Ib txwm rov qab transcriptases tsis tuaj yeem tiv taus qhov kub thiab txias (qhov kub zoo rau kev ua haujlwm MMLV yog 37-50 ° C, thiab AMV yog 42-60 ° C).Cov kab mob sib kis ntau dua RNA tsis tuaj yeem thim rov qab tau zoo rau hauv cDNA ntawm qhov kub thiab txias, ua rau kev tshawb pom kev ua haujlwm txo qis.Ib txwm siv RT-qPCR feem ntau xav tau kev koom tes ntawm ob lub enzymes tseem ceeb (rov qab transcriptase thiab DNA polymerase), uas ua rau nws tsis yooj yim rau kev ua haujlwm ntawm cov tshuaj tiv thaiv kab mob thiab txo tus nqi.Ntawm no peb yuav qhia ob lub siab kub-tiv thaiv rov qab transcriptases, TtH thiab RevTaq.Ob lub enzymes no kuj muaj cov haujlwm ntawm DNA polymerase, yog li lawv hu ua bifunctional enzymes.

TTH DNA polymerase

Koj yuav tsum tau hnov ​​​​txog TtH, uas yog muab los ntawm cov kab mob thermophilic Thermus thermophilus HB8.Nyob rau hauv lub xub ntiag ntawm divalent cations xws li Mg2 +, nws muaj DNA polymerase kev ua.Nws tau dav siv hauv PCR cov tshuaj tiv thaiv zoo li Taq enzyme, tab sis nws muaj kev kub ntxhov ntau dua li Taq enzyme, yog li nws kuj muaj txiaj ntsig zoo rau PCR nrog cov qauv GC siab.

· Cov enzyme no yeej tsis muaj 3′→ 5′ exonuclease kev ua thiab 5′→ 3′ exonuclease kev ua, yog li nws kuj siv tau rau Dideoxy sequencing.

· Cov enzyme no muaj RTase kev ua haujlwm.Hauv lub xub ntiag ntawm Mn2+, RTase kev ua haujlwm yuav raug txhim kho.Siv cov yam ntxwv no, nws tuaj yeem siv los ua cov tshuaj tiv thaiv rov qab thiab PCR cov tshuaj tiv thaiv hauv tib lub raj, uas yog, ib kauj ruam RT-PCR.Txawm li cas los xij, nyob rau hauv lub xub ntiag ntawm Mn2+, qhov tseeb ntawm RT-PCR tsis siab.Kev ua RT tsis muaj dab tsi cuam tshuam nrog rnaase H kev ua.

· Kev ua kom muaj zog ntawm Tth-DNA polymerase (pH9, optimum +55 ℃ ~ + 70 ℃, siab tshaj + 95 ℃) overcomes cov teeb meem tshwm sim los ntawm RNA theem nrab qauv.Cov txiaj ntsig cDNA tuaj yeem nthuav dav los ntawm PCR nrog tib lub enzyme nyob rau hauv muaj Mg2 + ions.

· Lub peev xwm ntawm Tth-DNA polymerase ua rov qab transcription thiab DNA amplification ntawm qhov kub thiab txias ua rau cov enzyme no muaj txiaj ntsig zoo rau ntau yam RT-PCR, cloning, thiab gene qhia tsom xam ntawm cellular thiab viral RNA.
· Tth-DNA polymerase yog siv rau RT-PCR los ua kom RNA mus txog 1kb.

Nta thiab qhov zoo

DNA polymerase:

• Ua kom pom tseeb qhov zoo tshaj plaws polymerase saw cov tshuaj tiv thaiv (PCR) cov khoom loj, tsawg kawg yog 1000 bp hauv RT-PCR cov tshuaj tiv thaiv

• Txais hloov kho deoxyribonucleoside triphosphate ua substrate

• Tsis muaj feem cuam tshuam rau RNase H kev ua

• Muaj thermal stability siab kom kov yeej cov teeb meem, feem ntau muaj feem xyuam nrog cov qauv theem nrab uas muaj nyob hauv RNA

RevTaq RT-PCR DNA polymerase

Lub tshav kub-resistant DNA polymerase nrog rov qab transcriptase kev ua

RevTaq-RT-PCR-DNA polymerase yog ib qho kev tsim kho, kub-tiv taus, dual-functional enzyme nrog thim rov qab transcriptase thiab DNA polymerase cov dej num tau los ntawm kev qhia thiab kev hloov pauv.

· Lub neej ib nrab ntawm RevTaq RT-PCR DNA polymerase ntawm 95 ° C yog ntau dua 40 feeb.

· RevTaq RT-PCR DNA polymerase tso cai rau qhov kub thiab txias rov qab hloov pauv ncaj qha los ntawm RNA template, thiab cov kauj ruam rov qab tuaj yeem rov ua dua ntau zaus los tsim ntau cDNA templates.

· RevTaq RT-PCR DNA polymerase tso cai rau "zero-step" RT-PCR (tsis muaj isothermal reverse transcription step), vim hais tias nyob rau hauv lub cyclic PCR extension kauj ruam, rov qab transcription thiab DNA amplification tshwm sim ib txhij.Qhov no kuj txhawb kev rov qab cov tshuaj tiv thaiv ntawm qhov kub thiab txias, yog li txo cov teeb meem uas tau ntsib hauv melting lub zog theem nrab hauv RNA ntawm qhov kub thiab txias.

· Vim lub aptamer-raws li kub-pib mis, RevTaq RT-PCR DNA polymerase yuav ua tau zoo dua thaum lub annealing thiab extension kub siab tshaj 57 ° C.

· Txij li thaum lub enzyme yog tshav kub-resistant, nws yog pom zoo kom tsim primers thiab soj ntsuam nrog heev melting ntsiab lus (> 60 ° C).

· Nws raug nquahu kom ua kom zoo dua qhov kub ntawm qhov annealing / txuas ntxiv cov kauj ruam los ntawm qhov kub thiab txias thaum lub sij hawm teeb tsa cov tshuaj tiv thaiv.

· Qhov kub thiab txias, qhov siab dua ntawm PCR.Lub voj voog rov qab rov qab feem ntau ua tiav ntawm qhov kub siab dua li lub voj voog PCR, vim tias DNA Primer: RNA Template hybridization feem ntau muaj cov ntsiab lus siab dua li DNA Primer: cDNA Template duplex.

· RevTaq RT-PCR DNA polymerase yog genetic engineered thiab optimized, thiab amplicon loj yog nyob nruab nrab ntawm 60-300 bp.

RevTaq RT-PCR DNA polymerase kuaj pom txwv txog 4 daim ntawv luam /

Qhov zoo tshaj plaws tshuaj tiv thaiv kab mob (kev tsim kho ntawm high melting point primers) yog qhia nyob rau hauv daim duab.RevTaq RT-PCR DNA polymerase-tsav RT-PCR qhia tau hais tias zoo dua rhiab heev dua li TaqPath 1-kauj ruam RT-qPCR master mix, thiab qis dua cov qauv dilution gradient.

Ntau qhov zoo:

Pib ua haujlwm nrawm → Thawj kauj ruam thermal denaturation tuaj yeem hla.

Kub-pib aptamer formula → Muab 100% enzyme kev ua haujlwm tam sim ntawd thiab tiv thaiv tsis muaj qhov tshwj xeeb amplification ntawm qhov kub qis (<57 ° C).

Cleavage muaj nuj nqi → RNA rho tawm cov kauj ruam raug tshem tawm, vim RevTaq RT-PCR DNA polymerase kuj tuaj yeem ua cov tshuaj tiv thaiv tsis zoo.Nws tuaj yeem rhuav tshem cov cell membranes ntawm eukaryotes, cov kab mob thiab cov kab mob hauv lub voj voog kub RT-PCR.

IVD raw khoom qib → cov qauv zoo thiab cov nqi sib tw heev