Izici zokuphazamiseka ekuphenduleni kwe-PCR

Ngesikhathi sokuphendula kwe-PCR, ezinye izinto eziphazamisayo zivame ukuhlangana.
Ngenxa yokuzwela okuphezulu kakhulu kwe-PCR, ukungcoliswa kubhekwa njengenye yezinto ezibaluleke kakhulu ezithinta imiphumela ye-PCR futhi kungakhiqiza imiphumela emihle.
Okubucayi ngokulinganayo yimithombo ehlukahlukene eholela emiphumeleni engemihle. Uma izingxenye eyodwa noma eziningi ezibalulekile zengxube ye-PCR noma ukusabela kwe-Amplication uqobo kuyavinjelwa noma kuphazanyiswe, ukuxilonga okutholakalayo kungaphazanyiswa. Lokhu kungaholela ekunciphiseni kahle kanye nemiphumela engemihle engamanga.
Ngaphezu kokuvinjwa, ukulahleka kobuqotho be-nucleic acid kungenzeka ngenxa yokuthumela kanye / noma izimo zokugcina ngaphambi kokulungiswa kwesampula. Ikakhulu, amazinga okushisa aphezulu noma isitoreji esinganele kungaholela ekulimaleni kwamaseli nama-nucleic acid. Ukulungiswa kweseli nokwelapha izicubu kanye nopharafini ukushumeka izimbangela zabantu abaziwayo be-DNA Fgvermation kanye nenkinga ephikelelayo (bheka izibalo 1 no-2). Kulezi zimo, ngisho nokuhlukaniswa kahle nokuhlanza ngeke kusize.

Umdwebo 1 | Umphumela wokungasebenzi kahle kubuqotho be-DNA
I-Agarose Gel Electrophoresis ikhombisile ukuthi ikhwalithi ye-DNA eyenziwe ngezigaba zikaphalafini zama-autopsies ahlukahlukene kakhulu. I-DNA yobude obuhlukile bokuhlukaniswa kwengxenye yayikhona ekukhishweni kuya ngendlela yokulungiswa. I-DNA yagcinwa kuphela lapho ihlelwe kumasampula omdabu aqandisiwe nasekuhlutweni okungathathi hlangothi. Ukusetshenziswa kokulungiswa kwe-acidic acidic acidic noma okungekho emthethweni, okuqukethe i-acid-acid-acid kuholele ekulahlekelweni okukhulu kwe-DNA. Ingxenyana esele ihlukaniswe kakhulu.
Ngakwesobunxele, ubude bezingcezwana buvezwa kumabili ama-Kilobase (KBP)

Umdwebo 2 | Ukulahlekelwa Ubuqotho Bezinkomo ze-nucleic acid
. I-Synthesis ye-DNA isazokwenzeka kwingxenye encane. Kodwa-ke, uma isiza se-Primer Anveling silahlekile kwingxenye ye-DNA, kuvela kuphela ukulandisa okuqondile. Esimweni esihle kunazo zonke, izingcezwana zingabeka kabusha, kepha izivuno zizoba zincane futhi zingaphansi kwamazinga okutholwa.
. Ngesikhathi sesigaba sokufudumala esiphakeme, ama-primers azoncibilika kude ne-matrix DNA futhi ngeke anenzuzo ngisho nangaphansi kwezimo ezinzima.
(c) Izisekelo ze-thymine eziseduze ne-thymine zakha umcibisholo we-tt.
Enye inkinga evamile evame ukwenzeka ekuxilongeni kwama-molecular ukukhishwa okuncane kakhulu kokukhishwa okuphezulu kwama-nucleic acid kuqhathaniswa nokukhishwa kwe-phenol-chloroform. Ezimweni ezimbi kakhulu, lokhu kungahlotshaniswa nokubi kwamanga. Isikhathi esiningi singasindiswa ngokushayela i-lysis ebilayo noma ukugaya kwe-enzymatic of cell debris, kepha le ndlela ivame ukuphumela ekusokeni kwe-PCR okuphansi ngenxa yokukhishwa kwe-nucleic acid okunganele.

Ukuvinjwa komsebenzi we-polymerase ngesikhathi sokukhuliswa

Ngokuvamile, kusetshenziswa ukuvimbela njengomqondo wesitsha ukuchaza zonke izinto eziholela emiphumeleni ye-PCR ye-supopTimal. Ngomqondo oqinile we-biochemical, ukuvinjezelwa kukhawulelwe emsebenzini we-enzyme, okungukuthi, kunciphisa noma kuvimbela ukuguqulwa komkhiqizo okukhona ngokuxhumana nge-DNA DNA Polymerase).
Izakhi kusampula noma ama-buffers ahlukahlukene kanye nokukhishwa okuqukethe ama-reagents kungavimbela ngokuqondile i-enzyme noma i-cofactive yayo (isib.
Kodwa-ke, ukusebenzisana okuningi phakathi kwezakhi zokuphendula kanye nama-nucleic acid aqukethe ama-nucleic nawo aqokwa njenge-'Pcronitors '. Lapho ubuqotho beseli buphazamiseka ngokwahlukaniswa kanye ne-nucleic acid kukhululiwe, ukusebenzisana phakathi kwesampula kanye nesixazululo sayo esiseduze nesigaba esiqinile kungenzeka. Isibonelo, 'ama-scavenger' angabopha i-DNA eyodwa noma ekhuphuke kabili ngokusebenzisana okungewona ama-covanded futhi aphazamise ukwahlukaniswa nokuhlanzwa ngokunciphisa inani lamatshe agcina afika e-PCR reaction Vesel.
Ngokuvamile, ama-PCR inhibitors akhona kumanzi amaningi omzimba kanye nama-reagents asetshenziselwa ukuhlolwa kokuxilonga emtholampilo (urea in herpin egazini), amafutha okudla, amafutha, ama-ca2 tilals) nezakhi ezisendaweni (ama-phenols, izinsimbi ezisindayo)

Ama-PRCED PCRED PCCKER angatholakala kuma-bacterium kanye namaseli e-eukaryotic, i-DNA engahlosiwe, ama-macromoleks e-DNA-ahlanganisa ama-macromoleks ama-tish maticic kanye nemishini yelebhu efana namagilavu ​​namapulasitiki. Ukuhlanzwa kwama-acid ama-nucleic ngesikhathi noma ngemuva kokukhishwa kuyindlela ekhethwayo yokususa ama-PCR inhibitors.
Namuhla, imishini ehlukahlukene yokususa ezenzakalelayo ingangena esikhundleni sezinqubo eziningi zezincwadi, kepha ukululama okungu-100% kanye / noma ukuhlanzwa kwamatshe akuzange kutholakale. Ama-inhibitors okungenzeka angaba khona kuma-nucleic acid ahlanzekile noma kungenzeka ukuthi asevele eqala ukusebenza. Amasu ahlukile akhona ukunciphisa umthelela we-inhibitors. Ukukhethwa kwe-polymerase efanelekile kungaba nomthelela omkhulu emsebenzini woku-inhibitor. Ezinye izindlela ezifakazelwe zokunciphisa i-PCR Vividering zikhulisa ukugxila kwe-polymerase noma ukusebenzisa izengezo ezifana ne-BSA.
Ukuvinjwa kokuphendula kwe-PCR kungakhonjiswa ukusetshenziswa kwenqubo yekhwalithi yangaphakathi (IPC).
Ukunakekelwa kumele kuthathwe ukususa wonke ama-reagents kanye nezinye izixazululo ekhishini lokukhipha, njenge-ethanol, e-Edta, i-CETAB, i-SDS, i-soprop, kusuka ku-nucleic acid ukwahlukanisa ngesinyathelo esigcwele sokugeza. Ngokuya ngokugxila kwabo, bangasebenza noma bavimbele i-PCR.