Singakanani Isifanekiso Okufanele Sengeze Ekuphenduleni Kwami kwe-PCR?

Ngisho noma ngokombono, i-molecule eyodwa yesifanekiso izokwanela, amanani amakhulu kakhulu e-DNA ngokuvamile asetshenziselwa i-PCR yakudala, isibonelo, kufika ku-1 µg we-DNA yezilwane ezincelisayo ezincelisayo kanye nengxenye encane efinyelela ku-1 pg ye-plasmid DNA. Inani eliphelele lincike kakhulu enanini lamakhophi ochungechunge oluqondiwe, kanye nokuba yinkimbinkimbi kwalo.

Uma isifanekiso esincane kakhulu sisetshenziswa, ukwanda okuhambisanayo kwenani lemijikelezo yokukhulisa izwi kuyodingeka ukuze kutholwe inani elanele lomkhiqizo. I-Taq polymerase esetshenziselwa ukuhlola okuningi kwe-PCR ayinawo umsebenzi wokulungisa (3′-5′ umsebenzi we-exonuclease); ngakho-ke, amaphutha enzeka ngesikhathi sokukhulisa awakwazi ukulungiswa. Lapho inani lemijikelezo liphezulu, kuzokwanda ukwanda komkhiqizo onephutha. Uma, ngakolunye uhlangothi, inani lesifanekiso liphezulu kakhulu, amathuba okuba ama-primer axhunywe kokunye (hhayi amaphesenti ayikhulu okuncoma) ukulandelana, kanye nokwakhiwa kwama-primer dimers, azokhula, okuzoholela ekwandiseni ngemikhiqizo. Ezimweni eziningi, i-DNA ihlukanisiwe kumasiko amaseli noma kuma-microorganisms bese isetshenziswa njengesifanekiso se-PCR. Ukulandela ukuhlanzwa, kuyadingeka ukunquma ukugxila kwe-DNA ukuze ukwazi ukuchaza ivolumu edingekayo ekusethweni kwe-PCR. Nakuba i-agarose gel electrophoresis ingase inikeze isilinganiso, le ndlela ayinembile neze. I-spectrophotometry ye-UV-Vis isungulwe njengezinga legolide lokulinganisa ama-nucleic acid; le ndlela eqondile futhi ngakho-ke elula futhi esheshayo ikala ukumuncwa kwesampula ku-260 nm, futhi ukugxila kubalwa ngosizo lwesici sokuguqula.

Uma ukugxila kwe-DNA kuphansi kakhulu, nokho (< 1 µg/mL dsDNA), noma uma kungcoliswe izinto ezimunca futhi ku-260 nm range (isb. i-RNA, amaprotheni, usawoti), le ndlela izofinyelela ukulinganiselwa kwayo. Endabeni yokugxilisa okuphansi kakhulu, ukufundwa kuzobe kunganembile kakhulu ukuthi kungasetshenziswa, futhi ukungcoliswa kuzoholela (ngezinye izikhathi kukhulu) ekulinganisweni ngokweqile kwevelu yangempela. Kulokhu, ukulinganisa kwe-fluorescence kungase kubonise enye indlela. Le nqubo isuselwe ekusetshenzisweni kodayi we-fluorescent obophezela ngokuqondile ku-dsDNA kuphela inkimbinkimbi ehlanganisa i-nucleic acid nodayi ujatshuliswa ukukhanya, futhi ngemva kwalokho izokhipha ukukhanya kobude beza obuphakeme kancane. Lapha, ukushuba kwesignali ye-fluorescent ilingana nenani le-DNA, futhi ukuze kunqunywe ukugxilisa ingqondo kuyahlolwa ngokuhlobene nejika elijwayelekile. Izinzuzo zale ndlela zincike ekucacisweni kwebhondi, okungabandakanyi amathonya angaphandle alethwa ukungcola, kanye nasemandleni okuba umphumela wokuthola ukugxila okuphansi kakhulu kwe-DNA. Ukufaneleka kwanoma iyiphi indlela kuncike kakhulu ekugxilweni kwesampula nokuhlanzeka; ezimweni eziningi kungase kube kuhle ukusebenzisa izindlela zombili ngokuhambisana.


Isikhathi sokuthumela: Nov-30-2022