Izici zokuphazamiseka ekuphenduleni kwe-PCR

Ngesikhathi sokusabela kwe-PCR, ezinye izici eziphazamisayo zivame ukuhlangana nazo.
Ngenxa yokuzwela okuphezulu kakhulu kwe-PCR, ukungcola kubhekwa njengenye yezinto ezibaluleke kakhulu ezithinta imiphumela ye-PCR futhi kungaveza imiphumela emibi engamanga.
Okubaluleke ngokulinganayo yimithombo ehlukahlukene eholela emiphumeleni engemihle neyiphutha. Uma ingxenye eyodwa noma ngaphezulu ebalulekile yengxube ye-PCR noma ukusabela kokukhulisa ngokwako kuvinjelwa noma kuphazanyiswa, ukuhlolwa kokuxilonga kungavinjelwa. Lokhu kungaholela ekunciphiseni ukusebenza kahle ngisho nemiphumela engemihle neyiphutha.
Ngaphezu kokuvinjelwa, ukulahlekelwa ubuqotho be-nucleic acid eqondiwe kungenzeka ngenxa yezimo zokuthunyelwa kanye/noma zokugcina ngaphambi kokulungiswa kwesampula. Ikakhulukazi, amazinga okushisa aphezulu noma isitoreji esinganele kungaholela ekulimaleni kwamaseli nama-nucleic acid. Ukuqina kwamaseli nezicubu kanye nokushumeka kwe-parafini kuyizimbangela ezaziwayo zokuqhekeka kwe-DNA kanye nenkinga eqhubekayo (bheka Izibalo 1 no-2). Kulezi zimo, ngisho nokuhlukaniswa kanye nokuhlanzwa okufanele ngeke kusize.

Umfanekiso 1 | Umphumela wokungashukumi ekuthembekeni kwe-DNA
I-Agarose gel electrophoresis ibonise ukuthi ikhwalithi ye-DNA ehlukanisiwe nezingxenye ze-paraffin zokuhlolwa kwezidumbu yayihluka kakhulu. I-DNA yobude obuhlukene bezingcezu ezimaphakathi yayikhona ezikhishweni kuye ngendlela yokulungisa. I-DNA igcinwe kuphela uma iqinisiwe kumasampula eqandisiwe endawo kanye naku-buffered neutral formalin. Ukusetshenziswa kwe-formalin ene-acid enamandla noma engahlanganisiwe, equkethe i-formalin equkethe i-formalin kwaholela ekulahlekelweni okukhulu kwe-DNA. Ingxenye esele ihlukanisiwe kakhulu.
Ngakwesobunxele, ubude bezingcezu buvezwa ngama-kilobase pairs (kbp)

Umfanekiso 2 | Ukulahlekelwa ubuqotho bezinhloso ze-nucleic acid
(a) Igebe elingu-3′-5′ kuzo zombili izintambo lizoholela ekuqhekekeni kwe-DNA eqondiwe. Ukwenziwa kwe-DNA kusazokwenzeka engxenyeni encane. Kodwa-ke, uma indawo yokuqala yokudonsa ingekho engxenyeni ye-DNA, kuvele ukukhuliswa okuqondile kuphela. Esimweni esihle kakhulu, izingcezu zingase zigcwale kabusha, kodwa isivuno sizoba sincane futhi singaphansi kwamazinga okutholwa.
(b) Ukulahlekelwa yizisekelo, ikakhulukazi ngenxa yokususwa kokungcola kanye nokwakheka kwe-thymidine dimer, kuholela ekunciphiseni inani lama-H-bond kanye nokwehla kwe-Tm. Ngesikhathi sesigaba sokufudumala eside, ama-primer azoncibilika ku-matrix DNA futhi ngeke ancibilike ngisho nangaphansi kwezimo ezinzima kakhulu.
(c) Izisekelo ze-thymine eziseduze zakha i-TT dimer.
Enye inkinga evamile evame ukwenzeka ekuxilongweni kwama-molecule ukukhishwa okungaphansi kokufanele kwama-nucleic acid aqondiwe uma kuqhathaniswa nokukhishwa kwe-phenol-chloroform. Ezimweni ezimbi kakhulu, lokhu kungahlotshaniswa ne-false negative. Isikhathi esiningi singalondolozwa ngokubilisa i-lysis noma ukugaya ama-enzyme ama-debris amaseli, kodwa le ndlela ivame ukuphumela ekuzweleni okuphansi kwe-PCR ngenxa yokukhululwa okunganele kwe-nucleic acid.

Ukuvinjelwa komsebenzi we-polymerase ngesikhathi sokukhulisa

Ngokuvamile, ukuvimbela kusetshenziswa njengomqondo wesitsha ukuchaza zonke izici eziholela emiphumeleni ye-PCR engaphansi kwe-efficient. Ngomqondo we-biochemical kuphela, ukuvimbela kunqunyelwe emsebenzini we-enzyme, okungukuthi, kunciphisa noma kuvimbela ukuguqulwa komkhiqizo we-substrate ngokusebenzisana nendawo esebenzayo ye-DNA polymerase noma i-cofactor yayo (isb., i-Mg2+ ye-Taq DNA polymerase).
Izingxenye ezikusampula noma kuma-buffer ahlukahlukene kanye neziqephu eziqukethe ama-reagent zingavimbela ngqo i-enzyme noma zibambe ama-cofactor ayo (isb. i-EDTA), ngaleyo ndlela zivimbele i-polymerase futhi okuholela emiphumeleni ye-PCR engemihle noma engamanga.
Kodwa-ke, ukusebenzisana okuningi phakathi kwezingxenye zokusabela kanye nama-nucleic acid aqukethe okuqondiwe nakho kubizwa ngokuthi 'ama-PCR inhibitors'. Uma ubuqotho beseli buphazanyiswa ukuhlukaniswa futhi i-nucleic acid ikhishwe, ukusebenzisana phakathi kwesampula kanye nesisombululo sayo esizungezile kanye nesigaba esiqinile kungenzeka. Isibonelo, 'abahlaseli' bangabopha i-DNA eyodwa noma ephindwe kabili ngokusebenzisana okungekhona kwe-covalent futhi baphazamise ukuhlukaniswa kanye nokuhlanzwa ngokunciphisa inani lama-target agcina efinyelele emkhunjini wokusabela we-PCR.
Ngokuvamile, ama-PCR inhibitors akhona oketshezini oluningi lomzimba kanye nama-reagents asetshenziselwa ukuhlolwa kokuxilonga kwezokwelapha (i-urea emchameni, i-hemoglobin kanye ne-heparin egazini), izithasiselo zokudla (izingxenye eziphilayo, i-glycogen, amafutha, ama-ion e-Ca2+) kanye nezingxenye ezisendaweni ezungezile (ama-phenol, izinsimbi ezisindayo)

Ama-PCR inhibitors avame kakhulu angatholakala kuma-bacteria namaseli e-eukaryotic, i-DNA engeyona i-target, ama-macromolecules ahlanganisa i-DNA yama-tissue matrices kanye nemishini yelabhorethri efana namagilavu ​​​​kanye nepulasitiki. Ukuhlanzwa kwama-nucleic acid ngesikhathi noma ngemuva kokukhishwa kuyindlela ekhethwayo yokususa ama-PCR inhibitors.
Namuhla, imishini ehlukahlukene yokukhipha ezenzakalelayo ingathatha indawo yezinqubo eziningi ezenziwe ngesandla, kodwa ukubuyiselwa kanye/noma ukuhlanzwa kwezinhloso eziyi-100% akukaze kufezwe. Izithibi ezingaba khona zingase zibe khona kuma-nucleic acid ahlanziwe noma kungenzeka ukuthi sezivele zisebenza. Kukhona amasu ahlukene okunciphisa umthelela wezithibi. Ukukhetha i-polymerase efanele kungaba nomthelela omkhulu emsebenzini wezithibi. Ezinye izindlela eziqinisekisiwe zokunciphisa ukuvinjelwa kwe-PCR ukwandisa ukuhlushwa kwe-polymerase noma ukusebenzisa izithasiselo ezifana ne-BSA.
Ukuvinjelwa kokusabela kwe-PCR kungabonakaliswa ngokusebenzisa ukulawulwa kwekhwalithi yenqubo yangaphakathi (i-IPC).
Kumelwe kuqashelwe ukususa wonke ama-reagents nezinye izixazululo kukhithi yokukhipha, njenge-ethanol, i-EDTA, i-CETAB, i-LiCl, i-GuSCN, i-SDS, i-isopropanol kanye ne-phenol, kusuka ku-nucleic acid isolate ngesinyathelo sokuwasha ngokuphelele. Kuye ngokuthi zigxile kangakanani, zingase zisebenze noma zivimbele i-PCR.