PCR. Binyuze mu ntambwe zo gutandukana, annealing, kwaguka, nibindi, inzira yo muri vitro yigana umukobwa wumugozi ADN yuzuzanya nicyitegererezo cyababyeyi ADN irashobora kwihuta kandi byumwihariko kwagura ADN iyo ari yo yose muri vitro.
1. Gutangira bishyushye PCR
Igihe cyo gutangira amplification muri PCR isanzwe ntabwo ari ugushira imashini ya PCR mumashini ya PCR, hanyuma gahunda itangira kwiyongera.Iyo sisitemu iboneye irangiye, amplification iratangira, ishobora gutera amplification idasanzwe, kandi hot-start PCR irashobora gukemura iki kibazo.
Niki gitangiye PCR?Sisitemu yo kubyitwaramo imaze gutegurwa, moderi ya enzyme irekurwa mubushyuhe bwinshi (mubisanzwe hejuru ya 90 ° C) mugihe cyambere cyo gushyushya cya reaction cyangwa "hot start", kugirango polymerase ya ADN ikore.Igihe nyacyo cyo gukora nubushyuhe biterwa na miterere ya ADN polymerase na moderi ishyushye-itangira.Ubu buryo bukoresha cyane cyane abahindura nka antibodies, affinity ligands, cyangwa imiti ihindura imiti kugirango ibuze ibikorwa bya ADN polymerase.Kubera ko ibikorwa bya ADN polymerase bibujijwe ku bushyuhe bwicyumba, tekinoroji yo gutangiza itanga uburyo bworoshye bwo gutegura sisitemu nyinshi za reaction ya PCR mubushyuhe bwicyumba utitanze umwihariko wibikorwa bya PCR.
2. RT-PCR
RT-PCR.Uburyo bwikigereranyo nugukuramo RNA yose mubice cyangwa selile mbere, koresha Oligo (dT) nka primer, koresha transcriptase kugirango uhindure cDNA, hanyuma ukoreshe cDNA nkicyitegererezo cyo kongera PCR kugirango ubone gene igenewe cyangwa umenye imvugo ya gene.
3. Fluorescent yuzuye PCR
Fluorescent yuzuye PCR (Igihe nyacyo PCR,RT-qPCR) bivuga uburyo bwo kongeramo amatsinda ya fluorescent muri sisitemu ya reaction ya PCR, ukoresheje kwegeranya ibimenyetso bya fluorescente kugirango ukurikirane inzira yose ya PCR mugihe nyacyo, hanyuma amaherezo ukoreshe umurongo usanzwe kugirango ugereranye umubare wuzuye.Uburyo bukoreshwa muburyo bwa qPCR burimo SYBR Icyatsi I na TaqMan.
4. PCR yatowe
Nested PCR bivuga gukoresha ibice bibiri bya PCR primers kumirongo ibiri ya PCR yongerewe imbaraga, naho ibicuruzwa byongera icyiciro cya kabiri nigice cya gen.
Niba bidahuye na couple ya mbere ya primers (primers yo hanze) itera ibicuruzwa bidasanzwe byongerewe imbaraga, birashoboka ko akarere kamwe kadasanzwe kamenyekana na kabiri ya primers kandi ugakomeza kwiyongera ni nto cyane, bityo rero amplification by kabiri ya primers, umwihariko wa PCR watejwe imbere.Inyungu imwe yo gukora ibice bibiri bya PCR nuko ifasha kongera ibicuruzwa bihagije kuva ADN itangiye.
5. Gukoraho PCR
Touchdown PCR nuburyo bwo kunoza umwihariko wa PCR muguhindura ibipimo bya PCR.
Mugukoraho PCR, ubushyuhe bwa annealing kubice bike byambere bishyirwaho dogere nkeya hejuru yubushyuhe ntarengwa (Tm) bwa primers.Ubushyuhe bwo hejuru bwa annealing burashobora kugabanya neza amplification idasanzwe, ariko mugihe kimwe, ubushyuhe bwo hejuru bwa annealing buzamura itandukaniro rya primers hamwe nurutonde rwateganijwe, bigatuma umusaruro wa PCR ugabanuka.Kubwibyo, mubice bike byambere, ubushyuhe bwa annealing busanzwe bugabanukaho 1 ° C kuri buri cyiciro kugirango byongere ibikubiye muri gen igenewe muri sisitemu.Iyo ubushyuhe bwa annealing bwamanuwe kugeza ku bushyuhe bwiza, ubushyuhe bwa annealing bugumaho kumuzingo usigaye.
6. PCR itaziguye
PCR itaziguye yerekeza ku kwagura ADN igamije biturutse ku cyitegererezo bidakenewe kwigunga aside nucleic no kwezwa.
Hariho ubwoko bubiri bwa PCR itaziguye:
uburyo butaziguye: fata urugero ruto hanyuma wongere kuri PCR Master Mix kugirango imenye PCR;
uburyo bwo kumena: nyuma yo gutoranya icyitegererezo, ongera kuri lysate, lyse kugirango urekure genome, fata akantu gato ka lysed supernatant hanyuma uyongere kuri PCR Master Mix, kora PCR.Ubu buryo bworoshya ibikorwa byubushakashatsi, bigabanya igihe, kandi birinda gutakaza ADN mugihe cyo kweza.
7. SOE PCR
Gukwirakwiza gene ukoresheje kwaguka kwinshi PCR (SOE PCR) ikoresha primers ifite impera zuzuzanya kugirango ibicuruzwa bya PCR bibe urunigi rwuzuzanya, kuburyo mubyakurikiyeho amplification reaction, binyuze mukwagura iminyururu irenze, amasoko atandukanye ya tekinike aho ibice byongerewe byuzuye. hanyuma atera hamwe.Ubu buryo bwikoranabuhanga bufite ibyerekezo bibiri byingenzi bikoreshwa: kubaka ingirabuzimafatizo;gene site iyobowe na mutation.
8. IPCR
Inverse PCR (IPCR) ikoresha primers yuzuzanya yibanze kugirango yongere ibice bya ADN bitari primers ebyiri, kandi byongera urutonde rutazwi kumpande zombi z'igice kizwi cya ADN.
IPCR yabanje gukorwa kugirango hamenyekane urukurikirane rw'uturere tutazwi, kandi ikoreshwa cyane mukwiga urutonde rwa porotokoro;oncogenic chromosomal rearrangements, nka gene fusion, guhinduranya no guhinduranya;na virusi ya virusi ihuza, nayo irakoreshwa nonaha Kurubuga ruyobowe na mutagenezesi, kora plasmid hamwe na mutation wifuza.
9. DPCR
Digital PCR (dPCR) ni tekinike yo kugereranya byimazeyo molekile ya acide nucleic.
Hano hari uburyo butatu bwo kugereranya molekile ya acide nucleic.Photometrie ishingiye ku kwinjiza molekile ya acide nucleique;igihe nyacyo fluorescent PCR (Real Time PCR) ishingiye ku gaciro ka Ct, naho agaciro ka Ct bivuga umubare wizunguruka uhuye nagaciro ka fluorescence ishobora kugaragara;Digital PCR nubuhanga bugezweho bwa Quantitative bushingiye kuburyo bwa molekile imwe ya PCR yo kubara ingano ya acide nucleic nuburyo bwuzuye bwo kubara.
Igihe cyo kohereza: Jun-13-2023