Traditional reverse transcriptases ha e khone ho mamella mocheso o phahameng (mocheso o nepahetseng bakeng sa ts'ebetso ea MMLV ke 37-50 ° C, 'me AMV ke 42-60 ° C).RNA e rarahaneng haholo ea kokoana-hloko e ke ke ea fetoleloa ka mokhoa o nepahetseng ho cDNA ka mocheso o tlase, e leng se bakang katleho ea ho lemoha Phokotso.Traditional RT-qPCR ka kakaretso e hloka ho kenya letsoho ha li-enzyme tse peli tsa bohlokoa (reverse transcriptase le DNA polymerase), e leng se etsang hore ho se khonehe ho nolofatsa ts'ebetso ea sistimi ea karabelo le ho fokotsa litšenyehelo.Mona re tla hlahisa lingoloa tse peli tse hanyetsanang le mocheso o phahameng, TT le RevTaq.Li-enzyme tsena tse peli le tsona li na le mosebetsi oa DNA polymerase, kahoo li bitsoa li-enzyme tse sa sebetseng hantle.

TH DNA polymerase

E tlameha ebe u utloile ka TH, e nkiloeng ho baktheria ea thermophilic Thermus thermophilus HB8.Ka pel'a li-cations tsa divalent tse kang Mg2 +, e na le mosebetsi oa DNA polymerase.E sebelisoa haholo liketsong tsa PCR joalo ka Taq enzyme, empa e na le khanyetso e phahameng ea mocheso ho feta Taq enzyme, ka hona e na le phello e ntle ho PCR e nang le litempele tse phahameng tsa GC.

· Enzyme ena ha e na tšebetso ea 3′→5′ exonuclease le 5′→3′ exonuclease, kahoo e ka sebelisoa hape bakeng sa tatellano ea didooxy.

Enzyme ena e na le ts'ebetso ea RTase.Ka pel'a Mn2+, ts'ebetso ea RTase e tla ntlafatsoa.U sebelisa tšobotsi ena, e ka sebelisoa ho etsa reverse transcript reaction le PCR ka har'a tube e le 'ngoe, ke hore, mohato o le mong oa RT-PCR.Leha ho le joalo, ka pel'a Mn2 +, ho nepahala ha RT-PCR ha ho phahameng.Ts'ebetso ea RT ha e amane le ts'ebetso ea rnaase H.

· Ts'ebetso e ntseng e eketseha ea Tth-DNA polymerase (pH9, optimum +55℃～+70℃, boholo +95℃) e hlola mathata a bakoang ke sebopeho sa bobeli sa RNA.CDNA e hlahisoang e ka holisoa ke PCR ka enzyme e tšoanang ka pel'a li-ion tsa Mg2+.

· Bokhoni ba Tth-DNA polymerase ea ho etsa transcript le DNA amplification ka mocheso o phahameng o etsa hore enzyme ena e be molemo bakeng sa tlhahlobo ea RT-PCR, cloning, le gene expression ea RNA ea cellular le vaerase.
· Tth-DNA polymerase e sebelisetsoa RT-PCR ho holisa RNA ho fihla ho 1kb.

Likarolo le melemo

Tth DNA polymerase:

• Netefatsa boholo ba sehlahisoa sa polymerase chain reaction (PCR), bonyane 1000 bp ho RT-PCR

• Amohela deoxyribonucleoside triphosphate e fetotsoeng e le substrate

• Ha e amane le mosebetsi oa RNase H

• E na le botsitso bo phahameng ba mocheso ho hlōla mathata, hangata a amanang le sebopeho se phahameng sa bobeli se teng ho RNA

RevTaq RT-PCR DNA polymerase

DNA polymerase e thibelang mocheso e nang le ts'ebetso ea reverse transcriptase

RevTaq-RT-PCR-DNA polymerase ke enzyme e entsoeng ka boenjiniere, e thibelang mocheso haholo, e sebetsang habeli e nang le reverse transcriptase le mesebetsi ea DNA polymerase e fumanoang ka ho iphetola ha lintho ka tataiso le ka maiketsetso.

· Halofo ea bophelo ea RevTaq RT-PCR DNA polymerase ka 95°C e kholo ho feta metsotso e 40.

· RevTaq RT-PCR DNA polymerase e lumella ho ngoloa ha mocheso o phahameng ka ho toba ho tloha template ea RNA, 'me mohato oa khatiso o ka morao o ka phetoa ka makhetlo a mangata ho hlahisa li-template tse ngata tsa cDNA.

· RevTaq RT-PCR DNA polymerase e lumella "zero-step" RT-PCR (ha ho na isothermal reverse transcript step), hobane mohatong oa katoloso ea PCR ea cyclic, transcript le DNA amplification li etsahala ka nako e le 'ngoe.Sena se boetse se ts'ehetsa maikutlo a morao-rao a transcript lithempereichareng tse phahameng, ka tsela eo, ho fokotsa mathata a hlahang ha ho qhibiliha sebopeho se matla sa RNA ka mocheso o phahameng.

· Ka lebaka la mokhoa oa ho qala o chesang o thehiloeng ho aptamer, RevTaq RT-PCR DNA polymerase e tla hlahisa liphetho tse betere ha mocheso oa anneal le katoloso o phahame ho feta 57°C.

· Kaha enzyme e hanela mocheso, ho kgothaletswa ho etsa li-primers le probes tse nang le lintlha tse phahameng haholo tsa ho qhibiliha (> 60 ° C).

· E kgothaletswa ho optimize mocheso oa annealing / katoloso mohato ka mocheso gradient nakong ea karabelo seta tshebetso.

· Ha mocheso o phahame, ho ba le boemo bo phahameng ba PCR.Phetolelo ea morao-rao ea transcript hangata e etsoa ka mocheso o phahameng ho feta oa PCR, hobane DNA Primer:RNA Template hybridization hangata e na le ntlha e phahameng ea ho qhibiliha ho feta DNA Primer:cDNA Template duplex.

· RevTaq RT-PCR DNA polymerase e entsoe ka liphatsa tsa lefutso le ho ntlafatsoa, ​​'me boholo ba amplicon bo pakeng tsa 60-300 bp.

RevTaq RT-PCR DNA polymerase moeli oa ho lemoha o fihla likopi tse 4 /

Sistimi e ntlafalitsoeng ea karabelo (ho theha li-primers tse phahameng tsa ho qhibiliha) e bonts'oa setšoantšong.RevTaq RT-PCR DNA polymerase-driven RT-PCR e bonts'a kutlo e betere ho feta TaqPath 1-mohato o 1 oa motsoako o moholo oa RT-qPCR, le ho lemoha ho tlase Mohlala oa dilution gradient.

Melemo e meng:

Mosebetsi oa ho qala ka potlako → Mohato oa pele oa ho fetola mocheso o ka fetisoa.

Ho qala ka mokhoa o chesang oa aptamer formula → Fana ka ts'ebetso ea 100% ea enzyme hang-hang 'me u thibele ho holisa ka mokhoa o sa tobang mochesong o tlase (<57°C).

Ts'ebetso ea Cleavage → Mohato oa ho ntša RNA ha oa qheleloa ka thoko, hobane RevTaq RT-PCR DNA polymerase le eona e ka sebetsana le lisampole tsa karabelo e mpe.E ka senya hang-hang li-membrane tsa lisele tsa eukaryote, libaktheria le livaerase ka potoloho e chesang ea RT-PCR.

Boemo ba thepa e tala ea IVD → maemo a boleng bo holimo le litheko tse hlolisang haholo