Ubuchwepheshe bokuxilongwa kwamangqamuzana busebenzisa izindlela zebhayoloji yamangqamuzana ukuthola ukuvezwa nokwakheka kwezakhi zofuzo zomzimba womuntu kanye namagciwane ahlukahlukene, ukuze kufezwe inhloso yokubikezela nokuxilonga izifo.

Eminyakeni yamuva nje, ngokuthuthukiswa nokuphindaphindwa kobuchwepheshe bokuxilonga amangqamuzana, ukusetshenziswa komtholampilo kokuxilongwa kwamangqamuzana kuye kwanda kakhulu futhi kwajula, futhi imakethe yokuxilonga yamangqamuzana ingene esikhathini sokuthuthuka ngokushesha.

Umbhali ufingqa ubuchwepheshe obujwayelekile bokuxilonga amangqamuzana emakethe, futhi ihlukaniswe izingxenye ezintathu: ingxenye yokuqala yethula ubuchwepheshe be-PCR, ingxenye yesibili yethula ubuchwepheshe be-nucleic acid isothermal amplification, kanti ingxenye yesibili yethula ubuchwepheshe bokulandelana.

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Ingxenye I: PCR Technology

Ubuchwepheshe be-PCR

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

Ubuchwepheshe be-PCR baphayona ngo-1983 nguKary Mullis waseCetus, e-USA.U-Mullis wafaka isicelo selungelo lobunikazi le-PCR ngo-1985 futhi washicilela iphepha lokuqala lezemfundo le-PCR leSayensi ngawo lowo nyaka.UMulis wawina uMklomelo KaNobel kuKhemistry ngo-1993.

Izimiso Eziyisisekelo ze-PCR

I-PCR ingakhulisa izingcezu ze-DNA eziqondiwe izikhathi ezingaphezu kwesigidi esisodwa.Isimiso siwukuthi ngaphansi kwe-catalysis ye-DNA polymerase, i-DNA yomucu womzali isetshenziswa njengesifanekiso, futhi i-primer ethile isetshenziswa 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.Amabhondi e-hydrogen phakathi kwemicu ephindwe kabili ye-DNA aphulwa emazingeni okushisa aphezulu (93-98°C).

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

3. Isandiso: I-DNA polymerase iqala ukuhlanganisa imicu ehambisanayo 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 izakhi zofuzo eziqondiwe ezihlukene, ukuze izingcezu zofuzo eziqondiwe zikhuliswe 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:

-Ithambekele ekukhuliseni okungaqondile kanye nemiphumela emihle engamanga.

-Ukutholwa kuthatha isikhathi eside futhi ukusebenza kunzima.

-Ukuhlolwa kwekhwalithi kuphela okungenziwa.

I-PCR yesizukulwane sesibili se-fluorescence quantitative

I-Fluorescence quantitative PCR (I-PCR Yesikhathi Sangempela), eyaziwa nangokuthi i-qPCR, isetshenziselwa ukuqapha ukuqoqwa kwemikhiqizo ekhulisiwe ngokunqwabelana kwamasignali we-fluorescent ngokungeza ama-fluorescent probes angabonisa ukuqhubeka kwesistimu yokusabela, nokwahlulela imiphumela ngejika le-fluorescence elikwazi ukugoba, futhi Ikwazi ukugoba okujwayelekile.

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 probes, amabhakhoni amangqamuzana kanye nodayi be-fluorescent.

1) I-TaqMan fluorescent probe:

Ngesikhathi sokukhulisa i-PCR, i-probe ethize ye-fluorescent iyengezwa ngenkathi kwengezwa ipheya yeziqalo.I-probe iyi-oligonucleotide, futhi iziphetho ezimbili zilebulwe ngokulandelana kweqembu 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) Odayi be-fluorescent be-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) Izibani zamangqamuzana

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, ngeke ikhiqize i-fluorescence.

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 akunembile.

Kunomthelela wevelu yangemuva, futhi umphumela usengozini 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 kwe-endpoint 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: i-microfluidic, i-chip kanye ne-droplet systems.