I-PCR, amaningi I-PCR, Itholakala ku-PCR, Shintshanisa amasheya PCR, I-RT-PCR, qPCR (1)-I-PCR

Sizohlunga imiqondo, izinyathelo kanye nemininingwane ye-PCR ehlukahlukene

Ⅰ. I-PCR

I-Polymerase Chain Reaction, ebizwa ngokuthi i-PCR, ubuchwepheshe bebhayoloji yamangqamuzana obusetshenziselwa ukukhulisa izingcezu ezithile ze-DNA.Kungathathwa njengokuphindaphinda okukhethekile kwe-DNA in vitro.I-DNA polymerase (i-DNA Polymerase I) yatholwa ekuqaleni kuka-1955, futhi i-Klenow Fragment ka-E. Coli, enenani lokuhlola nokusebenza, yatholwa nguDkt. H. Klenow ekuqaleni kwawo-1970, kodwa ngenxa yokuthi le enzyme ayikubekezeleli ukushisa, izinga lokushisa eliphezulu lingakwazi ukuwohloka, ngakho alihlangabezani ne-polymerase degeneration chain reaction.Ama-enzyme asetshenziswa namuhla (abizwa ngokuthi i-Taq polymerase), ahlukaniswa ne-Thermus aquaticus, i-bacterium yasentwasahlobo eshisayo ngo-1976. Isici sayo ukuthi ingakwazi ukumelana nokushisa okuphezulu futhi iyi-enzyme efanelekile, kodwa isetshenziswa kabanzi ngemva kweminyaka yawo-1980.Umqondo wangempela we-prototype yakudala ye-PCR ufana nokulungisa izakhi zofuzo nokukopishwa, okwahlongozwa uDkt. KJell Kleppe ngo-1971. Washicilela ikhophi yokuqala elula neyesikhathi esifushane yofuzo (efana nokusabela kwemijikelezo emibili yokuqala ye-PCR).I-PCR eyakhiwe namuhla yathuthukiswa uDkt. Kary B. Mullis ngo-1983. UDkt. Mullis wasebenzela izinkampani ze-PE ngalowo nyaka, ngakho i-PE inesimo esikhethekile embonini ye-PCR.UDkt. Mullis washicilela ngokusemthethweni iphepha lokuqala elihlobene no-Saiki nabanye ngo-1985. Kusukela ngaleso sikhathi, ukusetshenziswa kwe-PCR izinkulungwane zamamayela ngosuku, futhi ikhwalithi yamaphepha ahlobene kungathiwa yenza ezinye izindlela eziningi zocwaningo zinganambiki.Kamuva, ubuchwepheshe be-PCR busetshenziswa kabanzi ocwaningweni lwesayensi yebhayoloji nasekusetshenzisweni komtholampilo, buba ubuchwepheshe obubaluleke kakhulu ocwaningweni lwebhayoloji yamangqamuzana.UMulis uphinde wawina uMklomelo KaNobel kuChemistry wango-1993.

I-PCRIsimiso

Umgomo oyisisekelo wobuchwepheshe be-PCR ufana nenqubo yemvelo yokuphindaphinda kwe-DNA, futhi ukucaciswa kwayo kuncike ku-oligonucleotide primer ehambisana nazo zombili iziphetho zokulandelana okuqondiwe.I-PCR yakhiwe ukuwohloka-annealing-ukunweba izinyathelo ezintathu eziyisisekelo zokusabela: ①Ukuwohloka kwesifanekiso se-DNA: Ngemva kokuthi isifanekiso se-DNA sishisiswe sibe cishe ku-93°C isikhathi esithile, isixazululo se-DNA embaxambili se-dual-chain DNA esakhiwe yi-PCR yokukhulisa isifanekiso se-DNA Ephumayo, ukuze silungiselele ukusabela okukodwa okulandelayo.②I-annealing (inhlanganisela) yesifanekiso se-DNA kanye ne-primer: Ngemva kokuthi isifanekiso se-DNA sishisisiwe futhi sonakala saba iketango elilodwa, izinga lokushisa liyehla lifinyelele cishe ku-55°C.Ukulandelana okuhambisanayo kwe-primer kanye nesifanekiso se-DNA single-chain.③Ukunwetshwa kwe-primer: Isifanekiso se-DNA-isibopho sokuqala sisekelwe esenzweni se-TaqDNA polymerase, nge-dNTP njengokusabela kwempahla eluhlaza.Gcina umgomo wokuphindaphinda, hlanganisa uchungechunge olusha lwekhophi egodliwe kancane oluhambisana nochungechunge lwe-DNA yesifanekiso, futhi ukuphinda umjikelezo wokuwohloka-annealing-extension izinqubo ezintathu zingathola "uchungechunge lwamakhophi abekelwe eceleni", futhi lolu chungechunge olusha luyatholakala futhi Iba isifanekiso somjikelezo olandelayo.Kuthatha i-2-4min ukuqedela iluphu, isakhi sofuzo esiqondiwe singakhuliswa izikhathi eziyizigidi ezimbalwa emahoreni angu-2-3.

OkujwayelekileI-PCRIsistimu yokusabela

Izinto ezinhlanu zokusabela kwe-PCR

Kukhona ikakhulukazi izinhlobo ezinhlanu zezinto ezibandakanyekayo ekuphenduleni kwe-PCR, okuyi-primer, i-enzyme, i-dNTP, isifanekiso kanye ne-buffer (i-Mg2+ iyadingeka).[Inqubo ye-PCR]

Inqubo ye-PCR ejwayelekile ihlukaniswe ngezinyathelo ezintathu

1. Ukonakala kwe-DNA (90°C-96°C): Izifanekiso ze-DNA ezinochungechunge olukabili ngaphansi kwesenzo esishisayo, i-hydrogen bond break, yakhe i-DNA yeketango elilodwa.

2. I-Annealing (25℃ -65℃): Izinga lokushisa lesistimu liyancishiswa, i-primer ihlanganiswa nesifanekiso se-DNA ukuze kwakheke i-dual-chain yendawo.

3. Isandiso (70℃ -75℃): Ngaphansi kwesenzo se-enzyme ye-Taq (cishe 72°C, umsebenzi ongcono kakhulu), i-dNTP isetshenziswa njengempahla eluhlaza, inwebe kusukela ekupheleni kuka-5′ kwe-primer → 3′ ekupheleni, ukuhlanganisa kanye nesifanekiso kuhambisana neketango le-DNA.

Umjikelezo ngamunye uyakhishwa, uyanwetshwa futhi uyanwetshwa, okuphindwe kabili okuqukethwe kwe-DNA.Njengamanje, ngenxa yendawo emfushane yokukhulisa, enye i-PCR ingaphindaphindwa ngesikhathi esifushane kakhulu ngisho noma umsebenzi we-enzyme ye-Taq ungalungile, ngakho-ke ingashintshwa ibe yizinyathelo ezimbili, okungukuthi, ukukhishwa kwe-annealing nokwandiswa kungenziwa ku-60 ° C-65 ° C ngesikhathi esifanayo.Ukuze unciphise inqubo yokuphakamisa nokupholisa futhi uthuthukise isivinini sokuphendula.

Izici ze-PCR Reaction

● Ukucacisa Okuphezulu

Izinto ezinqumayo eziqondile zempendulo ye-PCR yilezi: ①Inhlanganisela ethile ye-primer kanye ne-DNA yesifanekiso.②Umgomo wokumatanisa okuyisisekelo.③Ukwethembeka kokusabela kwe-TaqDNA polymerase synthesis.④Ukucaciswa nokugcinwa kofuzo oluqondiwe.

Inhlanganisela efanele yeziqalo nezifanekiso ingukhiye.Ukubophezelwa kwe-primer nesifanekiso kanye nokunwetshwa kochungechunge lwe-primer kusekelwe kumgomo wokumatanisa kwesisekelo se-alkali.Ukwethembeka kokusabela kwe-polymerase synthesis kanye nokumelana nezinga lokushisa eliphezulu kwe-Taq DNA polymerase ukwenza ukubopha (inhlanganisela) yesifanekiso kanye ne-primer ekuphenduleni kungenziwa ezingeni lokushisa eliphezulu.Ukucaciswa kwenhlanganisela kuyanda kakhulu.Isiqeshana singagcina izinga eliphezulu lokulunga.Ngokukhetha isifunda sofuzo esiqondiwe esinokulondolozwa okuphezulu kanye nokulondolozwa okuphezulu, ukucaciswa kwayo kuphakeme.

● Ukuzwela Okuphezulu

Ivolumu yokukhiqiza yemikhiqizo ye-PCR inyuswa ngenkomba, enganweba ithempulethi yokuqala ye-Picker (PG=10-12) ukukhulisa izinga le-microcontroller ezingeni lama-micrograms (μg= -6).Amaseli okuhlosiwe angatholwa kumaseli ayisigidi esingu-1;ekutholeni amagciwane, ukuzwela kwe-PCR kungafinyelela ku-3 RFUs (amayunithi angenalutho akhiwe amayunithi);isilinganiso esincane sokutholwa kusayensi yebhaktheriya amabhaktheriya ama-3.

● Kulula futhi Kuyashesha

I-PCR reflection isebenzisa i-Polymerase ye-Taq DNA ephezulu yokushisa, enezela isisombululo sokusabela ngesikhathi esisodwa, okungukuthi, ukusabela kokuwohloka kwe-anneal-extension kusixazululo se-DNA amplification kanye nebhodwe lokugeza amanzi.Ngokuvamile, ukusabela kwe-amplification kuqedwa emahoreni angu-2 kuya kwangu-4.Imikhiqizo engathandwa kwabathelisi esikubona ngokuvamile ihlaziywa ngenkemba kagesi, futhi akudingeki ukuthi isebenzise ama-isotopes, akukho ukungcoliswa kwemisebe, nokukhuthazwa okulula.

● Ukuhlanzeka kwesifanekiso kuphansi

Asikho isidingo sokuhlukanisa amagciwane noma amabhaktheriya namaseli amasiko.Imikhiqizo engahluziwe ye-DNA kanye ne-RNA ingasetshenziswa njengama-amplifiers.Ukutholwa kokukhulisa i-DNA kungasetshenziswa ngokuqondile kusetshenziswa izibonelo zomtholampilo ezifana negazi, uketshezi lomzimba, uketshezi lokugeza ukukhwehlela, izinwele, amaseli, nezicubu eziphilayo.

I-PCRizinkinga ezivamile

● I-negethivu yamanga, awekho amabhendi akhulisiwe

Izigaba ezibalulekile zokusabela kwe-PCR zifaka: ① ukulungiswa kwesifanekiso se-nucleic acid, ② ikhwalithi nokucaciswa kweziqalo, ③ ikhwalithi yama-enzyme ④ izimo zomjikelezo we-PCR.Ukuthola isizathu nakho kufanele kuhlaziywe futhi kufundwe ngezixhumanisi ezingenhla.

Izifanekiso: ① Isifanekiso siqukethe iphrotheni ehlukahlukene, ② Isifanekiso siqukethe i-Taq enzyme inhibitor, ③ Iphrotheni kusifanekiso ayikhishiwe, ikakhulukazi iphrotheni yeqembu kuchromosome.⑤ Ukuwohloka kwe-nucleic acid ye-deminer akuphelele.Uma ikhwalithi yama-enzyme kanye ne-primers iyinhle, alikho ibhande lokukhulisa, okungenzeka kakhulu liyindlela yokwelapha yokugaya yezibonelo.Kukhona okungalungile ngenqubo yokukhipha i-nucleic acid yesifanekiso, ngakho-ke ukulungiselela isisombululo sokugaya esisebenzayo nesizinzile, inqubo yaso kufanele ilungiswe futhi ingashintshwa ngokungenasisekelo.

Ukungasebenzi kwe-enzyme: i-enzayimu entsha noma womabili ama-enzyme amadala namasha kufanele asetshenziswe ndawonye ukuze kuhlaziywe ukuthi umsebenzi we-enzayimu ulahlekile noma awenele, okuholela ekungangeni kahle.Kufanele kuqashelwe ukuthi i-Taq enzyme noma i-ethidium bromide ngezinye izikhathi iyakhohlwa.

I-Primer: ikhwalithi ye-primer, ukugxila kwe-primer, kanye nokuthi ukugxila kwama-primers amabili ku-symmetrical.Kuyisizathu esivamile sokwehluleka kwe-PCR noma ibhendi ekhulayo ayilungile futhi ijwayele ukusabalala.Kunezinkinga ngekhwalithi yeziqalo zezinombolo zenqwaba.Ama-primers amabili ane-concentration ephezulu kanye ne-concentration ephansi, okubangela ukuthuthukiswa okuphansi kwe-asymmetric.Izinyathelo zokuphikisa yilezi: ① Khetha isiqalo esihle ukuze uhlanganise amayunithi.② Ukugxila kwe-primer akuncikile kuphela enanini le-OD, kodwa futhi kunaka uketshezi lwangempela lwe-primer ukwenza i-agar sugar gel electrophoresis.Kumelwe kube khona indawo ye-primer strip, futhi ukukhanya kweziqalo ezimbili kufanele kuhambisane ngokujwayelekile.Ibhande, i-PCR ingase yehluleke ngalesi sikhathi, futhi kufanele ixazululwe ngeyunithi yokuqala yokuhlanganisa.Uma i-primer iphakeme, ukukhanya kuphansi, futhi ukugxila kwayo kufanele kulinganiswe lapho kuhlanjululwe.③ I-primer kufanele ikhokhwe futhi igcinwe ekugxilweni okuphezulu ukuvimbela izingxenye eziningi zesiqandisi ezibandayo noma zesikhathi eside zesiqandisi, okuzokwenza ukuthi isiqandisi siwohloke futhi siwohloke.④ Idizayini ye-primer ayinangqondo, njengokuthi ubude be-primer abanele, futhi i-di cluster yakheka phakathi kwama-primer.

I-Mg2+concentration: I-Mg2+ion concentration inomthelela omkhulu ekusebenzeni kahle kokukhulisa i-PCR.Ukugxilisa ingqondo ngokweqile kunganciphisa ubulili obuhlukile bokukhulisa i-PCR.Uma ukugxilisa ingqondo kuphansi kakhulu, okukhiphayo kokukhulisa i-PCR kuzokwenza ngisho nokwehluleka kokukhulisa i-PCR ngaphandle kwebhendi yokunweba.

Ukushintsha kwevolumu yokusabela: Ivolumu esetshenziswa ekukhuliseni i-PCR ngu-20ul, 30ul, kanye no-50ul noma 100uL, umthamo omkhulu wesicelo sokukhulisa i-PCR usethwe ngokwezinjongo ezahlukene zocwaningo lwesayensi nokuhlolwa komtholampilo.Ngemuva kokwenza imiqulu emincane efana ne-20ul, kuyadingeka ukwenza isimo sentambo lapho wenza usayizi, kungenjalo uzohluleka.

Izizathu ezibonakalayo: Ukuguqulwa kubaluleke kakhulu ekukhuliseni i-PCR.Uma izinga lokushisa lokuwohloka liphansi, isikhathi sokuwohloka sifushane, kungenzeka ukuthi kwenzeke ezingezinhlelweni zamanga;Izinga lokushisa eliphansi kakhulu le-annealing lingabangela ukukhulisa okungaqondile futhi kunciphise ukusebenza kahle kokukhulisa i-amplification.Kuthinta kakhulu inhlanganisela yeziqalisi nezifanekiso ukuze kwehliswe ukusebenza kahle kokukhulisa i-PCR.Ngezinye izikhathi kuyadingeka ukusebenzisa ama-thermometers ajwayelekile ukuze kutholwe ukuguquguquka, ukulinganisa kanye nokushisa okunwetshiwe ku-extension noma umpheki oncibilikayo wamanzi, okungesinye sezizathu zokwehluleka kwe-PCR.

Okuhlukile kokulandelana okuqondiwe: Uma ukulandelana okuqondiwe kwenzeka, ukuguqulwa noma ukususwa, inhlanganisela yesibonelo nesifanekiso kuhlanganisiwe, noma ngenxa yokuntuleka kokulandelana okuqondiwe, isiqalo nesifanekiso kuzolahlekelwa ukulandelana okuhambisanayo, futhi ukukhuliswa kwayo kwe-PCR ngeke kuphumelele.

● Okungelona iqiniso

Ibhendi yokukhulisa i-PCR ibonakala ihambisana nebhendi yokulandelana okuqondiwe, futhi ngezinye izikhathi ibhendi yayo iba ngobunono nangaphezulu.

Idizayini yokuqala ayilungile: ukulandelana kokukhulisa okukhethiwe kanye nokulandelana kokukhulisa okungezona inhloso kune-homologous, ngakho-ke lapho i-PCR ikhuliswa, imikhiqizo ye-PCR ekhulisiwe iwukulandelana okungenanjongo.Ukulandelana okuqondiwe kufushane kakhulu noma i-primer imfushane kakhulu, futhi ithambekele ekubeni nephozithivu engamanga.Idinga ukuklanywa kabusha.

Ukungcoliswa okuphambanayo kokulandelana kwethagethi noma imikhiqizo yokukhulisa amandla: Kunezizathu ezimbili zalokhu kungcola: Esokuqala, ukungcoliswa okunqamula i-genome yonke noma amasegimenti amakhulu, okuholela emiphumeleni engamanga.Lolu hlobo lwephothizithi engamanga lungaxazululwa ngezindlela ezilandelayo: Qaphela futhi ube mnene ngesikhathi sokusebenza ukuze uvimbele ukulandelana okuhlosiwe ukuthi kungahogeli kusibhamu esiyisampula noma kuphume ishubhu elimaphakathi.Ngaphandle kwama-enzyme nezinto ezingakwazi ukumelana namazinga okushisa aphezulu, wonke ama-reagents noma izinto zokusebenza kufanele zibulawe amagciwane ngokucindezela okukhulu.Amapayipi we-centrifugal namasampuli kufanele asetshenziswe ngesikhathi esisodwa.Uma kunesidingo, ngaphambi kokwengeza ama-specimens, ishubhu lokusabela kanye ne-reagent ivezwa emisebeni ye-ultraviolet ukubhubhisa i-nucleic acid ekhona.Okwesibili, izingcezu ezincane ekungcoleni komoya.Lezi zingcezu ezincane zifushane kunokulandelana okuqondiwe, kodwa zine-homology ethile.Ingahlanganiswa nomunye nomunye.Ngemuva kokugcwalisa ama-primers, umkhiqizo we-PCR unganwetshwa, okuzodala ukukhiqizwa okuhle okungamanga.Ingasetshenziswa ukunciphisa noma ukuqeda indlela ye-PCR yesidleke.

● Ibhendi yokukhulisa engacacisiwe

Amabhendi avele ngemva kokukhulisa i-PCR awahambisani nosayizi olindelekile, noma omkhulu noma omncane, noma ngesikhathi esifanayo, noma ngesikhathi esifanayo, amabhendi athile okukhulisa izwi namabhande angacacisiwe wokukhulisa umsindo.Ukuvela kwamabhendi angacacisiwe ukuthi: Okokuqala, ama-primers awaphelele ahambisana nokulandelana okuqondiwe, noma i-polymerization ye-primer ukuze kwakhe i-di cluster.Okwesibili ukuthi ukugcwala kwama-MG2+ion kuphezulu kakhulu, izinga lokushisa le-annealing liphansi kakhulu, futhi inani lemijikelezo ye-PCR lihlobene.Okwesibili, izinga kanye nenani lama-enzyme.Imvamisa, ama-enzyme eminye imithombo athambekele kumabhande angakhethekile futhi ama-enzyme womunye umthombo awekho.Kwesinye isikhathi ukukhuliswa okungaqondile kwama-enzyme nakho kwenzeka.Izinyathelo zokulwa yilezi: ezikhangayo eziklanywe kabusha uma kunesidingo.Yehlisa inani le-enzyme noma shintsha i-enzyme yomunye umthombo.Yehlisa inani lesisekelo, khulisa inani lezifanekiso ngokufanelekile, futhi unciphise inani lemijikelezo.Khulisa ngendlela efanele izinga lokushisa lokufafaza noma sebenzisa indlela yamaphuzu okushisa amabili (93°C degeneration, annealing and extended at 65°C).

● I-tow engabonakali noma i-smear tape

Ukukhulisa i-PCR kwesinye isikhathi kubonakala kusetshenziswa noma igobolondo noma ibhande elifana nekhaphethi.Ngenxa yesizathu, ngenxa yenani eliningi lama-enzyme noma izinga eliphansi le-enzyme, ukugxila kwe-dNTP kuphezulu kakhulu, ukugxila kwe-Mg2+ kuphezulu kakhulu, izinga lokushisa le-anneal liphansi kakhulu, futhi inani lemijikelezo liningi kakhulu.Izinyathelo zokulwa yilezi: ①Yehlisa inani lama-enzyme, noma ushintshe i-enzyme yomunye umthombo.②Yehlisa ukugxila kwe-dNTP ③Yehlisa kahle ukugxilisa kwe-Mg2+.④Khulisa ubuningi bezifanekiso futhi unciphise inani lemijikelezo.

Imikhiqizo Ehlobene

I-PCR Heroᵀᴹ (With Dye)

◮ Ukwethembeka okuphezulu: izikhathi ezingu-6 kune-enzyme ye-Taq evamile;

◮ Isivinini sokukhulisa esisheshayo

◮ Ukuvumelana nezimo kwesifanekiso

◮ Ukusebenza kahle kwe-amplification ephakeme

◮ Ukubekezelela imvelo kunamandla: kubekwe ku-37°C isonto lonke, kugcina umsebenzi ongaphezu kuka-90%;

◮ Inomsebenzi ongu-5'→3' we-DNA polymerase kanye nomsebenzi we-exonuclease ongu-5'→3', ngaphandle komsebenzi we-exonuclease ongu-3'→5'.

I-PCR Easyᵀᴹ (Ngedayi)

Isistimu yokusabela eyingqayizivele kanye nokusebenza kahle okuphezulu kwe-Taq DNA Polymerase kwenza ukusabela kwe-PCR kube nokusebenza kahle kokukhulisa ukuphakama, ukucacisa nokuzwela.

RT-qI-PCR Easyᵀᴹ (Isinyathelo esisodwa)-SYBR Green I

◮ Ikhithi yesinyathelo esisodwa yenza ukuloba okuhlanekezelwe kanye ne-qPCR ukusabela okubili kushubhu efanayo, idinga kuphela ukwengeza isifanekiso se-RNA, ama-primers e-PCR athile kanye ne-RNase-Free ddH₂O.

◮ Ikhithi ingakwazi ukuhlaziya ngokushesha nangempumelelo i-RNA yegciwane noma ilandelele i-RNA.

◮ Ikhithi isebenzisa i-Foregene reverse transcription reagent eyingqayizivele kanye ne-Foregene HotStar Taq DNA Polymerase ehlanganiswe nesistimu yokusabela ehlukile ukuze kuthuthukiswe ngempumelelo ukusebenza kahle kokukhulisa nokucaciswa kokusabela.

◮ Isistimu yokusabela ethuthukisiwe yenza ukusabela kube nokuzwela kokutholwa okuphezulu, ukuzinza okuqinile kokushisa, nokubekezelelana okungcono.

◮ RT-qPCR Easy^TM(One Step) -SYBR Green I kit iza nodayi wereferensi wangaphakathi we-ROX, ongasetshenziswa ukuqeda ingemuva lesignali namaphutha esignali phakathi kwemithombo, okulula ukuthi amakhasimende ayisebenzise kumamodeli ahlukene wamathuluzi e-PCR amaningi.

I-RT Easy^TMII (I-Master Premix ye i-first-strand cDNA synthesis forI-PCR yesikhathi sangempela)

-Ikhono elisebenzayo lokususa i-gDNA, engasusa i-gDNA kusifanekiso phakathi nemizuzu emi-2.

-Isistimu yokubhala ehlehlayo esebenza kahle, kuthatha imizuzu eyi-15 kuphela ukuqeda ukuhlanganiswa kwe-cDNA yomucu wokuqala.

-Izifanekiso eziyinkimbinkimbi: izifanekiso ezinokuqukethwe okuphezulu kwe-GC nesakhiwo sesibili esiyinkimbinkimbi nazo zingahlehliswa ngokusebenza kahle okuphezulu.

-I-High-sensitivity reverse transcription system, izifanekiso zezinga le-pg nazo zingathola i-cDNA yekhwalithi ephezulu.

-Isistimu yokubhala ngokuhlehlayo inokuqina okuphezulu kwe-thermal, izinga lokushisa elilungile lokusabela ngu-42℃, futhi isenokusebenza okuhle kokulotshiweyo okuhlehlayo kokuthi 50℃.