PCR???? Introduction to PCR Technology

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PCR????Introduction to PCR
Technology
??????????????? PCR??? ??
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????
?. DNA????? ?. PCR 1. PCR???? 2. PCR????
3. PCR?? ?. ????PCR??HBV 1. ????PCR??
2. ????PCR???? 3. ????PCR???? 4.
????PCR?? ?.???????? HPV23??????
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DNA?????
DNA??
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??
A??? G??? C??? T????
??
??
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DNA?????
???DNA??
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1.DNA?????
2.?????RNA??
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3.DNA??? ????
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?????????

• 1985?,??PE-Cetus???Mullis???????????(PCR)
• ???????????????DNA??
• ????E-coli DNA?????PCR,???????,???????,??,????
• ??DNA????????PCR???????,??PE-Cetus????????PCR?????
  ??
• 1993?,Mullis?????????????

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PCR ????
??????(Polymerase Chain
Reaction)??PCR,???????????????DNA???????????
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PCR ????
???? ? ? ? ? DNA??? ?
? ??? Mg2
???? ? ? ?
?(??) ??
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??DNA
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Taq?
??2
??1
Taq?
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?1???
?2???
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DNA??
DNA??
TaqDNA???
dNTP
???
Mg2
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MJ Research, Inc.
PTC-100
PTC-150
PTC-200
PTC-220
PTC-225
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ABS, Inc.
PE-2400
PE-2700
PE-9600
PE-9700
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????
??????PCR???????????????????
1.??????? 2. ??????? 3. ??????? 4. ???
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PCR??????
??13? 2530?
DNA?? ??2???
??DNA?? ??100????
???? DNA??
DNA?? ?????
DNA???
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?????
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????PCR (Real-time PCR)
????????PCR??,???PCR???????????,?????????????
?PCR??,???????????????????????
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????PCR????
1. TaqMan???? 2. LightCycler ????? 3. ??
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TaqMan????
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LightCycler ?????
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?????
? ? ? ? P C R
? ? ? ?
?????
?????
????
??????
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?????

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

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????????????????
????????,????????????????????
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?????????
?????????????????????????,????,???????????????????
?,??????????????????????
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???????????
???????????????????
HBV ???EDTA????200ul
TB ??
HPV CT
????????
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???????
????????,??,?????
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???????
????????????????????? ??????????????? ????????????
??????????????????????? ????????
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?????????
????????????,?????????? ??????????,???????????????
???? ???????,???250C??4????DEPC???,??????RNAase??
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????????

• ??DNA?????,????????????????,???????????
• ??RNA?????,??RNA??RNA????,????????,?????????????1
  4???????5mol/L GITC(??????)????,?????(?)??RNA?????
  ???????????,??????????????

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?????

• ??????????????
• ???????????????????(?
• ??DNA?????EDTA?????????
• RNA ??????GITC????????)?
• ???????????????????
• ??RNA?????,?????????,?
• ?????,???????

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?????

• ?????????/?????-70???????
• ??DNA????????????10mmol/L Tris,
• 1 mmol/L EDTA???(pH 7.5-8.0)?4???
• ??RNA????????????????-80?
• ???????????????????-20?
• ???GITC???RNA????????7?

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

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

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?????(? ?)

• ????????????????????? ????
• ????????????????? ???,????
• ???70?????????????
• ??????????????,??????????
• ??????????????????,?????,
• ????????????????????
• ??????????????????,??????
• ???????

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???????(? ?)
???? ???? ????? ???(??????) ??? ????
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????
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????(HBV)

• ?????1.0 103copies/ml HBV DNA 4.2
  109copies/ml,??????,?????????
• 2. ?????HBVDNAgt 4.2 109copies/ml,??????gt 4.2
  109copies/ml?
• ??????lt 1.0 103copies/ml,???????,???lt 1.0
  copies/ml
• ???? 6.666E066.666 106 copies/ml

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????PCR???
1. ??????????????????,??? ???????????????
2. ???????????????mRNA????
?????,??????????,?????? ????????,?????? 3.
??(?????????SNP???)
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HBV DNA
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??????
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HBV DNA???????
?????????????? ?????????? ?????????????
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??????????????

• ?????????????,??????????????????????
• ????????????????,??????????????,??????????,???????
  ?????,????????????????,??????????????????
• ??HBV?????????,??????????????????

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??????????
??????,???????????,??????? ??????,????? ??????,??
????
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?????????????
???????(??????????)?????????,???????????,?????????
????,????????????
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YMDD???
YMDD ??????????????????? YIDD ?????
??? YVDD????? ???
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? ? ? ? ? ? ? ?
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DNA???? ?????????? ??????? PCR??
????PCR PCR
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?????????
??
???
??
???
??????
DNA????? 1 ???????????????????? 2
????????????????????????,???????????
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??????
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DNA???????????PCR??
????
Biotin
Primer up
Primer down
Biotin
PCR??????????
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??????????????
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??????(Human Papillomaviruses, HPV)

• ??DNA?????
• ??DNA,?8Kb
• ???????5055nm?
• ?????????????????
• ???????????
• ?????????
• ????????????????

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• ?????
• ???100???????,????40????????????,?30???????
• ??????HPV?????????????
• 1)????????????,?????????????(HPV 6?11?42?)
• 2)????????????,?????????(HPV 16?
  18?31?33?35?39 ?45?51?52?53?56?58?59?66? 68?)
• ????????????

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HPV????

• ???????????
• ??????????,???????Regoni Stern????????????????????
  
• 1977?Laverty????????????????????????(Human
  papillomavirus,HPV)???
• ?89???,??.????????Keerti Shah???????HPV????????
• 1995??????????????(IARC)???????HPV??????????????

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HPV????
?????????HPV?????????????????

• HPV????????95??????
• ???HPV??????,??????????????????1.5?5???
• ????CIN3?????HPV?????99.7

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HPV?????

• HPV E6??????p53??
• HPV E7???RB????,RB?????? E2F???????????
• HPV16-E5????CDK???p27????????????????

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???????????
HPV??(-) ?????(-)
?
58??? HPV??()
?????() HPV??(-) ?????()
?
?
?????
????
?
?
?????
HPV??() ?????(-)

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

• ??
• ????HPV???,???HPV??
• ??????????????
• ?????
• ??PCR?????????????18?????
• 5????HPV
• ?????????????????

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HPV33?35
HPV16?43
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HPV????????????
HPV18
Primer up
HPV16
Primer down
HPV58
HPV16?43
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???????????(HPV)?????
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????????????????? ??HPV?????? ????????
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• ???--??????????
• ????????,?????50???????????,??????????5,??80
  ????????????????????????13.15?,?????????????28.8
  ,??????????????
• ?????????,???????????????????????70???10.28
  /10????90???3.25/10?,??????,?????????

?? ??? (/10?)
?? 8.00
?? 3.25
?? 2.54
?? 3.8
???? 36.00
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??????????(???????45?),???????????80??????? ???
??35?????4.8,?2000?????34.1
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• ?????????
• ?????????????????????????????
• ?????????????????????????
• ?????????????????????????

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?????
?????100???????(??DNA???????90,???????)????
?35????????????,?30???????11-37?????????????HPV?
1)????????????,?????????????(HPV 6?11?42?43?44)
2)??? ?????????,?????????(HPV 16? 18? 31?
33?35? 39 ?45? 51? 52? 53?56? 58?59?
66?68?73?83?MM4)3)????????
?????????????????????HPV45????????,HPV39?59?????
?????,HPV52,58?????? ????????????HPV16(?51),?????
???HPV18???56?39?
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? HPV???????
??HPV?? ????
6,11 ??,CIN I
16 CIN,
18 CIN
30,40,58,69 CIN
31,33,35,39 CIN
45,51,52,56 CIN I
42,43,44 CIN I
53 ??????
54 ????
55 ??????
59 VIN
61,62,64,67 VaIN
66 ??
70 ??????
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HPV???

• ???????(HPV6?11)
• ???????(HPV16?18?31?33?45?58 )

?????????HPV???
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? ??HPV??????????
  ?1.????????1153?,????????119?
?????????????,????????(???????? /
?????????)100, ?????????226? 2.
????????????????,HPV???????????????(??)???????
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2001??TBS(The Bethesda System)????????????????????
??????
???????--????????--??????? ASCUS(Atypical
Squamous Cells)????????? ASC-US??????????????? A
SC-H??????????????????????????? L-SIL?????????--
--HPV???CIN I H-SIL?????????----?CIN II?CIN
III???????? SCC????? AGC( Atypical Glandular
Cells)???????? AIS(Adenocarcinoma In
Situ)???? CS?? ???????? CIN(Cervical
Intraepithelial Neoplasm)??????? CIN I???????
CIN II??????? CINIII???????????
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???,80 ??8??
??????/HPV??
??lt30?
????
HPV??
?????? ??????
HPV
CIN I(???)
?????? ????415
??gt30?
CIN II/III(???)
??????
??624??
5-10?
????? ?????? ????? HPV?????
? HPV????????????
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??
??(??)
??(??)
??(??)
? CIN?????CC?????
?? ??HPV CIN I CIN II CIN III
???() 5 15 30 45
???() 15 37 35 lt56
???() 80 57 43 32
??CIN????,????????????
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?????????
????,?HPV????????????????????????????5-10????,
??????????????,???????????????????
?????????????????,???21???????????23?HPV????????
???,????????5-8????70?,?10????3????????????HPV????
?,???????????????????,??,??3???,????
????????????25-30?,65?????????
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?????????????????
?????? (?) 1 2 3 35 510 gt10
???????? 1 2 2.5 3 4 9
??13?????????,?????????,????????
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????????????

• ??????????????
• ????,????????????(50-70)
• ????????????????,???????? ??LCT? TCT?
• 1)???????????????????,??HPV??????????????????????
• 2)?????,????????????,???HPV??????????????????
• ?????HPV DNA??(PCR??),???????? ????????PCR?
• ?????????,?????? (98)

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?????????(CCRF)?????????
?????????????(????) ????pap smearHPV?? ????TC
THPV??
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• HPV???????
• ????????
• ????????????

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??HPV???3-5???????? HPV????? ???HPV??????????????
?
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????????????
CIN?? ??????9095,???10
?????????????4-5? ???8???
??????4-6??,??HPV??????????
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????????????, ???????????
??????????? ?????????,?????21????
?????????????,???????30?????? ???????1????
????????????? ??????? ??HIV(?????)???
???????????? ??????????????
??????????????????????????
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????????????????,???????????? ????????????
????????3???????????????? ??24?????????,?????
????????
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????