PCR Ibibazo bya PCR.

1Ibinyoma bibi, nta bande yongerewe igaragara Ibintu by'ingenzi bya reaction ya PCR ni

Gutegura acide nucleic aside

Quality primer quality and umwihariko

Ubwiza bwa enzyme kandi

Conditions Imiterere ya PCR. Kugirango ubone impamvu, isesengura nubushakashatsi nabyo bigomba gukorwa kumurongo wavuzwe haruguru.

Inyandiko:

Inyandikorugero irimo poroteyine zanduye

Inyandikorugero irimo Taq enzyme inhibitor

Proteins Poroteyine ziri mu nyandikorugero ntizishishwa kandi zivanyweho, cyane cyane amateka ya chromosomes

Byinshi byatakaye mugihe cyo gukuramo no gutegura inyandikorugero, cyangwa fenol yashizwemo

TemplateIcyitegererezo cya nucleic aside ntabwo itandukanijwe rwose. Iyo ubwiza bwa enzymes na primers ari byiza, niba bande ya amplification itagaragara, birashoboka cyane ko hari ibitagenda neza muburyo bwo gusya bwikigereranyo cyangwa uburyo bwo gukuramo aside nucleic. Kubwibyo, igisubizo cyingirakamaro kandi gihamye kigomba gutegurwa, kandi inzira igomba gukosorwa kandi ntigomba guhinduka uko bishakiye. . Enzyme idakora: Birakenewe gusimbuza enzyme nshya, cyangwa gukoresha enzymes zishaje nizishya icyarimwe kugirango dusesengure niba ibibi bibi biterwa no gutakaza cyangwa ibikorwa bya enzyme bidahagije. Twabibutsa ko rimwe na rimwe imisemburo ya Taq yibagirana.Ibiciro: Ubwiza bwibanze, kwibanda kuri primer, kandi niba kwibumbira hamwe kwa primers byombi ari impanvu ni impamvu zisanzwe zitera kunanirwa kwa PCR cyangwa imirongo ya amplification idashimishije no gukwirakwizwa byoroshye. Hano haribibazo byubwiza bwa primer synthesis mubice bimwe. Imwe muri primers ebyiri ifite kwibanda cyane naho indi ifite intumbero nkeya, bikavamo ubushobozi buke bwa asimmetric amplification.

Ingamba zo guhangana ni:

① Hitamo igice cyiza cya primer synthesis.

② Kwibanda kuri primers ntibigomba kureba gusa agaciro ka OD, ahubwo tunitondere igisubizo kibanza cya agarose gel electrophorei. Hagomba kubaho primer band, kandi umucyo wibice bibiri bya primer ugomba kuba hafi. Kurugero, primer imwe ifite bande naho indi primer ntigira bande. Kubice, PCR irashobora kunanirwa muriki gihe kandi igomba gukemurwa binyuze mubiganiro na primer synthesis unit. Niba primer imwe ifite umucyo mwinshi naho indi ikagira umucyo muke, kuringaniza ibitekerezo mugihe ugabanya primers.

③ Primers igomba kubikwa cyane kandi muke kugirango irinde gukonjeshwa no gukonja cyangwa kubika igihe kirekire muri firigo, ibyo bikaba bishobora gutuma kwangirika no kwangirika kwa primers.

DesignIgishushanyo mbonera nticyumvikana, nkuburebure bwa primer ntibihagije, ibipimo byakozwe hagati ya primers, nibindi. Mg2 + kwibanda: Mg2 + ion yibandaho bigira uruhare runini mubikorwa bya amplification ya PCR. Niba kwibandaho ari hejuru cyane, birashobora kugabanya umwihariko wa amplification ya PCR. Niba kwibumbira hamwe ari bike cyane, bizagira ingaruka kumusaruro wa PCR ndetse no gutuma PCR yongerera imbaraga kunanirwa nta kwongera imirongo. Impinduka mubunini bwa reaction: Mubisanzwe ingano ikoreshwa muri amplification ya PCR ni 20ul, 30ul, na 50ul. Cyangwa 100ul, ni ikihe gipimo kigomba gukoreshwa mu kongera ingufu za PCR gishyirwaho ukurikije intego zitandukanye z'ubushakashatsi bwa siyansi no gupima amavuriro. Nyuma yo gukora ingano ntoya, nka 20ul, hanyuma ugakora ingano nini, ugomba gukurikiza ibisabwa, bitabaye ibyo bikananirana byoroshye. Impamvu z'umubiri: Gutandukana ni ngombwa cyane kuri amplification ya PCR. Niba ubushyuhe bwo gutandukana buri hasi kandi igihe cyo gutandukana ni kigufi, ibibi bibi birashoboka cyane; ubushyuhe bwa annealing buri hasi cyane, bushobora gutera amplification idasanzwe no kugabanya imikorere yihariye. Ubushyuhe bwa annealing buri hejuru cyane. Ihindura cyane guhuza primers kubishusho kandi bigabanya imikorere ya PCR. Rimwe na rimwe, birakenewe gukoresha ibipimo bisanzwe bya termometero kugirango ugenzure ubushyuhe, annealing hamwe nubushyuhe bwagutse muri amplifier cyangwa inkono ikabura amazi. Iyi nayo ni imwe mu mpamvu zananira PCR. Itondekanya ryintego itandukanijwe: Niba intego ikurikiranye ihinduwe cyangwa yasibwe, bigira ingaruka kumurongo wihariye wa primer kurugero, cyangwa primer hamwe nicyitegererezo bitakaza urutonde rwuzuzanya bitewe no gusiba igice runaka cyurwego rwateganijwe, amplification ya PCR ntabwo bizagerwaho.

2.ibinyoma byiza PCR amplification band igaragara igaragara ihuye nintego ikurikiranye, kandi rimwe na rimwe umurongo uba ufite gahunda kandi urabagirana. Igishushanyo mbonera cya primer kidakwiye: Urutonde rwatoranijwe rwongerewe rufite homology hamwe nurutonde rutagamije gukwirakwizwa, bityo mugihe ukora PCR amplification, ibicuruzwa byongerewe PCR nibitari intego. Niba intego ikurikiranye ari ngufi cyane cyangwa primer ni ngufi cyane, ibyiza byibinyoma birashobora kubaho byoroshye. Ibanze bigomba gusubirwamo. Kwanduzanya kwanduza intego cyangwa ibicuruzwa byongerewe imbaraga: Hariho impamvu ebyiri zitera kwanduza: Icya mbere, kwanduzanya genome yose cyangwa ibice binini, biganisha kubintu byiza. Ibi byiza bibi birashobora gukemurwa nuburyo bukurikira: Witondere kandi witonda mugihe ukora kugirango wirinde urutonde rwintego gutwarwa nimbunda ntangarugero cyangwa gusohoka mu muyoboro wa centrifuge. Usibye imisemburo nibintu bidashobora kwihanganira ubushyuhe bwinshi, reagent cyangwa ibikoresho byose bigomba guhindurwa numuvuduko mwinshi. Imiyoboro yose ya centrifuge hamwe nicyitegererezo cyo gutera inshinge zigomba gukoreshwa rimwe. Nibiba ngombwa, imiyoboro ya reaction na reagent irabagirana nurumuri ultraviolet mbere yo kongeramo urugero rwo gusenya acide nucleic ihari. Iya kabiri ni kwanduza uduce duto twa acide nucleique mu kirere. Ibi bice bito ni bigufi kurenza uko bikurikirana, ariko bifite homologiya imwe. Zishobora guterwa hagati yazo, kandi nyuma yo kuzuzanya na primers, ibicuruzwa bya PCR birashobora kongerwa, bikavamo ibyiza bitari byo, bishobora kugabanuka cyangwa gukurwaho nuburyo bwa PCR bwatewe.

 

3.Ntibisobanuro byihariye bya amplification bande bigaragara Amatsinda agaragara nyuma ya PCR yongerewe imbaraga ntaho ahuriye nubunini buteganijwe, bwaba bunini cyangwa buto, cyangwa byombi byongerewe imbaraga hamwe na bande zidasanzwe zidasanzwe zigaragara icyarimwe. Impamvu zo kugaragara kwitsinda ridasanzwe ni: icya mbere, primers ntabwo yuzuzanya rwose nintego ikurikiranye, cyangwa primers yegeranya kugirango ibe ibipimo. Impamvu ya kabiri nuko Mg2 + ion yibanze cyane, ubushyuhe bwa annealing buri hasi cyane, kandi numubare wa PCR ni mwinshi cyane. Ikintu cya kabiri nubwiza nubunini bwa enzyme. Enzymes ziva mubisoko bimwe na bimwe usanga zikunze kwibasirwa na bande zidasanzwe ariko enzymes ziva ahandi ntizikora. Umubare munini wa enzymes urashobora rimwe na rimwe kuganisha kuri amplification idasanzwe. Ingamba zo guhangana zirimo: gusubiramo primers nibiba ngombwa. Mugabanye ingano ya enzyme cyangwa uyisimbuze indi soko. Mugabanye ingano ya primers, ongera umubare wicyitegererezo uko bikwiye, kandi ugabanye umubare wizunguruka. Ongera ubushyuhe bwa annealing muburyo bukwiye cyangwa ukoreshe uburyo bwubushyuhe bubiri (denatration kuri 93 ° C, annealing no kwaguka hafi 65 ° C).

 

4.Gukurura neza cyangwa gusiga bigaragara PCR amplification rimwe na rimwe igaragara nkimigozi isize, amabati ameze nkamabati cyangwa amabati asa na tapi. Impamvu akenshi ziterwa na enzyme nyinshi cyangwa ubuziranenge bwa enzyme, kwibanda cyane kuri DNTP, kwibanda cyane kwa Mg2 +, ubushyuhe buke bwa annealing, hamwe ninzinguzingo nyinshi. Ingamba zo guhangana zirimo: uce Kugabanya ingano ya enzyme, cyangwa gusimbuza enzyme nandi masoko. Kugabanya kwibanda kuri dNTP. Mugabanye bikwiye Mg2 + kwibanda. Ongera umubare wicyitegererezo kandi ugabanye umubare wizunguruka