Ii-enzymes ezininzi eziqhele ukusetyenziswa kwiimvavanyo ze-PCR

Polymerase chain reaction, efinyeziweyo njengeI-PCRngesiNgesi, bubuchule bebhayoloji yemolekyuli esetyenziselwa ukukhulisa amaqhekeza athile eDNA.Inokuthi ithathwe njenge-replication ekhethekileyo ye-DNA ngaphandle komzimba, enokunyusa kakhulu inani elincinci le-DNA.Ngexesha lonkeI-PCRinkqubo yokusabela, iklasi enye yezinto idlala indima ebalulekileyo - enzymes.

1. Taq DNA

Kwimifuniselo ngeentsuku zokuqala zeI-PCR, izazinzulu zasebenzisa i-Escherichia coli DNA polymerase I, Kodwa kukho ingxaki ngale enzyme: kufuneka izalise i-enzyme entsha rhoqo xa umjikelezo wenziwa, okwenza amanyathelo okusebenza abe nzima kwaye kunzima ukukhulisa ngokuzenzekelayo ngokuzenzekelayo.Le ngxaki yasonjululwa emva kokuba izazinzulu zahlula ngengozi i-Taq DNA polymerase kwi-Thermus aquaticus ngo-1988. Ukususela ngoko, ukukhulisa ngokuzenzekelayo kwe-DNA kuye kwaba yinyani.Ukufunyanwa kwale enzyme nako kwenzaI-PCRitekhnoloji itekhnoloji efanelekileyo, esebenzayo kunye neyendalo yonke.Okwangoku, i-Taq DNA polymerase yeyona polymerase ixhaphakileyo kwiikhithi zeDNA.

2. PfuDNA

Njengoko kukhankanyiwe ngasentla, i-Taq DNase ine-bug enkulu, ngoko ke izazinzulu ziye zaguqula i-Taq DNA polymerase ukuya kwinqanaba elithile ukuze kuthintelwe ukukhulisa okungangqalanga ngenxa yokungahambelani, okukhokelela kwiziphumo zovavanyo ezingachanekanga.Kodwa ukuguqulwa kwe-Taq DNA polymerase kunokuthintela umsebenzi we-DNA polymerase kwiqondo lokushisa.I-PfuDNA polymerase inokwenza kakuhle ezi zinto zingalunganga ngasentla ze-Taq DNA polymerase, ukwenzela ukuba i-PCR iphendule ngokuqhelekileyo, kwaye izinga lokuphumelela lokukhulisa i-gene ekujoliswe kuyo linokuphuculwa ngokufanelekileyo.

3. Reverse Transcriptase

I-reverse transcriptase yafunyanwa ngo-1970. Le enzyme isebenzisa i-RNA njenge-template, i-dNTP njenge-substrate, ilandela umgaqo wokubambisana kwesiseko, kwaye idibanise i-DNA enye ye-strand ehambelana ne-RNA template kwindlela ye-5'-3'.I-reverse transcriptase ixhomekeke ngokukodwa kwi-DNA polymerase umsebenzi ovela kwi-DNA okanye i-templates ze-RNA kwaye ngoko ke akukho msebenzi we-3'-5' we-exonuclease.Nangona kunjalo, inomsebenzi we-RNase H, onqanda ubude be-synthesis ye-reverse transcriptase ukuya kumlinganiselo othile.Ngenxa yokuthembeka okuphantsi kunye ne-thermostability ye-wild reverse transcriptase, izazinzulu nazo ziyilungisile.

KubaI-PCRimifuniselo, izinto ezisetyenziswayo eziphambili zezi:ityhubhu yePCR yomntu, ityhubhu ye-PCR eyi-4/8, iipleyiti zePCR.

ILabio'sPCR izinto ezisetyenziswayoyiba noku kulandelayoiingenelo:

Iipleyiti zePCR: Ukuhambelana kwe-thermal cycler;Ukuchasana okuphezulu, ukuchonga kakuhle kakuhle;kakuhle fluorescence reflection;kulungileukuhanjiswa kobushushu;I-DNase eqinisekisiweyo, i-RNase, i-DNA, i-PCR inhibitors, kunye novavanyo olungenayo i-pyrogen.

Iityhubhu zePCR zomntu ngamnye: Ukumelana nokuvela kwamanzi; kulungileukuhanjiswa kobushushu;ukucaca okugqwesileyo kwamehlo; I-DNase eqinisekisiweyo, i-RNase, i-DNA, i-PCR inhibitors, kunye ne-pyrogen evavanyiweyo.

Iityhubhu ze-PCR ze-4/8: Iindonga ezibhityileyo kakhulu;kucaca okuphezulu;ukukhanya okuhle kwe-fluorescence;inokusebenzisa kwishishini lamayeza, ishishini lokutya, kunye nebhayoloji yemolekyuli;umgangatho ophezulu, izinto ezinyulu zePP;I-DNase eqinisekisiweyo, i-RNase, i-DNA, i-PCR inhibitors, kunye ne-pyrogen-free.