• PCR nuburyo bukoreshwa mu kwagura ADN kuva ku gipimo gito cya ADN.RT-PCR ikoresha transcript yinyuma kugirango ikore ADN yerekana isoko ya RNA ishobora noneho kongerwa.
• PCR na RT-PCR mubisanzwe reaction yanyuma, mugihe qPCR na RT-qPCR bakoresha kinetics yikigereranyo cyibicuruzwa mugihe cya PCR kugirango babare umubare wicyitegererezo gihari.
• Uburyo bushya, nka PCR ya digitale, butanga umubare wuzuye wintangarugero ya ADN yambere, mugihe uburyo nka isothermal PCR bugabanya ibikenerwa nibikoresho bihenze kugirango bitange ibisubizo byizewe.

Urunigi rwa polymerase (PCR) nuburyo bworoshye kandi bukoreshwa cyane muburyo bwa biologiya tekinike yo kwagura no kumenya ADN na RNA bikurikirana.Ugereranije nuburyo gakondo bwo gukoroniza ADN no gukuza, bishobora gufata iminsi, PCR isaba amasaha make.PCR irakomeye cyane kandi isaba inyandikorugero ntoya yo gutahura no kongera urutonde rwihariye.Uburyo bwibanze PCR bwateye imbere kuva ADN yoroshye no kumenya RNA.Hasi, twatanze incamake yuburyo butandukanye bwa PCR hamwe na reagent dutanga muri Enzo Life Science kubyo ukeneye ubushakashatsi.Dufite intego yo gufasha abahanga kubona byihuse reagent ya PCR kugirango bakoreshe mumushinga wabo wubushakashatsi!

PCR

Kuri PCR isanzwe, icyo ukeneye ni polymerase ya ADN, magnesium, nucleotide, primers, inyandikorugero ya ADN kugirango yongerwe imbaraga, hamwe na thermocycler.Uburyo bwa PCR buroroshye nkintego yabyo: 1) ADN ifite imirongo ibiri (dsDNA) ni ubushyuhe bwerekanwe, 2) primers ihuza umurongo umwe wa ADN, na 3) primers yaguwe na polymerase ya ADN, bikavamo kopi ebyiri za umwimerere wa ADN.Gutandukana, guhuza, no kuramba hejuru yuruhererekane rwubushyuhe nibihe bizwi nkumuzingi umwe wo kongera imbaraga (Ishusho 1).

Buri ntambwe yumuzingi igomba gutezimbere icyitegererezo na primer set yakoreshejwe.Uru ruzinduko rusubirwamo inshuro zigera kuri 20-40, kandi ibicuruzwa byongerewe imbaraga birashobora gusesengurwa, mubisanzwe na agarose gel (Ishusho 2).

Nka PCR nuburyo bworoshye cyane kandi ingano ntoya irakenewe kugirango umuntu agire icyo akora, harateganijwe gutegura igishushanyo mbonera cya reaction nyinshi.Igishushanyo mbonera kigomba kuvangwa neza hanyuma kigabanywa numubare wibisubizo, ukemeza ko buri reaction izaba irimo enzyme ingana, dNTPs, na primers.Abatanga isoko benshi, nka Enzo Life Science, nabo batanga PCR ivanze isanzwe irimo ibintu byose usibye primers hamwe nicyitegererezo cya ADN.

Uturere dukungahaye muri Guanine / Cytosine (GC ikungahaye) byerekana ingorane muburyo bwa PCR busanzwe.Urutonde rwa GC rukungahaye cyane kuruta urutonde hamwe nibiri munsi ya GC.Byongeye kandi, GC ikungahaye cyane ikunda gukora urwego rwa kabiri, nkumusatsi wogosha.Nkigisubizo, GC ikungahaye kumurongo ibiri iragoye gutandukana rwose mugihe cyo gutandukana.Kubwibyo, ADN polymerase ntishobora guhuza umurongo mushya nta nkomyi.Ubushyuhe bwo hejuru burashobora kunoza ibi, kandi guhinduka kugana ubushyuhe bwo hejuru bwa annealing hamwe nigihe gito cya annealing birashobora gukumira guhuza bidasanzwe kwa GC ikungahaye kuri primers.Reagent yinyongera irashobora kongera amplification ya GC ikungahaye.DMSO, glycerol, na betaine bifasha guhagarika ibyiciro bya kabiri biterwa n'imikoranire ya GC bityo bikorohereza gutandukanya imirongo ibiri.

Gutangira bishyushye PCR

Amplification idasobanutse nikibazo gishobora kubaho mugihe cya PCR.Polymerase nyinshi za ADN zikoreshwa muri PCR zikora neza mubushyuhe bugera kuri 68 ° C kugeza 72 ° C.Enzyme irashobora, ariko, gukora cyane mubushyuhe bwo hasi, nubwo kurwego rwo hasi.Ku bushyuhe buri munsi yubushyuhe bwa annealing, primers irashobora guhambira bidasanzwe kandi biganisha kuri amplification idasanzwe, kabone niyo reaction yaba yashyizwe kurubura.Ibi birashobora gukumirwa ukoresheje inhibitori ya polymerase itandukana na ADN polymerase gusa iyo ubushyuhe runaka bumaze kugerwaho, niyo mpamvu ijambo rishyushye gutangira PCR.Inhibitor irashobora kuba antibody ihuza polymerase na denaturasi yubushyuhe bwambere (95 ° C mubisanzwe).

Polymerase Yinshi

Mugihe ADN polymerase yongerewe neza neza muburyo bwambere bwikitegererezo, amakosa yo guhuza nucleotide arashobora kubaho.Kudahuza mubisabwa nka cloni birashobora kuvamo inyandiko-mvugo zaciwe, hamwe na poroteyine zidahinduwe neza cyangwa zidakora neza.Kugira ngo wirinde uku kudahuza, polymerase hamwe nigikorwa cya "kugenzura" cyamenyekanye kandi cyinjizwa mubikorwa.Polymerase ya mbere yerekana neza, Pfu, yamenyekanye mu 1991 muri Pyrococcus furiosus.Iyi enzyme ya Pfu ifite ibikorwa bya 3 'kugeza 5' exonuclease.Nkuko ADN yongerewe imbaraga, exonuclease ikuraho nucleotide idahuye kuri 3 'impera yumurongo.Nucleotide ikwiye noneho irasimburwa, kandi synthesis ya ADN irakomeza.Kumenyekanisha urutonde rwa nucleotide rutari rwo rushingiye ku guhuza isano ya nucleoside triphosphate ikwiye hamwe na enzyme, aho guhuza kudakora bidindiza synthesis kandi bigatuma habaho gusimburwa neza.Igikorwa cyo kugenzura ibikorwa bya Pfu polymerase bivamo amakosa make muburyo bwa nyuma ugereranije na Taq ADN polymerase.Mu myaka yashize, hamenyekanye izindi misemburo isuzuma, kandi hahinduwe enzyme yumwimerere ya Pfu kugirango irusheho kugabanya igipimo cyamakosa mugihe cyo kongera ADN.

RT-PCR

Guhindura inyandiko mvugo PCR, cyangwa RT-PCR, yemerera gukoresha RNA nkicyitegererezo.Intambwe yinyongera yemerera kumenya no kongera RNA.RNA ihindurwa yandukurwa muri ADN yuzuzanya (cDNA), ikoresheje transcriptque.Ubwiza nubuziranenge bwicyitegererezo cya RNA nibyingenzi kugirango intsinzi ya RT-PCR.Intambwe yambere ya RT-PCR ni synthesis ya ADN / RNA ivanze.Guhindura inyandiko-mvugo nayo ifite imikorere ya RNase H, itesha agaciro igice cya RNA cya Hybrid.Molekile ya ADN imwe imwe noneho irangizwa nigikorwa cya ADN gishingiye kuri ADN polymerase yibikorwa bya reaction transcriptase muri cDNA.Imikorere yimyitwarire yambere irashobora kugira ingaruka kumikorere.Kuva hano, uburyo busanzwe bwa PCR bukoreshwa mukwongera cDNA.Ibishoboka byo guhindura RNA muri cDNA na RT-PCR bifite ibyiza byinshi, kandi bikoreshwa cyane cyane mubisesengura rya gene.RNA ni umurongo umwe kandi udahungabana cyane, bigatuma bigorana gukorana nayo.Mubisanzwe ikora nkintambwe yambere muri qPCR, igereranya inyandiko za RNA murugero rwibinyabuzima.

qPCR na RT-qPCR

Umubare PCR (qPCR) ukoreshwa mugushakisha, kuranga no kugereranya acide nucleic ya progaramu nyinshi.Muri RT-qPCR, inyandiko-mvugo ya RNA ikunze kugereranywa no kuyisubiza muri cDNA mbere, nkuko byasobanuwe haruguru, hanyuma qPCR igakorwa nyuma.Nko muri PCR isanzwe, ADN yongerewe intambwe eshatu zisubiramo: gutandukana, guhuza, no kuramba.Ariko, muri qPCR, label ya fluorescent ituma ikusanyamakuru nkuko PCR itera imbere.Ubu buhanga bufite inyungu nyinshi bitewe nurwego rwuburyo na chimisties bihari.

Mu irangi rishingiye kuri qPCR (mubisanzwe icyatsi), label ya fluorescent ituma umubare wa molekile ya ADN yongerewe imbaraga ukoresheje ikoreshwa rya dsDNA ihuza irangi.Muri buri cyiciro, hapimwa fluorescence.Ikimenyetso cya fluorescence cyiyongera ugereranije nubunini bwa ADN yigana.Niyo mpamvu, ADN ibarwa mu “gihe nyacyo” (Ishusho 3).Ingaruka zo gusiga irangi qPCR ni uko intego imwe yonyine ishobora gusuzumwa icyarimwe kandi ko irangi rizahuza na ds-ADN iyo ari yo yose iri muri sample.

Muri probe ishingiye kuri qPCR, intego nyinshi zishobora kumenyekana icyarimwe muri buri cyitegererezo, ariko ibi bisaba gutezimbere no gushushanya intego yihariye ya probe (s) ikoreshwa hiyongereyeho primers.Ubwoko butandukanye bwubushakashatsi burahari, ariko ubwoko bukunze kugaragara ni hydrolysis probe, ikubiyemo fluorophore na quencher.Fluorescence resonance ihererekanyabubasha (FRET) irinda gusohora fluorophore ikoresheje kuzimya mugihe iperereza ridahwitse.Ariko, mugihe PCR yabyitwayemo, iperereza iba hydrolyz mugihe cyo kwagura primer no kwongerera urutonde rwihariye.Gutandukanya iperereza bitandukanya fluorophore nu kuzimya kandi bigatuma kwiyongera kwa florescence byiyongera (Ishusho 4).Rero, ibimenyetso bya fluorescence biva mubushakashatsi bushingiye kuri qPCR buringaniye nubunini bwikurikiranabikorwa ryikurikiranabikorwa biboneka muri sample.Kuberako iperereza rishingiye kuri qPCR risobanutse neza kuruta irangi rishingiye ku irangi rya qPCR, akenshi ni ikoranabuhanga rikoreshwa muri qPCR rishingiye ku gusuzuma.

Kwiyongera kwa Isothermal

PCR yavuzwe haruguru isaba ibikoresho bihenze bya termocycling kugirango izamure neza kandi igabanuke ubushyuhe bwicyumba kugirango habeho gutandukana, guhuza, no kwagura intambwe.Tekinike zitari nke zateguwe zidakeneye ibikoresho nkibyo kandi zishobora gukorerwa mu bwogero bworoshye bwamazi cyangwa no muri selile zinyungu.Ubu buhanga bwiswe hamwe kwitwa isothermal amplification hamwe nakazi gashingiye kumyerekano, umurongo, cyangwa casade.

Ubwoko buzwi cyane bwa isothermal amplification ni loop-medrated isothermal amplification, cyangwa LAMP.LAMP ikoresha amplification yerekanwe kuri 65⁰C kugirango yongere inyandikorugero ya ADN cyangwa RNA.Iyo ukora LAMP, primers enye kugeza kuri esheshatu zuzuzanya mukarere ka ADN igenewe gukoreshwa hamwe na polymerase ya ADN muguhuza ADN nshya.Babiri muri aba primers bafite urutonde rwishimwe rumenya urutonde rwizindi primers kandi rukabahuza, bigatuma imiterere ya "loop" ibaho muri ADN nshya yashizwemo hanyuma igafasha primer annealing mubyiciro byakurikiyeho bya amplification.LAMP irashobora kugaragara muburyo bwinshi, harimo fluorescence, agarose gel electrophorei, cyangwa colorimetry.Ubworoherane bwo kwiyumvisha no kumenya ahari ibicuruzwa cyangwa kutaboneka ukoresheje ibara ryamabara no kubura ibikoresho bihenze bisabwa byatumye LAMP ihitamo uburyo bwo kwipimisha SARS-CoV-2 mubice aho ibizamini bya laboratoire bitari byoroshye kuboneka, cyangwa kubika no gutwara ingero ntibyashobokaga, cyangwa muri laboratoire zitari zifite ibikoresho bya PCR bya termocycling.