Fluorescence yuzuye PCR (izwi kandi nka TaqMan PCR, nyuma yiswe FQ-PCR) ni tekinoroji nshya ya acide nucleic aside igizwe na PE (Perkin Elmer) muri Amerika muri 1995. Iri koranabuhanga rishingiye kuri PCR isanzwe hiyongeraho fluorescent yanditseho probe.Ugereranije na PCR ihindagurika, FQ-PCR ifite ibyiza byinshi byo kumenya imikorere yabyo.Iyi ngingo igamije gusobanura muri make ibiranga, amahame, uburyo, nuburyo bukoreshwa bwikoranabuhanga.
1 Ibiranga
FQ-PCR ntabwo ifite gusa sensibilité yo hejuru ya PCR isanzwe, ariko nanone kubera ikoreshwa rya probe ya fluorescent, irashobora kumenya neza ihinduka ryikimenyetso cya fluorescent mugihe cyo kongera ingufu za PCR binyuze muri sisitemu yo gutwara amafoto kugirango kibone ibisubizo byinshi, bikanesha amakosa menshi ya PCR isanzwe, bityo ikaba ifite umwihariko wo kuvanga ADN hamwe nubuhanga buhanitse bwa tekinoroji ya spekitroscopi.
Kurugero, ibicuruzwa rusange bya PCR bigomba kubahirizwa na agarose gel electrophorei na etidium bromide yandujwe numucyo ultraviolet cyangwa na polyacrylamide gel electrophoreis hamwe na silver.Ibi ntibisaba ibikoresho byinshi gusa, ahubwo bisaba igihe n'imbaraga.Ikirangantego cyakoreshejwe na Ethidium bromide cyangiza umubiri wumuntu, kandi ubwo buryo bugoye bwo kugerageza butanga amahirwe yo guhumana nibyiza bibi.Ariko, FQ-PCR ikeneye gukingura umupfundikizo rimwe gusa mugihe cyo gupakira icyitegererezo, kandi inzira ikurikiraho ni gufunga-tube rwose, bidasaba PCR nyuma yo gutunganywa, birinda ibibi byinshi mubikorwa bisanzwe bya PCR.Ubushakashatsi busanzwe bukoresha ABI7100 PCR yumuriro wamashanyarazi wakozwe na sosiyete ya PE.
Igikoresho gifite ibintu bikurikira: application Gukoresha byinshi: Irashobora gukoreshwa mugupima ibicuruzwa bya ADN na RNA PCR, ubushakashatsi bwerekana imiterere ya gene, gutahura indwara ya virusi, no kunoza imiterere ya PCR.Principle Ihame ridasanzwe ryo kubara: Ukoresheje fluoresc yanditseho probe, ingano ya fluorescence izegeranya na cycle ya PCR nyuma yo gushimishwa na laser, kugirango ugere ku ntego yo kubara.Effective Gukora neza cyane: Yubatswe muri 9600 PCR yumuriro wumuriro, mudasobwa yagenzuye amasaha 1 kugeza kuri 2 kugirango irangize amplification no kugereranya ingero 96 byikora kandi icyarimwe.④ Ntibikenewe kuri gel electrophorei: Ntibikenewe ko uhinduranya na electrophoreis icyitegererezo, koresha iperereza ryihariye kugirango umenye neza muri reaction.ONta mwanda uhari: Umuyoboro udasanzwe wuzuye wuzuye hamwe na sisitemu yo gutwara amashanyarazi, bityo rero nta mpamvu yo guhangayikishwa n’umwanda.ResultsIbisubizo byororoka: ingano yingirakamaro yingirakamaro igera kuri bitanu yubunini.Kubwibyo, kuva iryo koranabuhanga ryatezwa imbere neza, ryahawe agaciro nabashakashatsi benshi ba siyanse kandi ryakoreshejwe mubice byinshi.
2 Amahame nuburyo
Ihame ryakazi rya FQ-PCR nugukoresha ibikorwa 5 ′ → 3 ′ exonuc Please ibikorwa bya Taq enzyme kugirango wongereho fluoresc yanditseho probe kuri sisitemu ya PCR.Iperereza rirashobora kuvanga byumwihariko hamwe na ADN yerekana icyitegererezo gikurikiranye.5′end ya anketi yanditseho gene yoherejwe na fluorescence gene FAM (6-carboxyfluorescein, impanuka ya florescence yoherezwa kuri 518nm), naho 3′end yanditseho itsinda rya TAMRA (6-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, impanuka ya florescence iva kuri probe ya progaramu ya 3).Iyo iperereza rigumye kuba ntamakemwa, itsinda ryo kuzimya rihagarika imyuka ya fluorescence yitsinda risohora.Itsinda risohora rimaze gutandukana nitsinda rizimya, kubuzwa gukurwaho, kandi ubwinshi bwa optique kuri 518nm bwiyongera kandi bikamenyekana na sisitemu yo gutahura fluorescence.Mu cyiciro cyo guhindura ibintu, iperereza rivanga na ADN yerekana icyitegererezo, kandi enzyme ya Taq mugice cyo kwaguka igenda ikurikiza icyitegererezo cya ADN hamwe no kwagura primer.Iyo iperereza ryaciwe, ingaruka zo kuzimya zirekurwa kandi ibimenyetso bya fluorescent birekurwa.Igihe cyose inyandikorugero yimuwe, iperereza riracibwa, riherekejwe no gusohora ibimenyetso bya fluorescent.Kubera ko hari umubano umwe-umwe hagati yumubare wa fluorofore yasohotse numubare wibicuruzwa bya PCR, ubu buhanga burashobora gukoreshwa mukugereranya neza inyandikorugero.Igikoresho cyubushakashatsi gikoresha muri rusange ABI7100 PCR yumukino wamashanyarazi wateguwe nisosiyete ya PE, nabandi basiganwa ku magare nabo barashobora gukoreshwa.Niba uburyo bwa reaction ya ABI7700 bwakoreshejwe mubigeragezo, nyuma yo gukora ibisubizo birangiye, ibisubizo byuzuye birashobora gutangwa muburyo bwisesengura rya mudasobwa.Niba ukoresha andi magare yubushyuhe, ugomba gukoresha disiketi ya fluorescence kugirango upime ibimenyetso bya fluorescence mumiyoboro ya reaction icyarimwe kugirango ubare RQ +, RQ-, △ RQ.RQ + yerekana ikigereranyo cyimbaraga za luminescence zitsinda rya florescent yangiza ya groupe yicyitegererezo hamwe nuburemere bwa luminescence bwitsinda ryizimya, RQ- igereranya ikigereranyo cyibiri mumiyoboro yubusa, △ RQ (△ RQ = RQ + -RQ-) yerekana umubare wibimenyetso bya fluorescence mugihe cya PCR Nyuma yo gutunganya amakuru, ibisubizo byinshi bishobora kuboneka.Bitewe no kwinjiza fluorescent probe, umwihariko wubushakashatsi wateye imbere cyane.Igishushanyo mbonera kigomba kuba cyujuje ibi bikurikira: lengthUburebure bwa probe bugomba kuba hafi 20-40 kugirango hamenyekane umwihariko wo guhuza.ContentIbirimo shingiro rya GC biri hagati ya 40% na 60% kugirango wirinde kwigana nucleotide imwe.Irinde kuvanga cyangwa guhuzagurika na primers.Stability Guhagarara kwihuza hagati yubushakashatsi nicyitegererezo birarenze ihame ryimikorere ihuza primer nicyitegererezo, bityo Tm agaciro ka probe igomba kuba byibuze 5 ° C hejuru ya Tm agaciro ka primer.Mubyongeyeho, kwibanda kuri probe, homology hagati yubushakashatsi hamwe nicyitegererezo gikurikiranye, hamwe nintera iri hagati ya probe na primer byose bigira ingaruka kubisubizo byubushakashatsi.
Ibicuruzwa bifitanye isano:
Ubushinwa Igihe Cyukuri PCR Byoroshyeᵀᴹ-Taqman Ihingura nuwitanga |Foregene (foreivd.com)
Igihe cyo kohereza: Ukwakira-15-2021
