Yiyiphi i-PCR enokuyenza kunye ne-DNA Ukulinganisa kunye neGenesis
Impendulo ye-polymerase chain ( PCR ) yindlela yokuvelisa i-molecular molecule ukwenza iikopi ezininzi ze-gene kwaye nayo iyinxalenye yenkqubo yokulandelana kwemfuza.
I-Polymerase Yenza Usebenze Kangakanani?
Iikopi ze-Gene zenziwe ngokusebenzisa isampuli ye-DNA, kwaye iteknoloji yanele ngokwaneleyo ukwenza iikopi ezininzi kwiikopi enye yefayile efunyenwe kwisampuli. Ukuphakanyiswa kwe-PCR yesigidi ukwenza izigidi zeekopi, kuvumela ukufumanisa kunye nokuchongwa kwemigangatho yokusebenzisa izixhobo ezisetyenzisiweyo esekelwe kubukhulu kunye nokuhlawula (+ okanye-) kwesiqwenga seDNA.
Ngaphansi kweemeko ezilawulwayo, amacandelo amancinci e-DNA aveliswa ngama-enzyme eyaziwa ngokuthi yi-DNA polymerases, eyongeza i-deoxynucleotides (dNTPs) eyongeziweyo kwisiqwenga se-DNA ebizwa ngokuba "itemplate." Iingcongolo ezincinci zeDNA, ezibizwa ngokuthi "i-primers" zisetyenziselwa ukuqala kwipolmerase. Iziqhumiso zincinci ze-DNA (ii-oligomers) ezincinci ezenziwe ngumntu, ngokuqhelekileyo ziphakathi kwe-15 ne-30 nucleotides ende. Zenziwa ngokwazi okanye ukuqwalasela ukulandelelana kwe-DNA efutshane kwiiphelo zomgca. Ngethuba le-PCR, i-DNA ilandelelana kwaye iyatshitshiswa ngokuphindaphindiweyo. Emva kokupholisa, izibophelelo zibophelwa kwi-template (ebizwa ngokuthi i-annealing) kwaye yakha indawo yokuba i-polymerase iqale.
I-PCR Technique
I-polymerase chain reaction (PCR) eyenziwe ngokufumaneka kwe-thermophiles kunye ne-thermophilic enzyme enzyme (i-enzymes egcina ubuqotho kunye nokusebenza emva kokufudumala kumaqondo aphezulu).
Amanyathelo abandakanyekayo kwinkqubo ye-PCR yile ndlela ilandelayo:
- Umxube uyadalwa, kunye neendawo ezicwangcisiweyo zeethemplate ze-DNA, i-polymerase enzyme, i-primers, kunye ne-DNTPs. Ukukwazi ukutshisa umxube ngaphandle kokutyhila i-enzyme kuvumela ukutyunjwa kwe-helix kabini ye-DNA isampuli kumaqondo athile kuma-degree degrees Celsius.
- Ukulandela i-denaturation, isampuli ikhuhlile kwibanga elingqameneyo, malunga no-54 degrees, oluququzelela ukuhlenga (ukubopha) kwembalo kwiimodeli ze-DNA esisodwa.
- Kwisinyathelo sesithathu somjikelezo, isampuli ihlaziywa ukuya kuma-degrees angama-72, iqondo lokushisa elifanelekileyo likaTaq DNA Polymerase, ukuze ilandelwe. Ngexesha elide, i-DNA polymerase isebenzisa i-DNA enye ye-DNA njengetemplate yokongeza i-DNTPs eyongezelelweyo kwimida yesi-3 ye-primer nganye kwaye ivelise icandelo le-DNA ephindwe kabini kwimimandla yomdla.
- Izibongo eziye zathintela kwi-DNA yokulandelelana ezingahambelani ngokufanayo azihlali zihluthwe kuma-degrees angama-72, ngaloo ndlela zinciphisa i-gene to interest.
Le nkqubo yokuchasisa, ukunqanda kunye nokuxilonga iphindwaphindwa rhoqo (30-40) amaxesha, ngokwandisa ngokukhawuleza inani leekopi zomnqweno oyifunayo kumxube. Nangona le nkqubo iya kuba yinto enzima xa yenza ngesandla, iisampuli zinokulungiswa kwaye zifakwe kwi-Thermocycler ehleliweyo, ngoku iindawo eziqhelekileyo kwii-laboratories ezininzi, kunye nokuphendula okupheleleyo kwe-PCR kungenziwa ngeeyure eziyi-3-4.
Inyathelo ngalinye lokutyhila liyeka inkqubo yokuguguzisa umjikelezo wangaphambili, ngaleyo ndlela igqithise i-DNA entsha ye-DNA kwaye igcine ibe malunga nobukhulu be-gene efunwayo.
Ubungakanani bomjikelezo we-elongation bungenziwa ixesha elifutshane okanye elifutshane ngokuxhomekeka kubukhulu bomthwalo wemithwalo, kodwa ekugqibeleni, ngokujikeleza ngokuphindaphindiweyo kwe-PCR, uninzi lweetemplates luya kugcinwa kwinqanaba lomthwalo wenzalo kuphela, njengoko ziya kuveliswa kwiimveliso zombini zezinto zokuqala.
Kukho iinkalo ezahlukeneyo ze-PCR ephumelelayo enokusetyenziswa ukuze kuphuculwe iziphumo. Indlela esetyenziswa ngayo kakhulu ukuvavanya ubukho be-PCR ye- agarose gel electrophoresis . Eyasetyenziselwa ukwahlula iinqununu ze-DNA ngokusekelwe kubukhulu kunye nentlawulo. Ezi ziqhekeza ziboniswa ngetekisi okanye ii-radiosotopes.
Ukuguquka kwemvelo
Ukususela ekufumaneni i-PCR, i-DNA polymerases ngaphandle kwe-Taq yasekuqaleni ifunyenwe. Ezinye zazo zilunge "ubuchule bokuphonononga" okanye zizinzile kwiindawo ezifudumeleyo, ngoku kuphucula uqobo lwe-PCR kunye nokunciphisa amaphutha ukususela kwi-DNTP engalunganga.
Ezinye iintlobo ze-PCR zenzelwe izicelo ezithile kwaye zisetyenziselwa rhoqo kwiibhutyuli zemizimba yamathambo. Ezinye zezi zi-PC-Real-Time PCR kunye ne-Reverse-Transcriptase PCR. Ukufumanisa kwe-PCR kukhokelela ekuphuhliseni ukulandelelana kwe-DNA, i- DNA yeminwe kunye nezinye iindlela ze-molecular.