Izici zokuphazamiseka ekuphenduleni kwe-PCR

Ngesikhathi sokusabela kwe-PCR, ezinye izici eziphazamisayo zivame ukuhlangana nazo.
Ngenxa yokuzwela okuphezulu kakhulu kwe-PCR, ukungcoliswa kuthathwa njengesinye sezici ezibaluleke kakhulu ezithinta imiphumela ye-PCR futhi kungaveza imiphumela emihle engamanga.
Okubucayi ngokulinganayo imithombo ehlukahlukene eholela emiphumeleni engemihle engamanga. Uma ingxenye eyodwa noma eziningi ezibalulekile zengxube ye-PCR noma ukusabela kokukhulisa ngokwako kuvinjelwe noma kuphazamisekile, ukuhlolwa kokuxilonga kungavinjelwa. Lokhu kungaholela ekunciphiseni ukusebenza kahle ngisho nemiphumela engemihle engamanga.
Ngaphezu kokuvinjelwa, ukulahlekelwa kobuqotho be-nucleic acid ehlosiwe kungase kwenzeke ngenxa yezimo zokuthutha kanye/noma zokugcina ngaphambi kokulungiswa kwesampula. Ikakhulukazi, amazinga okushisa aphezulu noma ukugcinwa okwanele kungaholela ekulimaleni kwamaseli nama-nucleic acid. Ukulungiswa kwamaseli nezicubu kanye nokushumeka upharafini yizimbangela ezaziwayo zokuhlukana kwe-DNA kanye nenkinga eqhubekayo (bheka Umfanekiso 1 no-2). Kulezi zimo, ngisho nokuhlukaniswa okuphelele nokuhlanzwa ngeke kusize.

Umfanekiso 1 | Umthelela we-immobilization kubuqotho be-DNA
I-Agarose gel electrophoresis ibonise ukuthi ikhwalithi ye-DNA ehlukanisiwe nezingxenye zikapharafini zokuhlolwa kwezidumbu zahluka kakhulu. I-DNA yesilinganiso esihlukile sobude besiqephu yayikhona kokukhishiwe kuye ngendlela yokulungisa. I-DNA yalondolozwa kuphela lapho igxilile kumasampuli omdabu aqandisiwe kanye naku-formalin evikelwe emaphakathi. Ukusetshenziswa kwe-Bouin ene-asidi eqinile noma engavinjelwe, i-formalin ene-asidi equkethe i-formalin kubangele ukulahlekelwa okukhulu kwe-DNA. Ingxenye esele ihlukaniswe kakhulu.
Kwesokunxele, ubude bezingcezwana zivezwa ngamapheya e-kilobase (kbp)

Umfanekiso 2 | Ukulahlekelwa ubuqotho bezinhloso ze-nucleic acid
(a) Igebe elingu-3′-5′ kuyo yomibili imicu lizophumela ekuqhekekeni kwe-DNA eqondiwe. ukwakheka kwe-DNA kusazokwenzeka ocezwini oluncane. Kodwa-ke, uma isizinda sokuhlanganisa i-primer singekho esiqeshini se-DNA, ukukhulisa umugqa kuphela okwenzekayo. Esimeni esivumayo kakhulu, izingcezu zingase zigcwale kabusha enye nenye, kodwa isivuno sizoba sincane futhi sibe ngaphansi kwamazinga okutholwa.
(b) Ukulahleka kwezisekelo, ikakhulukazi ngenxa ye-depurination kanye nokwakheka kwe-thymidine dimer, kuholela ekwehleni kwenani lamabhondi e-H kanye nokuncipha kwe-Tm. Phakathi nesigaba sokufudumala esinde, ama-primers azoncibilika asuke ku-matrix DNA futhi ngeke adonse ngisho nangaphansi kwezimo ezicindezela kancane.
(c) Izisekelo ze-thymine eziseduze zakha i-TT dimer.
Enye inkinga evamile evame ukwenzeka ekuxilongweni kwamangqamuzana ukukhululwa okuncane kunokwanele kwama-nucleic acid aqondiwe uma kuqhathaniswa nokukhipha i-phenol-chloroform. Ezimweni ezimbi kakhulu, lokhu kungahlotshaniswa nokubi okungamanga. Isikhathi esiningi singalondolozwa ngokubilisa i-lysis noma ukugaywa kwe-enzymatic kwemfucumfucu yamangqamuzana, kodwa le ndlela ngokuvamile iholela ekuzweleni okuphansi kwe-PCR ngenxa yokukhululwa kwe-nucleic acid enganele.

Ukuvinjelwa komsebenzi we-polymerase ngesikhathi sokukhulisa

Ngokuvamile, ukuvimbela kusetshenziswa njengomqondo wesiqukathi ukuchaza zonke izici eziholela emiphumeleni ye-PCR ephansi. Ngomqondo oqinile we-biochemical, ukuvimbela kunqunyelwe ekusebenzeni kwe-enzyme, okungukuthi, kunciphisa noma kuvimbela ukuguqulwa kwe-substrate-product ngokusebenzisana nesizinda esisebenzayo se-DNA polymerase noma i-cofactor yayo (isb, i-Mg2+ ye-Taq DNA polymerase).
Izingxenye zesampula noma amabhafa ahlukahlukene kanye nezingcaphuno eziqukethe ama-reagents zingavimbela ngokuqondile i-enzyme noma zicuphe ama-cofactors ayo (isb. i-EDTA), ngaleyo ndlela yenze i-polymerase isebenze bese kuholela emiphumeleni enciphile noma engemihle ye-PCR engamanga.
Kodwa-ke, ukusebenzisana okuningi phakathi kwezingxenye zokusabela kanye nama-nucleic acid aqukethe ithagethi kuphinde kuqokwe njenge-'PCR inhibitors'. Uma ubuqotho beseli buphazanyiswa ukuhlukaniswa futhi i-nucleic acid ikhishwa, ukusebenzisana phakathi kwesampula nesisombululo sayo esizungezile kanye nesigaba esiqinile singenzeka. Isibonelo, 'abadobi' bangabopha i-DNA enemicu eyodwa noma kabili ngokusebenzisana okungahlangani futhi baphazamise ukuhlukaniswa nokuhlanza ngokunciphisa inani lezinto ezihlosiwe ezigcina zifinyelele umkhumbi wokusabela we-PCR.
Ngokuvamile, ama-PCR inhibitors akhona emanzini amaningi omzimba kanye nama-reagents asetshenziselwa ukuhlolwa kokuxilonga (i-urea emchameni, i-hemoglobin ne-heparin egazini), izithako zokudla (izingxenye eziphilayo, i-glycogen, amafutha, i-Ca2+ ions) kanye nezingxenye zemvelo (phenols , izinsimbi ezisindayo)

Ama-PCR inhibitors avame kakhulu angatholakala kuma-bacterium namaseli e-eukaryotic, i-DNA engahlosiwe, ama-macromolecules abopha i-DNA kamatikuletsheni wezicubu kanye nemishini yaselabhorethri efana namagilavu ​​namapulasitiki. Ukuhlanzwa kwama-nucleic acid ngesikhathi noma ngemva kokukhipha kuyindlela ekhethwayo yokukhipha ama-PCR inhibitors.
Namuhla, okokusebenza okuhlukile okuzikhiphayo okuzenzakalelayo kungangena esikhundleni sezivumelwano eziningi ezenziwa ngesandla, kodwa ukutakula okungu-100% kanye/noma ukucwenga okuhlosiwe akukaze kufinyelelwe. Ama-inhibitor angaba khona kungenzeka ukuthi asesekhona kuma-nucleic acid ahlanzekile noma kungenzeka ukuthi aseqalile ukusebenza. Kukhona amasu ahlukene okunciphisa umthelela wama-inhibitors. Ukukhethwa kwe-polymerase efanelekile kungaba nomthelela omkhulu emsebenzini we-inhibitor. Ezinye izindlela ezifakazelwe zokunciphisa ukuvinjelwa kwe-PCR ukukhulisa ukugxila kwe-polymerase noma ukusebenzisa izithasiselo ezifana ne-BSA.
Ukuvinjelwa kokusabela kwe-PCR kungaboniswa ngokusebenzisa ukulawulwa kwekhwalithi yangaphakathi (IPC).
Ukunakekelwa kufanele kuthathwe ukuze kukhishwe wonke ama-reagents nezinye izixazululo kukhithi yokukhipha, njenge-ethanol, i-EDTA, i-CETAB, i-LiCl, i-GuSCN, i-SDS, i-isopropanol ne-phenol, kusukela ku-nucleic acid ihlukaniswe ngesinyathelo sokugeza ngokuphelele. Ngokuya ngokugxila kwabo, bangase basebenze noma bavimbele i-PCR.