????PCR??16SrDNA
??????
1?PCR

• ???????(polymerase chain reaction)??PCR??,?80?????
  ????????????DNA????????????, ?????????????????????
  DNA?????,PCR???????????, ??????????????????,??????
  ????????,????????, ??????,????????,???,???????????
  ????

2

• PCR??????????DNA????,????DNA, ???4???????????????D
  NA?????????,PCR??????????????DNA???????
  ????????(denaturation)???(annealing)???(ex
  tension),???????

3
PCR(Polymerase Chain Reaction) ???
4
??????
2? 16SrDNA???????
16SrDAN?????????????rRNA(16SrRNA)???,??????
??????????????? 16SrRNA???????,?????????
?????,16SrRNA????1500bp??,??????????????,???,?????
????? ?????????PCR??????16SrRNA????????,???
??
5
(No Transcript)
6
??23SrDNA???????,?????????,???????,?????????
?????10????(variable region)??????11????(constant
region),????????,??????????????????
7
??PCR??????

• DNA??
• ??
• ?????
• dNTP
• ddH2O
• ?????

8
??PCR??????

• 1.DNA?????DNA??50ng200ng????DNA????, ?????????
• 2.dNTP??????100µM200µM?
• 3.Mg2Mg2?Taq???????2.0mM??,????Taq????????????
  ???
• 4.????????????????????20????GC???40
  -70???????????????3??????
• 5.Taq???????????????DNA???,?95??30????50???

9
??PCR??????

• 6.?????9395???????????
• 7.????55???,?????????????????????, ?????????????
• 8.????7072???,?Taq????????

10
PCR??????
Step 1 DNA??? Step 2 ???? Step 3 ????
11

• ??
• ??DNA?94?????????? ?55???????????????????????????
  ??70??????DNA???Taq ????, ?4?dNTP????????????????
  ??? ????????DNA?????????,??????30?,????????DNA??2n
  ????,30??????DNA????106-7?????PCR?????????????,???
  ??????

12
???????PCR?????

• ????????????????PCR??
• ??? ???????PCR?????
• ?? ??????????????
• ??????????????????
• ?????????
• ?????,??????????,?????????

DNA??
? ?
13
???????PCR?????

• pH?,?????
• ???,???

??Buffer

• ????????
• ???????

Mg 2??

• ????
• ??????

dNTP Mixture

• pH???
• ????

ddH2O
14
??????????
?? eppendorf???????, ?? ?????, ????
????UVP???????PCR? ??16SrDNA????????DNA?Taq
Plus?dNTP? PCR ?????????
?TAE?????? Lamdar DNA/Hind? ?EB ?
?????
15
??????
(?)?PCR?? 1. ???????? 5µl 10PCR????? 1µl 4?dNTP
2µl ????(??1) 2µl ????(??2) 1µl ??DNA 0.5µl
Taq DNA??? 38.5µl H2O ?????5?? (?)???94???3??
, 94???1??,61???1??, 72???1??, ??30?,??PCR????????
??, ?72??????5??,?????????? (?)??????????(2?????
)
??????PCR??????
16
??PCR??????

• ????????

• ????????,?????

??

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

1. ???????,???
2. Buffer??????
3. ????????????
4. ??????????,??????

? ?
17
??PCR??????
?????????

• ??PCR?????????????????,????,????????????????????
  ?

18
?????????
??

1. ????????????PCR
2. ???????????
3. ??????
4. ???????
5. ?????????????????
6. ??????

1. ??????
2. ?????????
3. ????
4. Mg2????
5. ??????
6. ??????

? ?
19
??PCR??????
?????

• ?????????Smear???

20
?????
??

1. ????
2. Buffer???
3. ??????
4. ????
5. dNTP?Mg2????
6. ??????

1. ????
2. ??Buffer
3. ????????
4. ????
5. ????dNTP???????
6. ??????

? ?
21
??PCR??????
??????(??????????????)

• ??????????????

??

1. ????????,???????????????????
2. ????????????,?????????????????????????????????
3. ???????????,???????

? ?

• ?????????????

22
???????

• 1???PCR????
• 2????????PCR??????????
• 3????????????????????