Fluorescence quantitative PCR (tseem hu ua TaqMan PCR, hereinafter hu ua FQ-PCR) yog ib tug tshiab nucleic acid quantitative technology tsim los ntawm PE (Perkin Elmer) nyob rau hauv lub tebchaws United States nyob rau hauv 1995. Lub tshuab no yog raws li cov pa PCR los ntawm kev ntxiv fluorescent lo lo lo ntxhuav probes.Piv nrog rau PCR hloov tau yooj yim, FQ-PCR muaj ntau yam zoo kom paub txog nws qhov ntau npaum li cas.Kab lus no npaj siab piav qhia luv luv txog cov yam ntxwv, cov hauv paus ntsiab lus, txoj hauv kev, thiab kev siv tshuab.
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FQ-PCR tsis tsuas yog muaj lub siab rhiab heev ntawm PCR zoo tib yam, tab sis kuj vim yog daim ntawv thov ntawm fluorescent probes, nws tuaj yeem ncaj qha txheeb xyuas qhov kev hloov ntawm cov teeb liab fluorescent thaum lub sijhawm PCR amplification los ntawm photoelectric conduction system kom tau txais cov txiaj ntsig ntau, uas kov yeej ntau qhov tsis txaus ntawm cov qauv PCR, yog li nws kuj muaj qhov tshwj xeeb ntawm DNA high hybridization technology.
Piv txwv li, cov khoom PCR dav dav yuav tsum tau soj ntsuam los ntawm agarose gel electrophoresis thiab ethidium bromide staining nrog ultraviolet lub teeb lossis los ntawm polyacrylamide gel electrophoresis thiab nyiaj staining.Qhov no tsis tsuas yog xav tau ntau yam twj paj nruag, tab sis kuj siv sijhawm thiab siv zog.Cov stains uas siv Ethidium bromide yog teeb meem rau tib neeg lub cev, thiab cov txheej txheem kev sim nyuaj no muab sijhawm rau kev ua qias tuaj thiab qhov tsis zoo.Txawm li cas los xij, FQ-PCR tsuas yog xav tau qhib lub hau ib zaug thaum coj mus kuaj, thiab cov txheej txheem tom ntej yog kaw tag nrho cov raj ua haujlwm, uas tsis tas yuav tsum tau PCR tom qab ua tiav, zam ntau qhov tsis zoo hauv kev ua haujlwm PCR.Qhov kev sim feem ntau siv ABI7100 PCR thermal cycler tsim los ntawm PE tuam txhab.
Cov cuab yeej muaj cov yam ntxwv hauv qab no: ① Daim ntawv thov dav: Nws tuaj yeem siv rau DNA thiab RNA PCR cov khoom lag luam kom muaj nuj nqis, kev tshawb fawb noob caj noob ces, kuaj kab mob, thiab kev ua kom zoo ntawm PCR cov xwm txheej.② Lub hauv paus ntsiab lus tshwj xeeb: Siv cov tshuaj fluorescently sau npe sojntsuam, tus nqi ntawm cov fluorescence yuav sib sau nrog PCR lub voj voog tom qab laser excitation, thiaj li ua tiav lub hom phiaj ntawm quantification.③ Kev ua haujlwm siab: Ua-nyob rau hauv 9600 PCR thermal cycler, lub computer tswj 1 mus rau 2 teev kom tiav cov amplification thiab quantification ntawm 96 cov qauv txiav thiab synchronously.④ Tsis xav tau gel electrophoresis: Tsis tas yuav dilute thiab electrophoresis tus qauv, tsuas yog siv ib qho kev sojntsuam tshwj xeeb kom pom ncaj qha rau hauv cov tshuaj tiv thaiv raj.⑤ Tsis muaj kuab paug nyob rau hauv lub raj xa dej: Qhov tshwj xeeb ntim cov tshuaj tiv thaiv lub raj thiab photoelectric conduction system tau txais yuav, yog li tsis tas yuav txhawj xeeb txog kev ua qias tuaj.⑥ Cov txiaj ntsig tau rov ua dua: qhov ntau npaum li qhov muaj zog ntau txog li tsib qhov kev txiav txim siab.Yog li ntawd, txij li thaum lub tshuab no tau ua tiav tiav, nws tau muaj nuj nqis los ntawm ntau tus kws tshawb fawb thiab tau siv rau ntau qhov chaw.
2 Cov ntsiab lus thiab cov txheej txheem
Lub hauv paus ntsiab lus ua haujlwm ntawm FQ-PCR yog siv 5′ → 3′ exonuclease kev ua ntawm Taq enzyme ntxiv cov tshuaj tiv thaiv fluorescently sau rau PCR cov tshuaj tiv thaiv.Kev sojntsuam tuaj yeem tshwj xeeb hybridize nrog DNA qauv uas muaj nyob rau hauv cov txheej txheem primer.Lub 5'kawg ntawm qhov kev sojntsuam yog sau nrog cov fluorescence emission gene FAM (6-carboxyfluorescein, fluorescence emission ncov ntawm 518nm), thiab 3'end yog sau nrog Cov fluorescence quenching pawg TAMRA (6-carboxytetramethylrhodamine, fluorescence ntawm 518', fluorescence attenuation. phosphorylated los tiv thaiv kev sojntsuam los ntawm kev txuas ntxiv thaum lub sijhawm PCR amplification.Thaum qhov kev sojntsuam tseem nyob ruaj khov, pawg quencher suppresses fluorescence emission ntawm pawg emitting.Thaum cov emitting pawg tau sib cais los ntawm pawg quenching, qhov inhibition yog lifted, thiab optical ceev ntawm 518nm nce thiab raug kuaj pom los ntawm fluorescence detection system.Nyob rau hauv lub renaturation theem, cov sojntsuam hybridizes nrog tus qauv DNA, thiab cov Taq enzyme nyob rau hauv lub extension theem mus rau lub extension ntawm DNA template.Thaum qhov kev sojntsuam raug txiav tawm, cov nyhuv quenching raug tso tawm thiab cov teeb liab fluorescent raug tso tawm.Txhua zaus cov qauv luam tawm, ib qho kev sojntsuam raug txiav tawm, nrog rau kev tso tawm cov teeb liab fluorescent.Txij li thaum muaj ib qho kev sib raug zoo ntawm tus naj npawb ntawm cov fluorophores tso tawm thiab tus naj npawb ntawm cov khoom PCR, cov txheej txheem no tuaj yeem siv los txheeb xyuas cov qauv kom raug.Kev sim ntsuas feem ntau siv ABI7100 PCR thermal cycler tsim los ntawm PE lub tuam txhab, thiab lwm yam thermal cyclers kuj siv tau.Yog tias ABI7700 cov tshuaj tiv thaiv hom tshuaj tiv thaiv yog siv rau qhov kev sim, tom qab cov tshuaj tiv thaiv tiav, cov txiaj ntsig ntau tuaj yeem muab ncaj qha los ntawm kev tshuaj xyuas hauv computer.Yog tias koj siv lwm cov thermal cyclers, koj yuav tsum siv lub ntsuas fluorescence los ntsuas cov teeb liab fluorescence hauv cov raj tshuaj tiv thaiv tib lub sijhawm los xam RQ +, RQ-, △RQ.RQ + sawv cev rau qhov piv ntawm luminescence siv ntawm fluorescent emission pawg ntawm cov qauv raj mus rau lub luminescence siv ntawm pawg quenching, RQ- sawv cev rau qhov piv ntawm ob lub raj dawb paug, △RQ (△RQ = RQ + - RQ-) sawv cev rau tus nqi ntawm cov ntaub ntawv tau txais kev hloov pauv thaum lub sij hawm fluorescent PC.Vim qhov kev taw qhia ntawm fluorescent probes, qhov tshwj xeeb ntawm qhov kev sim tau zoo dua.Kev sojntsuam tsim yuav tsum feem ntau ua tau raws li cov xwm txheej hauv qab no: ① Qhov ntev ntawm qhov kev sojntsuam yuav tsum yog li 20-40 lub hauv paus kom ntseeg tau qhov tshwj xeeb ntawm kev khi.②Cov ntsiab lus ntawm GC hauv paus yog nyob nruab nrab ntawm 40% thiab 60% kom tsis txhob muaj kev sib tw ntawm ib qho nucleotide sequences.③ Tsis txhob hybridization lossis sib tshooj nrog primers.④ Kev ruaj ntseg ntawm kev sib txuas ntawm qhov kev sojntsuam thiab cov qauv yog ntau dua li qhov ruaj khov ntawm kev sib khi ntawm cov primer thiab cov qauv, yog li tus nqi Tm ntawm qhov kev sojntsuam yuav tsum yog tsawg kawg 5 ° C siab dua Tm tus nqi ntawm cov primer.Tsis tas li ntawd, qhov concentration ntawm kev sojntsuam, homology ntawm qhov kev sojntsuam thiab cov qauv sib lawv liag, thiab qhov kev ncua deb ntawm qhov kev sojntsuam thiab cov primer muaj feem cuam tshuam rau qhov kev sim.
Yam khoom:
Tuam Tshoj Real Time PCR Easyᵀᴹ-Taqman Chaw tsim tshuaj paus thiab tsum |Foregene (foreivd.com)
Post lub sij hawm: Oct-15-2021
