PCR, ndi polymerase unyolo anachita, kutanthauza Kuwonjezera dNTP, Mg2+, elongation zinthu ndi matalikidwe kuwongola zinthu ku dongosolo pansi catalysis wa DNA polymerase, ntchito DNA kholo monga Chinsinsi ndi zoyambira enieni monga poyambira kutambasuka , Kupyolera mu masitepe a denaturation, annealing, extension, etc., ndondomeko ya in vitro kutengera mwana wamkazi strand DNA yogwirizana ndi kholo strand template DNA akhoza mwamsanga ndi mwachindunji kukulitsa chandamale chilichonse DNA mu vitro.
1. Hot Start PCR
Nthawi yoyambira kukulitsa mu PCR wamba sikuyika makina a PCR mu makina a PCR, ndiyeno pulogalamuyo imayamba kukulitsa.Kukonzekera kwadongosolo kukamalizidwa, kukulitsa kumayamba, komwe kungayambitse kukulitsa kosakhazikika, ndipo PCR yotentha imatha kuthetsa vutoli.
Kodi hot Start PCR ndi chiyani?Pambuyo pokonzekera dongosolo, enzyme modifier imatulutsidwa kutentha kwambiri (nthawi zambiri kuposa 90 ° C) panthawi yotentha yoyambira kapena "kuyambira kotentha", kotero kuti DNA polymerase imatsegulidwa.Nthawi yeniyeni yotsegulira ndi kutentha zimatengera momwe DNA polymerase ilili komanso chosinthira choyambira.Njirayi makamaka imagwiritsa ntchito zosintha monga ma antibodies, affinity ligands, kapena ma modifiers amankhwala kuti alepheretse ntchito ya DNA polymerase.Popeza ntchito ya DNA polymerase imaletsedwa kutentha kwa chipinda, ukadaulo woyambira wotentha umapereka mwayi waukulu wokonzekera machitidwe angapo a PCR pa kutentha kwa chipinda popanda kusiya zomwe zimachitika pa PCR.
2. RT-PCR
RT-PCR (Reverse transcription PCR) ndi njira yoyesera yosinthira kuchokera ku mRNA kupita ku cDNA ndikuigwiritsa ntchito ngati template yokulitsa.Njira yoyesera ndikuchotsa RNA yonse m'matenda kapena ma cell, gwiritsani ntchito Oligo (dT) ngati choyambira, gwiritsani ntchito reverse transcriptase kupanga cDNA, kenako gwiritsani ntchito cDNA ngati template ya PCR amplification kuti mupeze jini yomwe mukufuna kapena kuzindikira jini.
3. Fluorescent kuchuluka kwa PCR
Fluorescent quantitative PCR (Real-time Quantitative PCR,RT-qPCR) amatanthauza njira yowonjezerera magulu a fulorosenti ku PCR reaction system, pogwiritsa ntchito kusonkhanitsa ma siginecha a fulorosenti kuti ayang'anire ndondomeko yonse ya PCR mu nthawi yeniyeni, ndipo potsiriza amagwiritsa ntchito mphira wokhazikika kuti ayese template mochulukira.Njira zomwe zimagwiritsidwa ntchito kwambiri za qPCR zimaphatikizapo SYBR Green I ndi TaqMan.
4. Nested PCR
Nested PCR imatanthawuza kugwiritsa ntchito ma seti awiri a PCR zoyambira pamizere iwiri yakukulitsa kwa PCR, ndipo zokulitsa pamzere wachiwiri ndi gawo la jini lomwe mukufuna.
Ngati kusagwirizana kwa zoyambira ziwiri zoyambirira (zoyambira zakunja) kumapangitsa kuti chinthu chomwe sichinatchulidwe chiwonjezeke, kuthekera kwa gawo lomwelo lomwe silinatchulidwe kuzindikirika ndi gulu lachiwiri la zoyambira ndikupitiliza kukulitsa kumakhala kochepa kwambiri, chifukwa chake kukulitsidwa ndi awiri oyambira awiri, kutsimikizika kwa PCR kwasinthidwa.Ubwino umodzi wochita maulendo awiri a PCR ndikuti umathandizira kukulitsa zinthu zokwanira kuchokera ku DNA yochepa yoyambira.
5. Touchdown PCR
Touchdown PCR ndi njira yosinthira mawonekedwe a PCR posintha magawo a PCR.
Pa touchdown PCR, kutentha kwapakati pamizere ingapo yoyambira kumayikidwa madigiri angapo pamwamba pa kutentha kwakukulu (Tm) kwa zoyambira.Kutentha kwakukulu kwa annealing kumatha kuchepetsa kukulitsa kosagwirizana kwenikweni, koma nthawi yomweyo, kutentha kwapamwamba kumakulitsa kulekanitsa kwa zoyambira ndikutsata zomwe mukufuna, zomwe zimapangitsa kuchepa kwa PCR.Choncho, m'mizere yoyambirira, kutentha kwa annealing nthawi zambiri kumachepetsedwa ndi 1 ° C pamzere uliwonse kuti muwonjezere zomwe zili mu jini yomwe mukufuna.Pamene kutentha kwa annealing kwatsitsidwa kuti pakhale kutentha koyenera, kutentha kwa annealing kumasungidwa kwa maulendo otsalawo.
6. Direct PCR
Direct PCR imatanthawuza kukulitsa kwa chandamale cha DNA mwachindunji kuchokera pachitsanzo popanda kufunikira kwa kudzipatula kwa nucleic acid ndi kuyeretsedwa.
Pali mitundu iwiri ya PCR yolunjika:
njira yolunjika: tengani chitsanzo chochepa ndikuchiwonjezera mwachindunji ku PCR Master Mix kuti mudziwe PCR;
njira yowonongeka: mutatha kuyesa chitsanzo, yonjezerani ku lysate, lyse kuti mutulutse majeremusi, tengani pang'ono lysed supernatant ndikuwonjezera ku PCR Master Mix, chitani chizindikiritso cha PCR.Njirayi imapangitsa kuti ntchito yoyesera ikhale yosavuta, imachepetsa kugwiritsira ntchito nthawi, komanso imapewa kutaya kwa DNA panthawi yoyeretsa.
7. SOE PCR
Kuphatikizika kwa ma gene polumikizirana PCR (SOE PCR) imagwiritsa ntchito zoyambira zomwe zili ndi malekezero owonjezera kuti zinthu za PCR zipange unyolo wolumikizana, kotero kuti pakukulitsa kotsatira, kudzera pakukulitsa unyolo womwe umadutsana, magwero osiyanasiyana a njira ya A momwe zidutswa zokulira zimaphatikizidwira. ndi kuphatikizana.Tekinolojeyi pakadali pano ili ndi njira ziwiri zazikulu zogwiritsira ntchito: kupanga ma gene ophatikizika;gene site-lolunjika masinthidwe.
8. IPCR
Inverse PCR (IPCR) imagwiritsa ntchito zoyambira zobwerera kumbuyo kukulitsa zidutswa za DNA kusiyapo zoyambira ziwirizo, ndikukulitsa mndandanda wosadziwika mbali zonse za chidutswa chodziwika cha DNA.
IPCR idapangidwa poyambilira kuti izindikire kutsatizana kwa madera oyandikana nawo osadziwika, ndipo imagwiritsidwa ntchito kwambiri pophunzira zotsatsira ma gene;kukonzanso kwa chromosomal oncogenic, monga gene fusion, translocation and transposition;ndi kuphatikiza kwa ma virus, amagwiritsidwanso ntchito kwambiri tsopano Kwa mutagenesis yoyendetsedwa ndi malo, lembani plasmid ndi kusintha komwe mukufuna.
9. dPCR
Digital PCR (dPCR) ndi njira yowerengera mtheradi wa ma nucleic acid mamolekyulu.
Pano pali njira zitatu zowerengera ma nucleic acid mamolekyulu.Photometry imachokera ku kuyamwa kwa ma nucleic acid mamolekyulu;nthawi yeniyeni ya fulorosenti yochuluka ya PCR (Real Time PCR) imachokera ku mtengo wa Ct, ndipo mtengo wa Ct umatanthawuza nambala yozungulira yomwe ikugwirizana ndi mtengo wa fluorescence womwe ungadziwike;PCR ya digito ndiukadaulo waposachedwa kwambiri wa Quantitative wotengera njira imodzi ya PCR yowerengera ma nucleic acid quantification ndi njira yochulukira.
Nthawi yotumiza: Jun-13-2023