Imibhalo yendabuko ehlanekezelwe ayikwazi ukubekezelela izinga lokushisa eliphezulu (izinga lokushisa elilungile lomsebenzi we-MMLV ngu-37-50°C, futhi i-AMV ingu-42-60°C).I-RNA yegciwane eyinkimbinkimbi ayikwazi ukuhlehliswa ngempumelelo ibe yi-cDNA emazingeni okushisa aphansi, okuholela ekusebenzeni kahle kokutholwa Ukuncipha.I-RT-qPCR yendabuko ngokuvamile idinga ukubamba iqhaza kwama-enzyme amabili abalulekile (i-reverse transcriptase ne-DNA polymerase), okwenza kungenzeki ukwenza lula ukusebenza kwesistimu yokusabela nokunciphisa izindleko.Lapha sizokwethula ama-reverse transcriptases amabili amelana nokushisa okuphezulu, i-TTH kanye ne-RevTaq.Lawa ma-enzyme amabili nawo anomsebenzi we-DNA polymerase, ngakho abizwa ngokuthi ama-enzyme angasebenzi.

TH DNA polymerase

Kumelwe ukuba uzwile nge-TtH, etholakala kubhaktheriya ye-thermophilic Thermus thermophilus HB8.Ebukhoneni be-divalent cations njenge-Mg2+, inomsebenzi we-DNA polymerase.Isetshenziswa kakhulu ekuphenduleni kwe-PCR njenge-Taq enzyme, kodwa inokumelana nokushisa okuphezulu kune-enzyme ye-Taq, ngakho iphinde ibe nomthelela ongcono ku-PCR enezifanekiso zokuqukethwe kwe-GC ephezulu.

· Le enzyme ngokuyisisekelo ayinawo u-3′→5′ umsebenzi we-exonuclease kanye nomsebenzi we-5′→3′ we-exonuclease, ngakho ingase isetshenziselwe ukulandelana kwe-dieoxy.

· Le enzyme inomsebenzi we-RTase.Ebukhoneni be-Mn2+, umsebenzi we-RTase uzothuthukiswa.Ngokusebenzisa lesi sici, singasetshenziswa ukwenza ukusabela okuhlanekezelwe kokulotshiweyo nokusabela kwe-PCR kushubhu efanayo, okungukuthi, isinyathelo esisodwa se-RT-PCR.Nokho, phambi kwe-Mn2+, ukunemba kwe-RT-PCR akuphakeme.Umsebenzi we-RT awuhlangene nomsebenzi we-rnaase H.

· Umsebenzi owandisiwe we-Tth-DNA polymerase (pH9, optimum +55℃～+70℃, esiphezulu +95℃) unqoba izinkinga ezibangelwa ukwakheka kwesibili kwe-RNA.I-cDNA ewumphumela ingakhuliswa yi-PCR nge-enzyme efanayo phambi kwama-ion we-Mg2+.

· Ikhono le-Tth-DNA polymerase ukwenza ukuloba okuhlanekezelwe kanye nokukhulisa i-DNA emazingeni okushisa aphezulu lenza le enzyme ibe wusizo ekuhlaziyeni kwe-RT-PCR yobuningi, i-cloning, kanye nokuhlaziywa kofuzo lwe-RNA yeselula negciwane.
· I-Tth-DNA polymerase isetshenziselwa i-RT-PCR ukuze ikhulise i-RNA ifike ku-1kb.

Izici nezinzuzo

I-DNA polymerase:

• Qinisekisa usayizi womkhiqizo we-polymerase chain reaction (PCR), okungenani u-1000 bp ekuphenduleni kwe-RT-PCR

• Yamukela i-deoxyribonucleoside triphosphate eguquliwe njenge-substrate

• Ayihlobene nomsebenzi we-RNase H

• Inokuqina kokushisa okuphezulu ukuze inqobe izinkinga, ngokuvamile ezihlobene nesakhiwo sesibili esiphezulu esikhona ku-RNA

I-RevTaq RT-PCR DNA polymerase

I-DNA polymerase emelana nokushisa enomsebenzi we-reverse transcriptase

I-RevTaq-RT-PCR-DNA polymerase iyi-enzyme eyakhiwe, imelana nokushisa kakhulu, esebenza kabili futhi ene-reverse transcriptase kanye nemisebenzi ye-DNA polymerase etholwe ngokuziphendukela kwemvelo okuqondisiwe nokokwenziwa.

· Ingxenye yempilo ye-RevTaq RT-PCR DNA polymerase ku-95°C inkulu kunemizuzu engama-40.

· I-RevTaq RT-PCR DNA polymerase ivumela ukulotshwa okuhlanekezela okuphezulu kwezinga eliphezulu ngokuqondile kusuka kusifanekiso se-RNA, futhi isinyathelo sokuhlehla sokuloba singaphindwa izikhathi eziningi ukuze kukhiqizwe izifanekiso ze-cDNA ezengeziwe.

· I-RevTaq RT-PCR DNA polymerase ivumela i-RT-PCR “eyizero-step” (asikho isinyathelo sokuloba esihlanekezela esihlanekezelwe yi-isothermal), ngoba esinyathelweni se-PCR esijikelezayo, ukuloba okuhlanekezelwe kanye nokukhulisa i-DNA kwenzeka ngesikhathi esisodwa.Lokhu kuphinda kuthuthukise ukusabela kokuhlehla kokulotshiweyo emazingeni okushisa aphezulu, ngaleyo ndlela kuncishiswe izinkinga okuhlangatshezwane nazo ekuncibilikiseni ukwakheka kwesibili okuqinile ku-RNA emazingeni okushisa aphezulu.

· Ngenxa yefomula esekelwe ku-aptamer-hot-start, i-RevTaq RT-PCR DNA polymerase izokhiqiza imiphumela engcono uma izinga lokushisa le-anneal kanye nesandiso lingaphezu kuka-57°C.

· Njengoba i-enzayimu imelana nokushisa, kuyanconywa ukuba uklame ama-primers nama-probe anamaphuzu aphezulu kakhulu ancibilika (>60°C).

· Kutuswa ukukhulisa izinga lokushisa lesinyathelo sokukhipha/isandiso ngokusebenzisa i-gradient yezinga lokushisa ngesikhathi senqubo yokusetha ukusabela.

· Uma izinga lokushisa liphezulu, kulapho izinga lokushisa liphezulu le-PCR.Umjikelezo wokuloba okuhlanekezelwe ngokuvamile wenziwa ezingeni lokushisa eliphakeme kunomjikelezo we-PCR, ngoba i-DNA Primer:RNA Template hybridization ngokuvamile inephuzu lokuncibilika eliphakeme kune-DNA Primer:cDNA Template duplex.

· I-RevTaq RT-PCR DNA polymerase yenziwe ngofuzo futhi yathuthukiswa, futhi usayizi we-amplicon uphakathi kuka-60-300 bp.

Umkhawulo wokutholwa kwe-RevTaq RT-PCR DNA polymerase ufinyelela kumakhophi angu-4/

Isistimu yokusabela eyenziwe kahle (ukusungulwa kweziqalo zokuncibilika okuphezulu) kuboniswa esithombeni.I-RevTaq RT-PCR DNA polymerase-driven RT-PCR ibonisa ukuzwela okungcono kune-TaqPath 1-step RT-qPCR master mix, kanye nokutholwa okuphansi kwesampula yokuhlanjululwa kwegradient.

Izinzuzo ezengeziwe:

Umsebenzi wokuqalisa ngokushesha → Isinyathelo sokuqala sokuguqula isimo esishisayo singeqiwa.

Ifomula ye-aptamer yokuqala eshisayo → Nikeza umsebenzi we-enzyme engu-100% ngokushesha futhi uvimbele ukukhulisa okungaqondile emazingeni okushisa aphansi (<57°C).

Umsebenzi wokukhipha i-cleavage → Isinyathelo sokukhipha i-RNA asifakiwe, ngoba i-RevTaq RT-PCR DNA polymerase ingaphinda icubungule amasampula okusabela aluhlaza.Ingabhubhisa ngokushesha ulwelwesi lwamaseli eukaryote, amagciwane namagciwane emjikelezweni oshisayo we-RT-PCR.

Ileveli yempahla eluhlaza ye-IVD → amazinga ekhwalithi ephezulu nezintengo ezincintisana kakhulu