Igihe nyacyo PCR, izwi kandi ku bwinshi bwa PCR cyangwa qPCR, ni uburyo bwo gukurikirana-igihe no gusesengura ibicuruzwa byongera PCR.
Kuberako PCR yuzuye ifite ibyiza byo gukora byoroshye, byihuse kandi byoroshye, ibyiyumvo byinshi, gusubiramo neza, hamwe nigipimo gito cyanduye, ikoreshwa cyane mugupimisha ubuvuzi, gusuzuma imikorere yibiyobyabwenge, ubushakashatsi bwerekana imiterere ya gene, ubushakashatsi bwa transgenji, kumenya gene, gutahura indwara, kumenya inyamaswa n’ibimera., gupima ibiryo nibindi bice.
Kubwibyo, waba ukora ubushakashatsi bwibanze mubumenyi bwubuzima, cyangwa abakozi ba societe yimiti, amasosiyete y’ubworozi, amasosiyete y’ibiribwa, ndetse n’abakozi bo mu biro byinjira-bisohoka n’ibiro by’akato, ishami rishinzwe gukurikirana ibidukikije, ibitaro n’ibindi bice, uzagerwaho cyane cyangwa bike Cyangwa ukeneye kumenya ubumenyi bwo kumenya PCR yuzuye.

Ihame ryigihe nyacyo PCR

Igihe nyacyo PCR nuburyo uburyo fluorescent yongewemo muri sisitemu ya reaction ya PCR, kandi ubukana bwibimenyetso bya fluorescence mugikorwa cya PCR bikurikiranwa mugihe nyacyo nigikoresho cya PCR, hanyuma amaherezo amakuru yubushakashatsi arasesengurwa kandi agatunganywa.

【Amplification curve】ni umurongo usobanura inzira ya PCR.Amplification curve ya PCR ntabwo mubyukuri isanzwe igaragara, ahubwo ni sigmoid curve.

[Icyiciro cya platform ya amplification curve]Hamwe no kwiyongera kwumuzenguruko wa PCR, kudakora polymerase ya ADN, kugabanuka kwa dNTPs na primers, hamwe no kubuza synthesis reaction na reaction yibicuruzwa byitwa pyrophosphate, nibindi, PCR ntabwo buri gihe yaguka kuburyo bugaragara., kandi amaherezo azinjira mu kibaya.

[Gukura Kwiyongera Akarere ka Amplification Curve]Nubwo icyiciro cya plateau gitandukana cyane, mukarere runaka kagace kerekana iterambere ryikura ryikwirakwizwa rya amplification curve, gusubiramo nibyiza cyane, nibyingenzi cyane kubisesengura ryinshi rya PCR.

[Agaciro ntarengwa na Ct agaciro]Twashyizeho imipaka ntarengwa yo gutahura fluorescence kumwanya ukwiye mukarere gakura kerekana imikurire ya amplification curve, aribyo agaciro kinjira (Threshold).Ihuriro ryagaciro ntarengwa hamwe na amplification curve ni Ct agaciro, ni ukuvuga, agaciro ka Ct bivuga umubare wizunguruka (Threshold Cycle) mugihe agaciro kateganijwe kageze.

Igishushanyo gikurikira kirerekana neza isano iri hagati yumurongo wambukiranya umurongo no kwongera umurongo, kurenga na Ct agaciro.

【Nigute dushobora kubara?】

Byagaragajwe nigitekerezo cyimibare ko Ct agaciro gafite umurongo uhuza umurongo na logarithm yumubare wambere wicyitegererezo.Igihe nyacyo PCR ikurikirana ibicuruzwa byongera PCR mugihe nyacyo kandi ikabigereranya mugihe cyicyerekezo cya amplification.

Kuri buri cyiciro cya PCR, ADN yiyongereye cyane inshuro 2, kandi bidatinze igera mu kibaya.

Dufashe ko ingano yo gutangira ADN ari A.₀ , nyuma ya n cycle, ingano yibitekerezo byibicuruzwa bya ADN bishobora kugaragazwa nka:

A _n =A ₀ × 2ⁿ

Noneho, uko umubare wambere wa ADN ya A 0 ari, niko byihuse ibicuruzwa byongerewe imbaraga bigera ku gaciro kerekana An, kandi umubare wizunguruka iyo ugeze kuri An ni Ct agaciro.Nukuvuga ko, uko ADN ibanza umubare A 0 ari, mbere ya amplification curve ya mpinga, kandi bihuye numubare ukenewe wa cycle n ni muto.

Turakora gahoro gahoro kugipimo cyibisanzwe bizwi kandi tukabikoresha nkicyitegererezo cyigihe nyacyo PCR, kandi urukurikirane rwimigozi ya amplification izaboneka mugihe kingana murwego rwo gutangira umubare wa ADN kuva kuri byinshi kugeza kuri bike.Ukurikije umurongo ugereranije hagati ya Ct agaciro na logarithm yumubare wo gutangira inyandikorugero, a[umurongo usanzwe] urashobora gushirwaho.

Mugusimbuza Ct agaciro ka sample hamwe nubushakashatsi butazwi muburyo busanzwe, umurongo wambere wicyitegererezo hamwe nicyerekezo kitazwi urashobora kuboneka, niryo hame ryumubare wigihe nyacyo PCR.

Uburyo bwo kumenya igihe nyacyo PCR

Igihe nyacyo PCR itahura ibicuruzwa byongera PCR mukumenya ubukana bwa fluorescence muri sisitemu yo kwitwara.

Ihame ryuburyo bwa Fluorescent】

Amabara ya Fluorescent, nka TB Green ®, irashobora guhuza bidasubirwaho ADN ikubye kabiri muri sisitemu ya PCR na fluoresce iyo ihambiriye.

Imbaraga za fluorescence muri sisitemu ya reaction yiyongereye cyane hamwe no kwiyongera kwa PCR.Mugushakisha ubukana bwa fluorescence, ingano ya ADN yongerewe imbaraga muri sisitemu ya reaction irashobora gukurikiranwa mugihe nyacyo, hanyuma umubare wicyitegererezo cyo gutangira murugero urashobora kugereranywa.

【Ihame ryuburyo bwa fluorescent probe】

fluorescent probeni aside nucleic ikurikirana hamwe nitsinda rya fluorescent kuri 5 ′ iherezo hamwe nitsinda ryo kuzimya kuri 3 ′, rishobora guhuza cyane nicyitegererezo.Iyo iperereza ridahwitse, fluorescence itangwa na fluorophore izimwa nitsinda rizimya kandi ntishobora fluoresce.Iyo iperereza ryangirika, ibintu bya fluorescent bizatandukana kandi bisohore fluorescence.

Iperereza rya fluorescent ryongewe kumuti wa PCR.Mugihe cya annealing, probe ya fluorescent izahuza kumwanya wihariye wicyitegererezo.Mugihe cyo kwagura, ibikorwa 5 ′ → 3 ′ exonuclease yibikorwa bya enzyme ya PCR irashobora kubora probe ya fluorescent ivanze nicyitegererezo, kandi ibintu bya fluorescent bigabanywa kugirango bisohore fluorescence.Mugutahura ubukana bwa fluorescence ya probe muri sisitemu yo kubyitwaramo, intego yo kugenzura umubare w’ibicuruzwa bya PCR irashobora kugerwaho.

【Guhitamo Uburyo bwa Fluorescence】

Niba ikoreshwa mugutandukanya urutonde hamwe na homologiya yo hejuru no gukora multiplex PCR gutahura nka SNP yandika, uburyo bwa fluorescent probe ntabwo busimburwa.
Kubindi bigeragezo nyabyo PCR, uburyo bworoshye, bworoshye kandi buhendutse bwa fluorescent chimera uburyo burashobora gukoreshwa.

ipererezaIbisobanuro byihariye, birashobora kugwiza PCR

Ikibazo

Ibisabwa byihariye byihariye kugirango byongerwe imbaraga;

multiplex PCR ntishobora gukorwaBikenewe gushushanya ibintu byihariye, igiciro kinini;

rimwe na rimwe igishushanyo mbonera kiragoye

Ibicuruzwa bifitanye isano: