Real-time Quantity PCR

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Real-time Quantity PCR
NJSunshine ?? 2005.11.17
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????PCR??
 ??PCR??PCR?????????????????????????PCR??????PCR??
???????????????????????PCR??,?????????????????????
???????????DNA????????????????????????????????????
??????????????????? ?
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????PCR???????
(1)????( threshold) ?PCR????15 ????????????????,?
????????3---15??????????????10 ??
(2)Ct???????,C??Cycle,t??threshold???PCR?????????
???????????????????????????????,??????????????????
??????????,??Ct???????
?????,Ct????DNA??????(dRn)??? ?
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????
??????PCR????????? (1)TaqMan?? (2)SYBR Green?
(3)LUX?? (4)Molecular beacon
(5)????????(FRET,Roche product)
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(No Transcript)
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1.TaqMan????????? ???????????Taq ??5'?3'?????
????PCR??????????????????TaqMan??5'?????????,3'???
???????????PCR?????????????????,3'???????5'???????
????PCR?????????????????DNA????????????????Taq????
?DNA????,??????,Taq???5'?3'???????,????????????,??
????????,?????????????????????????????????????,???
??????,?????????????????????????????PCR???????????
,?????????????????????,???????????????????????????
??,????????50150bp?
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• ????????????? 
• (1)GC?????40-60?
• (2) ?????????????,???G?
• (3)??5'??G, ??G??????????,???C????G?
• (4)????15--25?????,??????????
• (5)???Tm??????Tm??????10??
• ??,????????????, ?????????????????

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(2) SYBR Green I?????????
SYBR Green I???????DNA???????,???DNA?????,?????D
NA????????,???????????,??????,??????????DNA???????
??????????????DNA(??????)??,??????????????????????
?
?????????????qPCR?
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(No Transcript)
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(3)LUX??
LUX (light upon extention)
?????????????????????????????????????????,????????
???????????????3??????????????????????,???????,??
????,???????????????,???????,??????,?????????LUX??
,?????????,????????????????????Invitrogen????5????
?FAM,JOE,HEX,TET and Alexa Flour 546?
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(No Transcript)
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Lux??
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LUXTM ?????

• 1.??www.invitrogen.com/dlux-easy-to-use
  methode??????????LUXTM Primer?
• 2.??????,???LUXTM Primer-???????????(40)??????
• 3.???Invittrogen Custom LUXTM Primer??????,???????
  ???????????

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Invitrogen qPCR??
(1) superscript RT ??????cDNA??????????
(2) Platinum Taq DNA Polymerse?PCR????????????
??
(3) Supermixes ???Platinum Taq DNA
????UDG?dNTPs,ROX?????
(4) SYBR Green ? ?LUX??
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qPCR ???
1.??? (1)???ABI?? ? SuperScript ? Platinum
one-step qRT-PCR Kit w/ROX ?
SuperScript ? Platinum SYBR Green one-step
qRT-PCR Kit w/ROX (2)??????? ? Superscript ?
Platinum one-step qRT-PCR Kit ? SuperScript ?
Platinum SYBR Green one-step qRT-PCR Kit
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(3)RNA UltraSense One-step Quantitative RT- PCR
System ??????RNA????????QRT-PCR?? (4)Plati
num qRT-PCR Thermoscript One-step System
????????????RNA
2.??? (1)???ABI?? ? SuperScript ? Platinum
two-step qRT-PCR Kit w/ROX ? SuperScript ?
Platinum SYBR Green two-step qRT-PCR Kit
w/ROX
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(2) ??????? ? Superscript ? Platinum
one-step qRT-PCR Kit ? SuperScript ?
Platinum SYBR Green one-step qRT-PCR Kit (3)
Superscript ? Platinum CellsDirect ??? ?
Superscript ? Platinum CellsDirect Two-step
qRT-PCR Kit ? SuperScript ? Platinum
CellsDirect Two -step qRT-PCR Kit w/SYBR
Green
3. SuperScript ? 1st cDNA???? SuperScript
? First-Strand Synthesis superMix for qRT-PCR