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I-traditional reverse transcriptases ayikwazi ukunyamezela ukushisa okuphezulu (ubushushu obuphezulu bomsebenzi we-MMLV yi-37-50 ° C, kunye ne-AMV yi-42-60 ° C).I-RNA yentsholongwane entsonkothileyo ayinakuguqulelwa ngokufanelekileyo kwi-cDNA kumaqondo obushushu aphantsi, okukhokelela ekubhaqweni okusebenzayo Ukuncipha.I-RT-qPCR yesiNtu ifuna ngokubanzi ukuthatha inxaxheba kwee-enzyme ezimbini eziphambili (i-reverse transcriptase kunye ne-DNA polymerase), eyenza kube nzima ukwenza lula ukusebenza kwenkqubo yokusabela kunye nokunciphisa iindleko.Apha siza kwazisa ii-reverse transcriptases ezimbini zokumelana nobushushu obuphezulu, i-TTH kunye ne-RevTaq.Ezi enzyme zimbini zikwanomsebenzi we-DNA polymerase, ngoko zibizwa ngokuba zii-enzyme ezimbini.

TH DNA polymerase

Umele ukuba uvile nge-TtH, ephuma kwibhaktiriya ye-thermophilic i-Thermus thermophilus HB8.Phambi kwee-divalent cations ezifana ne-Mg2 +, inomsebenzi we-DNA polymerase.Isetyenziswa ngokubanzi kwiimpendulo ze-PCR njenge-enzyme ye-Taq, kodwa inokumelana nokushisa okuphezulu kune-Taq enzyme, ngoko ke inefuthe elingcono kwi-PCR kunye neetemplates eziphezulu ze-GC.

· Le enzyme ngokusisiseko ayinayo i-3′→5′ exonuclease activity kunye ne-5′→3′ exonuclease activity, ngoko ingasetyenziselwa ukulandelelana kwe-dieoxy.

· Le enzyme inomsebenzi weRTase.Phambi kwe-Mn2+, umsebenzi weRTase uya kuphuculwa.Ukusebenzisa eli nqaku, linokusetyenziselwa ukwenza i-reverse transcription reaction kunye ne-PCR reaction kwityhubhu enye, oko kukuthi, inyathelo elinye le-RT-PCR.Nangona kunjalo, phambi kwe-Mn2 +, ukuchaneka kwe-RT-PCR ayiphezulu.Umsebenzi we-RT awunanto yakwenza nomsebenzi we-rnaase H.

· Umsebenzi owandisiweyo we-Tth-DNA polymerase (pH9, optimum +55℃~+70℃, ubuninzi + 95℃) yoyisa iingxaki ezibangelwa yi-RNA yesakhiwo sesibini.Isiphumo se-cDNA sinokunyuswa yi-PCR kunye ne-enzyme efanayo phambi kwe-Mg2 + ion.

· Ikhono le-Tth-DNA polymerase ukwenza i-reverse transcription kunye ne-DNA amplification kumaqondo aphezulu okushisa kwenza le enzyme ibe luncedo kubuninzi be-RT-PCR, i-cloning, kunye nohlalutyo lwe-gene expression ye-RNA yeselula kunye ne-viral.
· I-Tth-DNA polymerase isetyenziselwa i-RT-PCR ukukhulisa i-RNA ukuya kwi-1kb.

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Iimpawu kunye neenzuzo

Tth DNA polymerase:

• Qinisekisa ubungakanani bemveliso yepolymerase chain reaction (PCR), ubuncinci 1000 bp kwi-RT-PCR reaction

• Yamkela i-deoxyribonucleoside triphosphate elungisiweyo njengenxalenye engaphantsi

• Ayinxulumananga nomsebenzi weRNase H

• Unozinzo oluphezulu lwe-thermal ukoyisa iingxaki, ngokuqhelekileyo ezinxulumene nolwakhiwo oluphezulu lwesibini olukhoyo kwi-RNA

I-RevTaq RT-PCR DNA polymerase

I-DNA polymerase ekwaziyo ukumelana nobushushu kunye nomsebenzi we-reverse transcriptase

I-RevTaq-RT-PCR-DNA polymerase yi-engineering, ukumelana nobushushu kakhulu, i-enzyme esebenza kabini kunye ne-reverse transcriptase kunye nemisebenzi ye-DNA polymerase efunyenwe ngokuziphendukela kwesikhokelo kunye nokufakelwa.

· Isiqingatha sobomi be-RevTaq RT-PCR DNA polymerase kwi-95 ° C inkulu kunemizuzu engama-40.

· I-RevTaq RT-PCR DNA polymerase ivumela i-high-temperature reverse transcript ngokuthe ngqo kwi-template ye-RNA, kwaye inyathelo lokukhuphela elibuyisela umva lingaphinda liphindwe kaninzi ukuvelisa itemplates ezininzi ze-cDNA.

· I-RevTaq RT-PCR DNA polymerase ivumela "inyathelo-zero" i-RT-PCR (akukho nyathelo le-isothermal reverse transcription), kuba kwinqanaba lokwandiswa kwe-PCR ye-cyclic, ukukhutshelwa umva kunye nokukhulisa i-DNA kwenzeka ngaxeshanye.Oku kukwakhuthaza ukuphendulwa kokukhutshelwa okubuyela umva kumaqondo obushushu aphezulu, ngokwenjenjalo kuncitshiswe iingxaki ekuhlangatyezwane nazo ekunyibilikiseni ulwakhiwo oluluqilima lwesibini kwi-RNA kumaqondo obushushu aphezulu.

· Ngenxa ye-aptamer-based hot-start formula, i-RevTaq RT-PCR DNA polymerase iya kuvelisa iziphumo ezingcono xa i-annealing kunye nobushushu bokwandisa bungaphezulu kwama-57°C.

· Ekubeni i-enzyme imelana nobushushu, kuyacetyiswa ukuba uyile iiprimers kunye neprobes ezinamaqondo aphezulu kakhulu anyibilikayo (>60 ° C).

· Kuyacetyiswa ukuba kunyuswe ubushushu benyathelo lokuthomalalisa/ulwandiso ngethambeka lobushushu ngexesha lenkqubo yokuseta impendulo.

· Ukuphakama kweqondo lobushushu, kokukhona kuphezulu ukuchana kwe-PCR.Umjikelo wokukhutshelwa umva uqhele ukwenziwa kubushushu obuphezulu kunomjikelo we-PCR, kuba i-DNA Primer:RNA Template hybridization idla ngokuba neqondo eliphezulu lokunyibilika kune-DNA Primer:cDNA Template duplex.

· I-RevTaq RT-PCR DNA polymerase yenziwe ngobunjineli kwaye iphuculwe, kwaye ubukhulu be-amplicon buphakathi kwe-60-300 bp.

I-RevTaq RT-PCR DNA i-polymerase yokufumanisa umda ifikelela kwiikopi ezi-4 /Zimbini (2)

Inkqubo yokusabela ephuculweyo (ukusekwa kweendawo eziphambili zokunyibilika) iboniswe kumzobo.I-RevTaq RT-PCR DNA polymerase-driven RT-PCR ibonisa ubuntununtunu obungcono kune-TaqPath 1-inyathelo le-RT-qPCR master mix, kunye nokubhaqwa okusezantsi kweSampule ye-dilution gradient.

Izibonelelo ezingakumbi:

Umsebenzi wokuqalisa ngokukhawuleza → Inyathelo lokuqala lokuchaza i-thermal linokutsitywa.

Ifomula ye-aptamer yokuqala eshushu → Nikezela nge-100% umsebenzi we-enzyme ngokukhawuleza kwaye uthintele ukukhulisa okungangqalanga kumaqondo obushushu aphantsi (<57°C).

Umsebenzi wokucoca → Inyathelo lokutsalwa kwe-RNA alifakwanga, kuba i-RevTaq RT-PCR DNA polymerase inokuphinda isebenze iisampulu ezikrwada zokusabela.Inokutshabalalisa ngokukhawuleza iiseli ze-eukaryotes, ibhaktheriya kunye neentsholongwane kumjikelo oshushu we-RT-PCR.

Inqanaba lemathiriyeli ekrwada ye-IVD → imigangatho yomgangatho ophezulu kunye namaxabiso akhuphisana kakhulu


Ixesha lokuposa: Aug-12-2021