PCR, byinshi PCR, Muri PCR, Hindura PCR, RT-PCR, qPCR （1）-PCR

Tuzatoranya ibitekerezo, intambwe nibisobanuro bya PCR zitandukanye

Ⅰ. PCR

Imyitwarire ya Polymerase, yitwa PCR, ni tekinoroji ya biologiya ikoreshwa mu kwagura ibice bya ADN byihariye.Irashobora gufatwa nka ADN idasanzwe yigana muri vitro.ADN polymerase (ADN Polymerase ya I) yavumbuwe mu 1955, naho Klenow Fragment ya E. Coli, ifite agaciro k'igeragezwa kandi ifatika, yavumbuwe na Dr. H. Klenow mu ntangiriro ya za 70, ariko kubera ko iyi misemburo itihanganira ubushyuhe, ubushyuhe bwo hejuru burashobora kuyangirika, bityo ntibujuje uburinganire bwa polymerase hamwe n'ubushyuhe bukabije.Imisemburo ikoreshwa muri iki gihe (yitwa Taq polymerase), yatandukanijwe na Thermus aquaticus, bagiteri ishyushye yo mu mpeshyi mu 1976. Ikiranga ni uko ishobora kurwanya ubushyuhe bwinshi kandi ni enzyme nziza, ariko ikoreshwa cyane nyuma ya za 1980.Igitekerezo cyumwimerere cya prototype yumwimerere ya PCR isa nogusana gene no kuyandukura, byasabwe na Dr. KJell Kleppe mumwaka wa 1971. Yasohoye kopi ya mbere yoroheje kandi ngufi -term (bisa nkibisubizo bibiri byambere bya PCR).PCR yateye imbere uyumunsi yatunganijwe na Dr. Kary B. Mullis mu 1983. Dr. Mullis yakoreye ibigo bya PE muri uwo mwaka, PE rero ifite umwanya wihariye mubikorwa bya PCR.Dr. Mullis yasohoye ku mugaragaro impapuro za mbere zifitanye isano na Saiki hamwe n’abandi mu 1985. Kuva icyo gihe, gukoresha PCR ni kilometero ibihumbi ku munsi, kandi ubwiza bw’impapuro zijyanye nabyo bushobora gutuma ubundi buryo bwinshi bw’ubushakashatsi budashimishije.Icyakurikiyeho, tekinoroji ya PCR ikoreshwa cyane mubushakashatsi bwa siyansi y’ibinyabuzima no mu mavuriro, ihinduka ikoranabuhanga rikomeye ry’ubushakashatsi ku binyabuzima.Mullis yatsindiye kandi igihembo cyitiriwe Nobel mu 1993.

PCRIhame

Ihame ryibanze ryikoranabuhanga rya PCR risa nuburyo bwo kwigana bisanzwe bya ADN, kandi umwihariko wacyo biterwa na oligonucleotide primer yuzuzanya kumpera zombi zikurikirana.PCR igizwe na degeneration-annealing-yagura intambwe eshatu zifatizo zifatika: egGusohora ibyitegererezo ADN: Nyuma yicyitegererezo ADN yashyutswe kugeza kuri 93 ° C mugihe runaka, igisubizo cya ADN ebyiri zuburyo bubiri bwa ADN zakozwe na PCR yongerewe icyitegererezo cya ADN Gusiga, bigakorwa hamwe na primer kugirango bitegure kuzakurikiraho.AnAnealing (compound) yicyitegererezo ADN na primer: Nyuma yicyitegererezo ADN ishyushye kandi igahinduka urunigi rumwe, ubushyuhe buramanuka bugera kuri 55 ° C.Urukurikirane rwuzuzanya rwa primer hamwe nicyitegererezo ADN imwe-urunigi.Kwagura primer: ADN yerekana icyitegererezo-guhuza primer bishingiye kubikorwa bya TaqDNA polymerase, hamwe na DNTP nkibikoresho fatizo byerekana.Komeza ihame ryo kwigana, shushanya urunigi rushya rwabigenewe rwuzuzanya rwuzuza inyandikorugero ya ADN, kandi usubiremo cycle degeneration-annealing-kwagura inzira eshatu zishobora kubona "urunigi rwabigenewe", kandi uruhererekane rushya ruraboneka wongere ube icyitegererezo cyizunguruka gikurikira.Bifata 2-4min kugirango urangize loop, gene intego irashobora kongerwa inshuro miriyoni mumasaha 2-3.

BisanzwePCRSisitemu yo Kwitabira

Ibintu bitanu byerekana PCR

Hariho ubwoko butanu bwibintu bigira uruhare mubikorwa bya PCR, aribyo primer, enzyme, dNTP, inyandikorugero na buffer (birakenewe Mg2 +).[Uburyo bwa PCR]

Inzira isanzwe ya PCR igabanijwemo intambwe eshatu

1. Kwangirika kwa ADN (90 ° C-96 ° C): Inyandikorugero zibiri za ADN zerekana ubushyuhe, imigozi ya hydrogène iracika, ikora ADN y'umunyururu umwe.

2. Annealing (25 ℃ -65 ℃): Ubushyuhe bwa sisitemu buragabanuka, primer ihujwe nicyitegererezo cya ADN kugirango ikore urunigi rwibanze.

3. Kwaguka (70 ℃ -75 ℃): Mubikorwa bya enzyme ya Taq (hafi 72 ° C, igikorwa cyiza), dNTP ikoreshwa nkibikoresho fatizo, iva kuri 5 ′ iherezo rya primer → 3 ′ iherezo, synthesis hamwe nicyitegererezo byuzuzanya urunigi rwa ADN.

Buri cyiciro kizengurutswe, gifatanye kandi kiragurwa, bikubye kabiri ADN.Kugeza ubu, kubera agace gato kongerewe imbaraga, PCR zimwe zishobora kwiganwa mugihe gito cyane nubwo ibikorwa bya Taq enzyme bitaba byiza, bityo birashobora guhinduka mubyiciro bibiri, ni ukuvuga ko annealing no kwaguka bishobora gukorwa kuri 60 ° C-65 ° C icyarimwe.Kugirango ugabanye inzira yo guterura no gukonjesha no kunoza umuvuduko wo gusubiza.

Ibiranga PCR

● Byihariye

Impamvu zifatika zifatika zo gusubiza PCR ni: combinationIhuza ryihariye rya primer hamwe nicyitegererezo ADN.PrincipleIhame ryo guhuza shingiro.LoyalUbudahemuka bwa TaqDNA polymerase synthesis reaction.SpecificUmwihariko no guharanira inyungu za gen.

Ihuriro ryukuri rya primers na templates nurufunguzo.Guhambira primer hamwe nicyitegererezo no kwagura urunigi rwa primer bishingiye ku ihame ryo guhuza alkaline.Ubudahemuka bwa synthesis ya polymerase hamwe nubushyuhe bwo hejuru bwa Taq ADN polymerase kugirango uhuze (compound) yicyitegererezo na primer mubitekerezo bishobora gukorwa mubushyuhe bwo hejuru.Umwihariko wo guhuza wiyongereye cyane.Clip irashobora kugumana urwego rwo hejuru rwukuri.Muguhitamo akarere ka genetike igamije hamwe no kwibumbira hamwe, hamwe nibidasanzwe.

Ens Kumva neza

Ingano yumusaruro wibicuruzwa bya PCR yongerewe nurutonde, rushobora kwagura icyitegererezo cyo gutangira Picker (PG = 10-12) kugirango wongere urwego rwa microcontroller kurwego rwa microgrammes (μg = -6).Ingirabuzimafatizo zishobora kugaragara kuva miriyoni 1;mugutahura virusi, sensitivite ya PCR irashobora kugera kuri RFU 3 (ibibanza byubusa byashizweho ibice);igipimo ntarengwa cyo kumenya muri siyanse ya bagiteri ni bagiteri 3.

● Byoroshye kandi byihuse

Imitekerereze ya PCR ikoresha ubushyuhe bwo hejuru bwa Taq ADN polymerase, yongeraho igisubizo icyarimwe, ni ukuvuga kwangirika-anneal-kwagura reaction kumuti wa amplification ya ADN hamwe ninkono yo koga.Mubisanzwe, amplification reaction irangira mumasaha 2 kugeza 4.Ibicuruzwa byongerewe imbaraga bisesengurwa ninkota yamashanyarazi, kandi ntibigomba gukoresha isotopi, nta mwanda uhumanya radio, no kuzamura byoroshye.

● Ubuziranenge bwikigereranyo buri hasi

Ntibikenewe gutandukanya virusi cyangwa bagiteri na selile yumuco.Ibicuruzwa bya ADN bitavanze na RNA birashobora gukoreshwa nka amplifier.Kumenyekanisha kwa ADN birashobora gukoreshwa hifashishijwe urugero rwamavuriro nkamaraso, amazi yumubiri, amazi yo gukaraba, umusatsi, selile, hamwe nuduce twizima.

PCRibibazo rusange

Negative Ibinyoma bibi, nta bande yongerewe

Ibyiciro byingenzi byimikorere ya PCR harimo: ① gutegura acide nucleic acide, ② ubwiza nibidasanzwe bya primers, ③ ubwiza bwimisemburo conditions Imiterere ya PCR.Gushakisha impamvu nabyo bigomba gusesengurwa no kwigwa kumirongo yavuzwe haruguru.

Inyandikorugero: ① Inyandikorugero irimo poroteyine zitandukanye, ② Inyandikorugero irimo Taq enzyme inhibitor, protein Poroteyine iri mu gishushanyo ntikurwaho, cyane cyane poroteyine yo mu itsinda muri chromosome.⑤ Kwangirika kwa aside nucleic aside ntabwo byuzuye.Iyo ubwiza bwa enzymes na primers ari byiza, nta bande ya amplification ibaho, bishoboka cyane ko igogorwa ryigogora ryikigereranyo.Hariho ibitagenda neza muburyo bwo gukuramo acide nucleic aside, kugirango utegure igisubizo cyiza kandi gihamye cyigogora, inzira yacyo igomba gukosorwa kandi ntigihindurwe uko bishakiye.

Enzyme idakora: enzyme nshya cyangwa enzymes zombi zishaje nizishya bigomba gukoreshwa hamwe kugirango dusesengure niba ibikorwa bya enzyme byatakaye cyangwa bidahagije, biganisha kubibi.Twabibutsa ko Taq enzyme cyangwa Ethidium bromide rimwe na rimwe yibagirana.

Primer: ubwiza bwa primer, kwibumbira hamwe kwa primer, no kumenya niba kwibumbira hamwe kwa primers byombi bihuye.Nimpamvu isanzwe yo kunanirwa kwa PCR cyangwa kwiyongera kwitsinda ntabwo ari byiza kandi bikunda gukwirakwira.Hano haribibazo byubwiza bwa primers ya numero imwe yicyiciro.Intangiriro zombi zifite kwibanda cyane hamwe no kwibanda cyane, bitera amplificatifike ya asimmetricike.Ingamba zo guhangana ni: ① Hitamo primer nziza kugirango uhuze ibice.② Kwibanda kwa primer ntibiterwa gusa nagaciro ka OD, ahubwo binita kumazi yambere ya primer kugirango akore agar isukari gel electrophorei.Hagomba kubaho primer strip zone, kandi umucyo wa primers zombi ugomba kuba muri rusange.Umukandara, PCR irashobora kunanirwa muriki gihe, kandi igomba gukemurwa na primer synthesis unit.Niba primer ari ndende, umucyo ni muke, kandi kwibanda kwayo bigomba kuringanizwa mugihe bivanze.③ Primer igomba kwishyurwa kandi ikabikwa murwego rwo hejuru kugirango hirindwe ubukonje bwinshi cyangwa burebure bwa firigo ndende ya firigo, bizatera primer kwangirika no kwangirika.Igishushanyo cya primer nticyumvikana, nkuburebure bwa primer ntibihagije, kandi di cluster iba hagati ya primers.

Mg2 + kwibanda: Mg2 + ion yibandaho bigira ingaruka zikomeye kumikorere ya PCR.Kwibanda cyane birashobora kugabanya abo mudahuje igitsina cya PCR.Niba kwibumbira hamwe ari bike cyane, ibisohoka bya PCR byongera umusaruro bizanatuma PCR yongerewe imbaraga idafite umurongo wagutse.

Guhindura ingano ya reaction: Ingano ikoreshwa muri amplification ya PCR ni 20ul, 30ul, na 50ul cyangwa 100uL, ingano nini yo gusaba PCR yongerewe ishyirwaho hakurikijwe intego zitandukanye zubushakashatsi bwa siyanse no gupima amavuriro.Nyuma yo gukora ibice bito nka 20ul, birakenewe gukora umugozi mugihe ukora ubunini, bitabaye ibyo bikananirana.

Impamvu zifatika: Guhinduka ni ngombwa cyane kuri amplification ya PCR.Niba ubushyuhe bwo kugabanuka ari buke, igihe cyo kwangirika ni gito, birashoboka ko bibaho mubi;ubushyuhe buke cyane bwa annealing burashobora gutera amplificateur idasanzwe kandi bikagabanya imikorere yihariye.Bigira ingaruka cyane kubihuza primers na templates kugirango ugabanye imikorere ya PCR.Rimwe na rimwe, birakenewe gukoresha ibipimo bya termometero kugirango tumenye itandukaniro, annealing hamwe nubushyuhe bwagutse mugihe cyo kwagura cyangwa gutekesha amazi-guteka, bikaba arimwe mubitera kunanirwa kwa PCR.

Intego zikurikirana: Niba intego ikurikiranye ibaye, ihinduka cyangwa gusiba, guhuza prototype hamwe nicyitegererezo byahujwe, cyangwa kubera kubura urutonde rwintego, primer hamwe nicyitegererezo bizabura ibyuzuzanya, kandi kwongera kwa PCR ntabwo bizagenda neza.

● Ibinyoma byiza

Itsinda rya amplification ya PCR rigaragara rihuye nintego ikurikiranye, kandi rimwe na rimwe umurongo wacyo uba mwiza kandi muremure.

Igishushanyo mbonera ntigikwiye: ibyatoranijwe byatoranijwe bikurikirana hamwe nuburyo butagamije gukwirakwiza amplifisione bifite homologique, iyo rero amplification ya PCR, ibicuruzwa byongerewe PCR ntabwo bikurikirana.Intego ikurikiranye ni ngufi cyane cyangwa primer ni ngufi cyane, kandi ikunda kwibeshya.Ukeneye gusubirwamo.

Kwanduza kwambukiranya ibicuruzwa bikurikirana cyangwa ibicuruzwa byongerewe imbaraga: Hariho impamvu ebyiri zitera uyu mwanda: Icya mbere, kwanduza-genome yose cyangwa ibice binini, biganisha ku byiza bibi.Ubu bwoko bw'ibinyoma bushobora gukemurwa nuburyo bukurikira: Witondere kandi witonda mugihe cyo gukora kugirango wirinde urutonde rwintego guhumeka mu mbunda ntangarugero cyangwa gusohoka mu muyoboro wa centrifugal.Usibye imisemburo nibintu bidashobora kwihanganira ubushyuhe bwinshi, reagent cyangwa ibikoresho byose bigomba kwanduzwa numuvuduko mwinshi.Imiyoboro ya centrifugal hamwe nicyitegererezo bigomba gukoreshwa icyarimwe.Iyo bibaye ngombwa, mbere yo kongeramo ingero, umuyoboro wa reaction na reagent uhura nimirasire ya ultraviolet kugirango isenye aside nucleique ihari.Icya kabiri, uduce duto mu kwanduza ikirere.Ibi bice bito ni bigufi kurenza uko byakurikiranye, ariko bifite homologiya runaka.Irashobora guterwa hamwe.Nyuma yo kuzuza primers, ibicuruzwa bya PCR birashobora kwagurwa, bizatera umusaruro mubi.Irashobora gukoreshwa mukugabanya cyangwa gukuraho uburyo bwa PCR icyari.

Kugaragara bande idafite akamaro

Amatsinda yagaragaye nyuma ya PCR yongerewe imbaraga ntaho ahuriye nubunini buteganijwe, cyangwa bunini cyangwa buto, cyangwa icyarimwe, cyangwa icyarimwe, imirongo yihariye ya amplification hamwe na bande idasanzwe.Kugaragara kwitsinda ridasanzwe ni: Icya mbere, primers ntabwo zuzuzanya zuzuzanya nintego zikurikirana, cyangwa polymerisation ya primer kugirango ikore di cluster.Iya kabiri ni uko kwibumbira hamwe kwa MG2 + ion ari hejuru cyane, ubushyuhe bwa annealing buri hasi cyane, kandi umubare wa cycle ya PCR urafitanye isano.Icya kabiri, ubwiza nubunini bwa enzymes.Akenshi, imisemburo yinkomoko imwe ikunda guhura na bande idasanzwe kandi imisemburo yandi masoko ntabwo ibaho.Rimwe na rimwe, amplification idasanzwe ya enzymes nayo ibaho.Ingamba zo guhangana nizo: kongera gushushanya ibikurura nibiba ngombwa.Mugabanye ingano ya enzyme cyangwa gusimbuza enzyme yandi masoko.Mugabanye umubare wibanze, ongera umubare wicyitegererezo uko bikwiye, kandi ugabanye umubare wizunguruka.Ongera neza ubushyuhe bwa annealing cyangwa ukoreshe uburyo bubiri bwubushyuhe (93 ° C degeneration, annealing kandi bugera kuri 65 ° C).

Kugaragara gukurura flake cyangwa gusiga kaseti

Kwiyongera kwa PCR rimwe na rimwe bigaragara ko byashyizwe mu bikorwa cyangwa bikingiwe cyangwa umukandara umeze nka tapi.Kubwimpamvu, kubera ubwinshi bwimisemburo cyangwa ubwiza buke bwa enzyme, intumbero ya DNTP iri hejuru cyane, Mg2 + yibanze cyane, ubushyuhe bwa annealing buri hasi cyane, kandi umubare wizunguruka ni mwinshi.Ingamba zo guhangana ni: edGabanya ingano ya enzymes, cyangwa uhindure enzyme yandi masoko.Kugabanya ubunini bwa dNTP reduceGabanya neza Mg2 + kwibanda.④Kongera ubwinshi bwicyitegererezo kandi ugabanye umubare wizunguruka.

Ibicuruzwa bifitanye isano

Intwari ya PCR (Hamwe n'irangi)

Ubudahemuka buhebuje: inshuro 6 z'umusemburo wa Taq usanzwe;

Speed ​​Kwihuta kwihuta

◮ Inyandikorugero nyinshi

Impinduka nziza yo kongera imbaraga

Erance Kwihanganira ibidukikije birakomeye: bishyirwa kuri 37 ° C icyumweru, bikomeza ibikorwa birenga 90%;

◮ Ifite ibikorwa bya 5 '→ 3' ADN polymerase na 5 '→ 3' ibikorwa bya exonuclease, nta bikorwa bya 3 '→ 5'.

PCR Byoroshyeᵀᴹ (Hamwe n'irangi)

Sisitemu idasanzwe ya reaction hamwe nubushobozi buhanitse Taq ADN Polymerase ituma PCR reaction ikora neza cyane, amplificateur na sensitivite.

RT-qPCR Byoroshyeᵀᴹ (Intambwe imwe) -SYBR Icyatsi I.

Kit Intambwe imwe yintambwe ikora transcription transcription na qPCR reaction ebyiri mumiyoboro imwe, gusa ukeneye kongeramo inyandikorugero RNA, primers yihariye ya PCR na ddH yubusa ya RNase.₂O.

Kit Igikoresho gishobora gusesengura vuba kandi neza muburyo bwo gusesengura virusi ya RNA cyangwa gukurikirana RNA.

Igikoresho gikoresha uburyo bwihariye bwa Foregene reverse transcription reagent hamwe na Foregene HotStar Taq ADN Polymerase ihujwe na sisitemu idasanzwe yo gukora kugirango tunoze neza imikorere ya amplification hamwe nibisobanuro bya reaction.

Sisitemu yogukora neza ituma reaction igira ibyiyumvo byo gutahura, gukomera kwumuriro, no kwihanganira neza.

◮ RT-qPCR Biroroshye^TM.

RT Biroroshye^TMII (Master Premix ya umurongo wa mbere cDNA synthesis yaIgihe nyacyo PCR)

-Ubushobozi buhagije bwo gukuraho gDNA, ishobora gukuraho gDNA mubishusho muminota 2.

-Uburyo bwiza bwo kwandukura sisitemu, bifata iminota 15 gusa kugirango urangize synthesis yumurongo wambere cDNA.

-Icyitegererezo cyoroshye: inyandikorugero zirimo ibintu byinshi bya GC hamwe nuburyo bugoye bwa kabiri birashobora kandi guhindurwa hamwe nubushobozi buhanitse.

-Ibihe byinshi-byunvikana sisitemu yo kwandukura, pg-urwego rwicyitegererezo nacyo gishobora kubona cDNA nziza.

-Uburyo bwo kwandukura sisitemu ifite ubushyuhe buhanitse, ubushyuhe bwiza bwa reaction ni 42 ℃, kandi buracyafite imikorere myiza yo kwandukura kuri 50 ℃.