Ukuzalwa kwe-PCR
I-PCR (i-Polymerase Chain Reaction)
Sekuyiminyaka engaphezu kwengu-30 kwasungulwa i-polymerase chain reaction.Eminyakeni engaphezu kwengu-30, ngemva kokuba osolwazi abaningi emhlabeni wonke beqhubeka nokwengeza nokwenza ngcono, ubuchwepheshe be-PCR buye baba indlela yocwaningo esetshenziswa kabanzi nevame ukusetshenziswa futhi ebaluleke kakhulu kuwo wonke umkhakha we-Life Sciences.
I-TouchDown PCR, i-Real-Time PCR, i-Multi PCR, njll. ethuthukiswe ngesisekelo sokusetshenziswa okubanzi kobuchwepheshe be-PCR bendabuko, kanye ne-Digital PCR esanda kuvela (i-PCR yedijithali), izicebise kakhulu izindlela zocwaningo zabacwaningi besayensi abaningi futhi yasheshisa kakhulu inqubo yokuthuthukiswa kweSayensi Yezokuphila yesimanje, ikakhulukazi i-molecular biology, ibe neqhaza elikhulu ekufundiseni isintu kanye nemvelo.
Amaphutha obuchwepheshe be-PCR bendabuko
Ukuhlukaniswa kwe-nucleic acid eyinkimbinkimbi kanyeukukhishwa:
★ Ubuchwepheshe be-PCR bendabuko: buyadingeka
★ PCR etholakala ubuchwepheshe: edingekayo
★ Amasampula e-DNA ne-RNA: umehluko omkhulu, izidingo zokusebenza ezinzima
★ Izingozi zomzimba: ama-reagents anobuthi alimaza umzimba
Ubuchwepheshe be-PCR bendabuko kanye nobuchwepheshe bokuphuma bunokwehlukaniswa nokuhlanzwa kwe-prerequisite-nucleic acid.
Noma iyiphi isampula yebhayoloji idinga ukudlula ochungechungeni lokucubungula isampula eyinkimbinkimbi futhi eyisicefe ukuze kutholwe amasampula e-nucleic acid ahlangabezana nezidingo zobuchwepheshe be-PCR.
Ukuhlukaniswa nokukhishwa kwe-DNA ne-RNA bekulokhu kuwumsebenzi oyisisekelo abacwaningi besayensi abafanelekile okudingeka bawuphindaphinde nsuku zonke.
Ngenxa yomehluko omkhulu phakathi kwamasampula, izinqubo zokuhlukanisa kanye nokukhipha i-DNA ne-RNA nazo zihluke kakhulu.Lo msebenzi udinga izinga eliphezulu lobuchwepheshe bokusebenza kubaqhubi.Izindlela zokuhlukanisa ezingokwesiko nezindlela zokukhipha zidinga ukuthintana isikhathi eside namanye ama-reagents amakhemikhali anobuthi obukhulu.Izodala umonakalo ongenakulungiseka emzimbeni womsebenzisi, futhi idale nomonakalo oqondile phakathi nokuhlolwa.
Ngesikhathi esifanayo, kulabo abanenani elikhulu lamasampula okutadisha, ukuhlukaniswa nokukhipha ama-nucleic acid kuwumsebenzi onzima kakhulu.
Ukuhlukaniswa kwe-Nucleic acid kanye namakhithi okukhipha emakethe manje asevuthiwe futhi kunezinhlobo eziningi zemikhiqizo, kodwa ziyafana.Kungakhathaliseki ukuthi ikhithi yekholomu ye-silica gel membrane centrifugal noma ikhithi yendlela yobuhlalu kazibuthe, kuthatha isikhathi esiningi futhi kuyabiza.Ngaphezu kwezindleko zekhithi, kunezidingo ezikhethekile zemishini yaselabhorethri.Indawo yokusebenza esetshenziswayo esetshenziswa ngendlela yobuhlalu kazibuthe iyisisetshenziswa sezinga elikhulu lenani eliphezulu, okuyizindleko ezinkulu zaselabhorethri.
Ngokufigqiwe
Ngaphambi kokwenza izivivinyo ze-PCR, ukuphathwa kusengaphambili kwamasampula kuyinto engenakugwemeka futhi ehlala ibuhlungu kubacwaningi.Ixazululwa kanjani le nkinga nokuthi ukuhlolwa kwe-PCR kungenziwa yini ngaphandle kokuhlukaniswa nokukhipha ama-nucleic acid bekulokhu kungukucabanga kweningi labacwaningi besayensi kanye nabasebenzi baselabhorethri yomtholampilo.
Isixazululo se-Foregene
Ngemva kweminyaka eminingi yocwaningo olubuhlungu ngobuchwepheshe be-Direct PCR namakhithi ahlobene, i-Forgene yadabula ngempumelelo amabhodlela amaningi futhi yazuza ngempumelelo i-PCR eqondile yezinhlobo eziningi zamasampula anokumelana okuqinile nokuzivumelanisa nezimo, okuvumela abacwaningi ukuba basuse ukuhlukana okunzima nokuyingozi nokukhipha ama-nucleic acid.Lokhu kuzonciphisa kakhulu izinga lomsebenzi wawo wonke umuntu, kusheshise inqubo yokuhlola, futhi konge izindleko zocwaningo lwesayensi nezindleko zokuhlola.
Ukuqonda kukaForgene nolwazi lwe-DirectPCR
Okokuqala, ubuchwepheshe be-DirectPCR ubuchwepheshe be-PCR obuqondile bezicubu ezihlukahlukene zesampula yezinto eziphilayo.Ngaphansi kwalesi simo sobuchwepheshe, asikho isidingo sokuhlukanisa nokukhipha ama-nucleic acid, futhi isampula yethishu isetshenziswa ngokuqondile njengento, futhi izakhi zofuzo ezihlosiwe zengezwe ekuphenduleni kwe-PCR.
Okwesibili, ubuchwepheshe be-DirectPCR akubona nje ubuchwepheshe bendabuko bokukhulisa isifanekiso se-DNA, kodwa futhi buhlanganisa i-RNA template reverse transcription PCR.
Okwesithathu, ubuchwepheshe be-DirectPCR abenzi kuphela ngokuqondile ukusabela kwekhwalithi ye-PCR kumasampula ezicubu, kodwa futhi buhlanganisa ukusabela kwe-QPCR Kwesikhathi Sangempela, okudinga isistimu yokusabela ukuthi ibe nekhono eliqinile lokumelana nokuphazanyiswa kwe-fluorescence yangemuva kanye nokuphikisa izicishi ze-endogenous fluorescence.
Okwesine, amasampula ezicubu ezihloswe ubuchwepheshe be-DirectPCR adinga kuphela ukukhululwa kwezifanekiso ze-nucleic acid futhi angasusi amaprotheni, ama-polysaccharides, ama-ion kasawoti, njll. aphazamisa ukusabela kwe-PCR.Lokhu kudinga i-nucleic acid polymerase kanye ne-PCR Mix ohlelweni lokusabela ukuze kube nokuhle kakhulu kokungaguquki nokuzivumelanisa nezimo, futhi kungaqinisekisa umsebenzi we-enzyme nokunemba kokuphindaphinda ngaphansi kwezimo eziyinkimbinkimbi.
Okwesihlanu, amasampula ezicubu eziqondiswe ubuchwepheshe be-DirectPCR awazange aphathwe noma yikuphi ukwelashwa okunothisa i-nucleic acid, futhi ubuningi besifanekiso buncane kakhulu, obudinga ukuzwela okuphezulu kakhulu kanye nokusebenza kahle kokukhulisa uhlelo lokusabela.
Isiphetho
Ubuchwepheshe be-DirectPCR bungenye yentuthuko ebaluleke kakhulu yezobuchwepheshe kanye nezinto ezintsha eminyakeni engama-30 edlule kusukela kwazalwa ubuchwepheshe be-PCR.UForgene une futhi usazoqhubeka nokuba yingqalabutho kanye nomsunguli walobu buchwepheshe.
Ithemba lokusetshenziswa kobuchwepheshe be-DirectPCR libanzi kakhulu.Ukuthuthukiswa okuqhubekayo nokukhuthazwa kwalobu buchwepheshe kuzoletha izinguquko eziphazamisayo ocwaningweni lwesayensi nomsebenzi wokuhlola.Lokhu ukuguquguquka kobuchwepheshe be-PCR.
Isikhathi sokuthumela: Feb-21-2017
