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 I-Fluorescence quantitative PCR (ekwaziwa ngokuba yi-TaqMan PCR, emva koku ekubhekiselwa kuyo njenge-FQ-PCR) bubuchwephesha obutsha be-nucleic acid bobungakanani obuphuhliswe ngu-PE (Perkin Elmer) e-United States ngo-1995. Le teknoloji isekwe kwi-PCR eqhelekileyo ngokongeza iiprobe ezibhalwe ngefluorescent.Xa kuthelekiswa ne-PCR eguquguqukayo, i-FQ-PCR ineenzuzo ezininzi zokuqaphela umsebenzi wayo wobungakanani.Eli nqaku lijonge ukuchaza ngokufutshane iimpawu, imigaqo, iindlela kunye nokusetyenziswa kobuchwephesha.

1 Iimpawu

I-FQ-PCR ayinabo ubuntununtunu obuphezulu be-PCR eqhelekileyo, kodwa nangenxa yokusetyenziswa kweeprobes ze-fluorescent, inokubona ngokuthe ngqo utshintsho lwesiginali ye-fluorescent ngexesha lokwandiswa kwe-PCR ngenkqubo ye-photoelectric conduction ukuze ifumane iziphumo zobuninzi, ezoyisa iintsilelo ezininzi ze-PCR eqhelekileyo, ngoko ke ikwanayo ncakasana ephezulu yokuchaneka kwe-DNA hybridization kunye nobuchwephesha obuphezulu.

Umzekelo, iimveliso ze-PCR ngokubanzi kufuneka zijongwe nge-agarose gel electrophoresis kunye ne-ethidium bromide staining ngokukhanya kwe-ultraviolet okanye nge-polyacrylamide gel electrophoresis kunye ne-silver staining.Oku akufuni kuphela izixhobo ezininzi, kodwa kuthatha ixesha kunye nomzamo.Amabala asetyenzisiweyo e-Ethidium bromide ayingozi kumzimba womntu, kwaye ezi nkqubo zovavanyo ezinzima zibonelela ngamathuba ongcoliseko kunye neziphumo zobuxoki.Nangona kunjalo, i-FQ-PCR idinga kuphela ukuvula isiciko kanye ngexesha lokulayishwa kwesampuli, kwaye inkqubo elandelayo ivaliwe ngokupheleleyo-i-tube operation, engadingi i-PCR post-processing, ukuphepha ukungahambi kakuhle kwimisebenzi ye-PCR eqhelekileyo.Uvavanyo ngokubanzi lusebenzisa i-ABI7100 PCR yebhayisekile yobushushu ephuhliswe yinkampani ye-PE.

Esi sixhobo sinezi mpawu zilandelayo: ① Usetyenziso olubanzi: Ingasetyenziselwa i-DNA kunye ne-RNA PCR ubungakanani bemveliso, uphando lwe-gene expression, ubhaqo lwe-pathogen, kunye nokulungiswa kweemeko ze-PCR.② Umgaqo wobungakanani owahlukileyo: Ukusebenzisa iiprobe ezibhalwe ngefluorescent, isixa sefluorescence siya kuqokelelana kunye nomjikelo wePCR emva kochulumanco lwelaser, ukuze kuphunyezwe injongo yobungakanani.③ Ukusebenza okuphezulu kokusebenza: Yakhelwe kwi-9600 PCR i-thermal cycler, ikhompyutha ilawulwa i-1 ukuya kwiiyure ze-2 ukugqiba ukukhulisa kunye nobungakanani beesampuli ze-96 ngokuzenzekelayo kunye ne-synchronously.④ Akukho mfuneko ye-gel electrophoresis: Akukho mfuneko yokunciphisa kunye ne-electrophoresis isampuli, sebenzisa nje i-probe ekhethekileyo ukuze ufumane ngokuthe ngqo kwi-tube yokusabela.⑤Akukho lungcoliseko kumbhobho: Iityhubhu yokusabela evaleleke ngokupheleleyo kunye nenkqubo yokwenziwa kombane yamkelwe, ngoko akukho mfuneko yakuzikhathaza ngongcoliseko.⑥Iziphumo ziyaphinda-phinda: uluhlu oluguquguqukayo lomyinge ukuya kuthi ga kwiiodolo ezintlanu zobukhulu.Ngoko ke, ekubeni obu bugcisa buphuhliswe ngempumelelo, buxatyiswe ngabaphandi abaninzi bezenzululwazi kwaye busetyenziswe kwiinkalo ezininzi.

2 Imigaqo kunye neendlela

Umgaqo osebenzayo we-FQ-PCR kukusebenzisa i-5′→3′ exonuclease umsebenzi we-Taq enzyme ukongeza iprobe ebhalwe ngefluorescently kwi-reaction system ye-PCR.I-probe inokuxutywa ngokukodwa kunye ne-template ye-DNA equlethwe ngokulandelelana kwe-primer.I-5′end yeprobe ibhalwe nge-fluorescence emission gene FAM (6-carboxyfluorescein, incopho yokukhutshwa kwe-fluorescence kwi-518nm), kwaye i-3′end ibhalwe nge Iqela lokucima i-fluorescence TAMRA (6-carboxytetramethylrhodance) i-phosphorylated ukukhusela i-probe ukuba yandiswe ngexesha lokwandiswa kwe-PCR.Xa i-probe ihleli, iqela le-quencher licinezela ukukhutshwa kwe-fluorescence yeqela elikhuphayo.Emva kokuba iqela elikhuphayo lihlulwe kwiqela lokucima, ukuvinjelwa kuphakanyiswe, kwaye ukuxinana kwe-optical kwi-518nm kwanda kwaye kuchongwa yinkqubo yokufumanisa i-fluorescence.Kwisigaba sokuguqulwa ngokutsha, i-probe idibanisa kunye ne-template ye-DNA, kwaye i-enzyme ye-Taq kwisigaba sokwandisa ihamba kunye ne-extension ye-DNA template kunye ne-prime template.Xa i-probe inqunyulwa, umphumo wokucima ukhululiwe kwaye umqondiso we-fluorescent ukhululiwe.Ngalo lonke ixesha i-template ikopishwa, i-probe iyanqunyulwa, ihamba kunye nokukhululwa kwesignali ye-fluorescent.Ekubeni kukho ubudlelwane obunye phakathi kwenani le-fluorophores ekhutshwe kunye nenani leemveliso ze-PCR, obu buchule bunokusetyenziselwa ukulinganisa ngokuchanekileyo itemplate.Isixhobo sokulinga ngokuqhelekileyo sisebenzisa i-ABI7100 PCR yebhayisekile yobushushu ephuhliswe yinkampani ye-PE, kunye nezinye iibhayisekile ezishushu nazo zingasetyenziswa.Ukuba inkqubo yokusabela yohlobo lwe-ABI7700 isetyenziselwa uvavanyo, emva kokuba impendulo igqityiwe, iziphumo zobungakanani zinokunikwa ngokuthe ngqo ngohlalutyo lwekhompyuter.Ukuba usebenzisa ezinye iibhayisikili ze-thermal, kufuneka usebenzise i-fluorescence detector ukulinganisa isignali ye-fluorescence kwityhubhu yokusabela ngaxeshanye ukubala i-RQ+, RQ-, △RQ.I-RQ + imele umlinganiselo we-luminescence intensity yeqela le-fluorescent ephumayo yetyhubhu yesampuli ukuya kwi-luminescence intensity yeqela lokucima, i-RQ- imele umlinganiselo wesibini kwi-tube engenanto, △RQ (△RQ = RQ + -RQ-) imele isixa sedatha ye-fluorescence emva kokuguqulwa kwedatha ye-fluorescence.Ngenxa yokuqaliswa kweeprobes ze-fluorescent, ukucaciswa kovavanyo kuphuculwe kakhulu.Uyilo lweprobe kufuneka lufezekise le miqathango ilandelayo ngokubanzi: ①Ubude beprobe kufuneka bube malunga neziseko ezingama-20-40 zokuqinisekisa ukuba ngqo kokubophelela.②Umxholo weziseko zeGC uphakathi kwe-40% kunye ne-60% ukuphepha ukuphindaphinda ukulandelelana kwe-nucleotide enye.③ Kuphephe ukuxutywa okanye ukugqithelana ngeeprimers.④ Ukuzinza kokubopha phakathi kweprobe kunye netemplate kukhulu kunozinzo lokubopha phakathi kwe-primer kunye netemplate, ngoko ixabiso le-Tm le-probe kufuneka libe ubuncinane be-5 ° C ngaphezu kwexabiso le-Tm le-primer.Ukongeza, ukugxininiswa kweprobe, i-homology phakathi kweprobe kunye nolandelelwano lwetemplate, kunye nomgama phakathi kweprobe kunye ne-primer zonke zinefuthe kwiziphumo zovavanyo.

Iimveliso eziyeleleneyo:

China Lnc-RT Heroᵀᴹ I(Nge gDNase)(Super Premix for first-strand cDNA synthesis from lncRNA) Umenzi kunye noMboneleli |I-Foregene (foreivd.com)

China Real Time PCR Easyᵀᴹ-Taqman Manufacturer kunye noMboneleli |I-Foregene (foreivd.com)


Ixesha lokuposa: Oct-15-2021