I-PCR (i-polymerase chain reaction) ingenye ye-in-vitro DNA amplification technologies, enomlando ongaphezu kweminyaka engama-30.

Ubuchwepheshe be-PCR basungulwa ngu-Kary Mullis wase-Cetus, e-USA ngo-1983. U-Mullis wafaka isicelo se-PCR patent ngo-1985 futhi washicilela iphepha lokuqala lezemfundo le-PCR leSayensi ngawo lowo nyaka.UMulis waklonyeliswa ngeNobel Prize kuchemistry ngo-1993 ngomsebenzi wakhe.

Izimiso Eziyisisekelo ze-PCR

I-PCR ingakhulisa izingcezu ze-DNA eziqondiwe izikhathi ezingaphezu kwesigidi esisodwa.Umgomo ungaphansi kwe-catalysis ye-DNA polymerase, kusetshenziswa umucu womzali i-DNA njengesifanekiso kanye ne-primer ethize njengendawo yokuqala yokunwetshwa.Iphindwaphindwa ku-vitro ngezinyathelo ezifana ne-denaturation, annealing, kanye nesandiso.Inqubo ye-DNA strand yendodakazi ehambisana ne-DNA yesifanekiso somucu womzali.

Inqubo ejwayelekile ye-PCR ihlukaniswe ngezinyathelo ezintathu:

1.I-Denaturation: Sebenzisa izinga lokushisa eliphezulu ukuze uhlukanise imicu ephindwe kabili ye-DNA.Isibopho se-hydrogen phakathi kwemicu ephindwe kabili ye-DNA sinqanyulwa kumazinga okushisa aphezulu (93-98℃).

2.Annealing: Ngemva kokuhlukaniswa kwe-DNA enemicu ekabili, yehlisa izinga lokushisa ukuze i-primer ikwazi ukuhlanganisa i-DNA enomucu owodwa.

3.Isandiso: I-DNA polymerase iqala ukuhlanganisa izintambo ezihambisanayo eduze nezintambo ze-DNA ezivela kuma-primers aboshwe lapho izinga lokushisa lehliswa.Lapho isandiso sesiqediwe, umjikelezo uyaqedwa, futhi inani lezingcezu ze-DNA liphinda kabili

Ngokubuyisela lezi zinyathelo ezintathu izikhathi ezingu-25-35, inani lezingcezu ze-DNA lizokhula ngokuphawulekayo.

Ubuhlakani be-PCR ukuthi ama-primer ahlukene angakhelwa izinhlobo ezahlukene zofuzo eziqondiwe, ukuze izingcezu zofuzo eziqondiwe zingakhuliswa ngesikhathi esifushane.

Kuze kube manje, i-PCR ingahlukaniswa ngezigaba ezintathu, okuyi-PCR evamile, i-fluorescent quantitative PCR kanye ne-digital PCR.

Isizukulwane sokuqala se-PCR ejwayelekile

Sebenzisa ithuluzi elivamile lokukhulisa i-PCR ukuze ukhulise isakhi sofuzo esiqondiwe, bese usebenzisa i-agarose gel electrophoresis ukuze uthole umkhiqizo, ukuhlaziya ikhwalithi kuphela okungenziwa.

Okubi okuyinhloko kwe-PCR yesizukulwane sokuqala:

1.Ijwayele ukukhuliswa okungaqondile kanye nemiphumela emihle engamanga.

2.Ukutholwa kuthatha isikhathi eside futhi ukusebenza kunzima.

3.Ukuhlolwa kwekhwalithi kuphela okungenziwa

I-PCR yesikhathi sangempela yesizukulwane sesibili

I-Real-Time PCR, eyaziwa nangokuthi i-qPCR, isebenzisa ama-fluorescent probes angabonisa ukuqhubeka kwesistimu yokusabela, futhi iqaphe ukunqwabelana kwemikhiqizo ekhulisiwe ngokunqwabelana kwamasignali e-fluorescent, futhi yahlulele imiphumela ngejika le-fluorescence.Ingalinganiswa ngosizo lwenani le-Cq kanye nejika elijwayelekile.

Ngenxa yokuthi ubuchwepheshe be-qPCR benziwa ngesistimu evaliwe, amathuba okungcola ayancishiswa, futhi isignali ye-fluorescence ingagadwa ukuze itholwe ubuningi, ngakho isetshenziswa kakhulu emisebenzini yomtholampilo futhi isibe ubuchwepheshe obuvelele ku-PCR.

Izinto ze-fluorescent ezisetshenziswa ku-PCR yesikhathi sangempela somthamo we-fluorescent zingahlukaniswa zibe: I-TaqMan fluorescent probe, amabhakhoni amangqamuzana kanye nodayi we-fluorescent.

1) I-TaqMan fluorescent probe:

Ngesikhathi sokukhulisa i-PCR, i-probe ethize ye-fluorescent yengezwa ngenkathi yengeza ipheya ye-primer.I-probe iyi-oligonucleotide, futhi zombili iziphetho zibhalwe ngeqembu lentatheli ye-fluorescent kanye neqembu le-quencher fluorescent.

Uma i-probe iphelele, isignali ye-fluorescent ekhishwa yiqembu lentatheli idonswa yiqembu lokucisha;ngesikhathi sokukhulisa i-PCR, umsebenzi we-5'-3′ we-exonuclease we-enzyme ye-Taq uklebhuka futhi wehlise i-probe, okwenza iqembu lentatheli ye-fluorescent kanye nesicisha Iqembu le-fluorescent lihlukaniswe, ukuze uhlelo lokuqapha lwe-fluorescence lukwazi ukuthola isignali ye-fluorescence, okungukuthi, ngaso sonke isikhathi lapho i-DNA strand ikhula, ikhuphuka futhi ikhuphuka i-fluorescent isignali ivumelaniswa ngokuphelele nokwakheka komkhiqizo we-PCR.

2) Udayi we-fluorescent we-SYBR:

Kuhlelo lokusabela lwe-PCR, kungezwe udayi we-fluorescent we-SYBR oweqile.Ngemuva kokuthi udayi we-fluorescent we-SYBR ungafakwanga ngokuqondile ku-DNA-double-strand, ukhipha isignali ye-fluorescent.I-molecule ye-SYBR kadayi engafakwanga ochungechungeni ngeke ikhiphe noma iyiphi isignali ye-fluorescent, ngaleyo ndlela iqinisekise isignali ye-fluorescent Ukwanda kwemikhiqizo ye-PCR kuvumelaniswa ngokuphelele nokwanda kwemikhiqizo ye-PCR.I-SYBR ibophezela kuphela ku-DNA enemicu ekabili, ngakho-ke ijika elincibilikayo lingasetshenziswa ukunquma ukuthi ukusabela kwe-PCR kuqondile yini.

3) Ukukhanya kwe-molecule:

Kuyi-stem-loop ebhalwe kabili ye-oligonucleotide probe eyakha isakhiwo se-hairpin esinezisekelo ezingaba ngu-8 emaphethelweni angu-5 no-3.Ukulandelana kwe-nucleic acid emikhawulweni yomibili kubhanqiwe ngokuhambisanayo, okwenza iqembu le-fluorescent kanye neqembu lokucisha kuqine.Vala, akukho fluorescence ezokhiqizwa.

Ngemuva kokuthi umkhiqizo we-PCR ukhiqiziwe, phakathi nenqubo yokuhlanganisa, ingxenye emaphakathi yebhakhoni yemolekyuli ibhangqwa nokulandelana okukhethekile kwe-DNA, futhi isakhi sofuzo se-fluorescent siyahlukaniswa nesakhi sofuzo ukuze sikhiqize i-fluorescence.

Okubi okuyinhloko kwe-PCR yesizukulwane sesibili:

Ukuzwela kusantuleka, futhi ukutholwa kwezibonelo zekhophi ephansi akulungile.

Kukhona umthelela wevelu yangemuva, futhi umphumela usengozini yokuphazamiseka.

Uma kukhona ama-PCR inhibitor ohlelweni lokusabela, imiphumela yokuthola isengozini yokuphazamiseka.

I-PCR yedijithali yesizukulwane sesithathu

I-Digital PCR (I-DigitalPCR, i-dPCR, i-Dig-PCR) ibala inombolo yekhophi yokulandelana okuqondiwe ngokutholwa kwendawo yokugcina, futhi ingathola ukutholwa kobuningi obunembile ngaphandle kokusebenzisa izilawuli zangaphakathi namajika ajwayelekile.

I-Digital PCR isebenzisa ukutholwa kwephoyinti lokugcina futhi ayincikile enanini le-Ct (umkhawulo womjikelezo), ngakho ukusabela kwe-PCR yedijithali akuthintwa kancane ukusebenza kahle kokukhulisa, futhi ukubekezelela i-PCR reaction inhibitors kuyathuthukiswa, ngokunemba okuphezulu nokukhiqiza kabusha.

Ngenxa yezici zokuzwela okuphezulu nokunemba okuphezulu, ayiphazanyiswa kalula yi-PCR reaction inhibitors, futhi ingafinyelela ukulinganisa kwangempela okuphelele ngaphandle kwemikhiqizo ejwayelekile, esiphenduke indawo yocwaningo kanye nesicelo.

Ngokwezinhlobo ezahlukene zeyunithi yokusabela, ingahlukaniswa ngezinhlobo ezintathu eziyinhloko: i-microfluidic, i-chip kanye ne-droplet systems.

1) I-Microfluidic digital PCR, mdPCR:

Ngokusekelwe kubuchwepheshe be-microfluidic, isifanekiso se-DNA siyahlukaniswa.Ubuchwepheshe be-microfluidic bungabona isampula yokuthuthukiswa kwe-nano noma ukukhiqizwa kwamaconsi amancane, kodwa amaconsi adinga indlela ekhethekile yokukhangisa bese ehlanganiswa nohlelo lokusabela lwe-PCR.mdPCR kancane kancane yamukelwe ezinye izindlela esikhundleni.

2) I-PCR yedijithali esekwe eyi-Droplet, i-ddPCR:

Sebenzisa ubuchwepheshe bokukhiqiza amaconsi emanzini ukuze ucubungule isampuli ibe amaconsi, futhi uhlukanise isistimu yokusabela equkethe ama-nucleic acid ama-nucleic amaconsi ezinkulungwane zamaconsi e-nanoscale, ngalinye lawo elingaqukethe i-nucleic acid target molecule ezotholwa, noma Iqukethe i-nucleic acid eyodwa kuya kuma-nucleic ambalwa okumele ahlolwe.

3) I-PCR yedijithali esekwe ku-chip, i-cdPCR:

Sebenzisa ubuchwepheshe obuhlanganisiwe bendlela yoketshezi ukuze uqophe amashubhu amaningi nama-microcavities kumawafa e-silicon noma ingilazi ye-quartz, futhi ulawule ukugeleza kwesixazululo ngamavalvu okulawula ahlukene, futhi uhlukanise uketshezi lwesampula lube ama-nanometers anosayizi ofanayo emithonjeni yokusabela ye-PCR Reaction yedijithali ukuze uzuze ukulinganisa okuphelele.

Okubi okuyinhloko kwe-PCR yesizukulwane sesithathu:

Imishini nama-reagents kuyabiza.

Izidingo zekhwalithi yesifanekiso ziphezulu.Uma ubuningi besifanekiso budlula ubuningi be-microsystem, ngeke kwenzeke ukulinganisa, futhi uma kuncane kakhulu, ukunemba kokulinganisa kuzoncishiswa.

Okuhle okungamanga nakho kungenziwa uma kukhona ukukhuliswa okungaqondile.