Traditional reverse transcriptases haigoni kushivirira tembiricha yepamusoro (tembiricha yakakwana yeMMLV chiitiko i37-50°C, uye AMV i42-60°C).Iyo yakanyanya kuomesesa hutachiona RNA haigone kudzoreredza zvinobudirira kunyorwa mu cDNA pakadzika tembiricha, zvichikonzera kugona kwekuona Kuderedza.Traditional RT-qPCR kazhinji inoda kutora chikamu kwema enzymes maviri akakosha (reverse transcriptase uye DNA polymerase), izvo zvinoita kuti zvisaite kurerutsa kushanda kweiyo reaction system uye kuderedza mutengo.Pano tichasuma maviri epamusoro-kupisa-anodzivirira reverse transcriptases, TTH uye RevTaq.Aya ma enzymes maviri anewo basa reDNA polymerase, saka anonzi bifunctional enzymes.

TH DNA polymerase

Unofanira kunge wakanzwa nezveTtH, iyo inotorwa kubva kubhakitiriya rethermophilic Thermus thermophilus HB8.Muhupo hwedivalent cations seMg2 +, ine DNA polymerase chiitiko.Iyo inoshandiswa zvakanyanya muPCR maitiro seTaq enzyme, asi ine yakanyanya kupisa kupisa kupfuura Taq enzyme, saka ine zvakare mhedzisiro iri nani paPCR ine yakakwira GC zvemukati matemplate.

Iri enzyme harina 3′→5′ exonuclease chiitiko uye 5′→3′ exonuclease chiitiko, saka rinogona zvakare kushandiswa kutevedzana kwedieoxy.

enzyme iyi ine RTase chiitiko.Pamberi peMn2 +, RTase chiitiko chichawedzerwa.Uchishandisa chimiro ichi, chinogona kushandiswa kuita reverse transcription reaction uye PCR kuita muchubhu imwechete, kureva, nhanho imwe RT-PCR.Nekudaro, pamberi peMn2 +, iko kurongeka kweRT-PCR hakuna kukwirira.RT chiitiko hachina chekuita nernaase H chiitiko.

· Iyo yakawedzera chiitiko cheTth-DNA polymerase (pH9, optimum +55 ℃～+70 ℃, yakanyanya +95 ℃) inokunda matambudziko anokonzerwa neRNA yechipiri chimiro.Iyo cDNA inoguma inogona kukwidziridzwa nePCR ine enzyme imwechete pamberi peMg2 + ions.

· Kugona kweTth-DNA polymerase kuita reverse transcription uye DNA amplification pakupisa kwakanyanya kunoita kuti enzyme iyi ibatsire pahuwandu hweRT-PCR, cloning, uye gene expression ongororo yemasero uye hutachiona RNA.
Tth-DNA polymerase inoshandiswa kuRT-PCR kukwidziridza RNA kusvika pa1kb.

Zvimiro uye zvakanakira

Tth DNA polymerase:

• Ita shuwa optimized polymerase chain reaction (PCR) chigadzirwa saizi, ingangoita 1000 bp mukuita kweRT-PCR

• Gashirai yakagadziridzwa deoxyribonucleoside triphosphate se substrate

• Hazvinei neRNase H chiitiko

• Iine kugadzikana kwepamusoro kwekushisa kukunda matambudziko, kazhinji ane chokuita nepamusoro pechipiri chimiro chiripo muRNA

RevTaq RT-PCR DNA polymerase

Iyo inodzivirira kupisa DNA polymerase ine reverse transcriptase chiitiko

RevTaq-RT-PCR-DNA polymerase injinjini, inodzivirira kupisa zvakanyanya, ine mbiri-inoshanda enzyme ine reverse transcriptase uye DNA polymerase zviitiko zvinowanikwa kuburikidza nekunangwa uye yekugadzira evolution.

· Iyo hafu yehupenyu hweRevTaq RT-PCR DNA polymerase pa95 ° C yakakura kupfuura maminetsi makumi mana.

RevTaq RT-PCR DNA polymerase inobvumira yakakwira-tembiricha reverse transcript yakananga kubva kuRNA template, uye reverse transcription nhanho inogona kudzokororwa kakawanda kugadzira akawanda cDNA matemplate.

RevTaq RT-PCR DNA polymerase inobvumira "zero-nhanho" RT-PCR (hapana isothermal reverse transcript nhanho), nekuti mune cyclic PCR yekuwedzera nhanho, reverse transcription uye DNA amplification inoitika panguva imwe chete.Izvi zvinosimudzira zvakare reverse transcription reaction pakupisa kwakanyanya, nekudaro kuderedza matambudziko anowanikwa mukunyungudutsa yakasimba yechipiri chimiro muRNA pakupisa kwakanyanya.

Nekuda kweiyo aptamer-based hot-start formula, RevTaq RT-PCR DNA polymerase ichaburitsa zvirinani kana tembiricha yekuwedzera uye yekuwedzera yakakwira kupfuura 57°C.

· Sezvo enzyme isingapise kupisa, inokurudzirwa kugadzira maprimers uye probes ane yakanyanya kunyungudika (> 60 ° C).

· Zvinokurudzirwa kukwidziridza tembiricha yeannealing/extension nhanho kuburikidza netembiricha gradient panguva yekuseta maitiro.

· Kukwirisa tembiricha, ndiko kukwira kwePCR chaiyo.The reverse transcription cycle inowanzoitwa patembiricha yepamusoro kupfuura PCR cycle, nekuti DNA Primer:RNA Template hybridization inowanzova nepamusoro pekunyungudika kupfuura DNA Primer:cDNA Template duplex.

RevTaq RT-PCR DNA polymerase yakagadzirwa nemajini uye yakagadziridzwa, uye saizi yeamplicon iri pakati pe60-300 bp.

RevTaq RT-PCR DNA polymerase yekuona muganho inosvika 4 makopi /

Iyo optimized reaction system (kugadzwa kweyakakwira melting point primers) inoratidzwa mumufananidzo.RevTaq RT-PCR DNA polymerase-inotyairwa RT-PCR inoratidza kunzwa kuri nani pane TaqPath 1-nhanho RT-qPCR master musanganiswa, uye yakaderera yekuona Sample dilution gradient.

Zvimwe zvakanakira:

Kurumidza kutanga basa → Yekutanga thermal denaturation nhanho inogona kutsikwa.

Hot-start aptamer formula → Ipa 100% enzyme chiitiko nekukasira uye kudzivirira kusiri-chaiyo amplification pakudziya kwakaderera (<57°C).

Cleavage basa → Iyo RNA yekubvisa nhanho inosiiwa, nekuti RevTaq RT-PCR DNA polymerase inogona zvakare kugadzirisa zvisina kufanira maitiro masampuli.Inogona kuparadza pakarepo masero eukaryotes, mabhakitiriya uye mavhairasi mune inopisa RT-PCR kutenderera.

IVD raw material level → high quality standards uye yakanyanya kukwikwidza mitengo