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Traditional reverse transcriptases sangathe kulekerera kutentha kwakukulu (kutentha koyenera kwa ntchito ya MMLV ndi 37-50 ° C, ndi AMV ndi 42-60 ° C).Ma virus ovuta kwambiri a RNA sangathe kusinthidwa kukhala cDNA pa kutentha kochepa, zomwe zimapangitsa kuti azindikire bwino Kuchepetsa.Traditional RT-qPCR nthawi zambiri imafuna kutenga nawo gawo kwa michere iwiri yayikulu (reverse transcriptase ndi DNA polymerase), zomwe zimapangitsa kuti zikhale zosatheka kufewetsa kachitidwe kachitidwe ndikuchepetsa mtengo.Apa tikuwonetsa zolemba ziwiri zosagwirizana ndi kutentha kwambiri, TTH ndi RevTaq.Ma enzyme awiriwa amagwiranso ntchito ya DNA polymerase, motero amatchedwa ma enzymes omwe amagwira ntchito mosiyanasiyana.

TH DNA polymerase

Muyenera kuti munamvapo za TH, yomwe imachokera ku bakiteriya yotchedwa Thermus thermophilus HB8.Pamaso pa ma divalent cations monga Mg2 +, ili ndi DNA polymerase ntchito.Amagwiritsidwa ntchito kwambiri pakuchita kwa PCR monga Taq enzyme, koma imakhala ndi kutentha kwakukulu kuposa Taq enzyme, kotero imakhala ndi zotsatira zabwino pa PCR yokhala ndi ma tempuleti apamwamba a GC.

· Enzyme iyi kwenikweni ilibe 3′→5′ exonuclease ntchito ndi 5′→3′ exonuclease ntchito, kotero itha kugwiritsidwanso ntchito pakutsatizana kwa didioxy.

enzyme iyi imakhala ndi ntchito ya RTase.Pamaso pa Mn2 +, ntchito ya RTase idzakulitsidwa.Pogwiritsa ntchito izi, zitha kugwiritsidwa ntchito popanga reverse transcript reaction ndi PCR reaction mu chubu lomwelo, ndiye kuti, gawo limodzi la RT-PCR.Komabe, pamaso pa Mn2 +, kulondola kwa RT-PCR sikwapamwamba.Zochita za RT sizikugwirizana ndi zochitika za rnaase H.

Kuchulukitsa kwa Tth-DNA polymerase (pH9, optimum +55 ℃~+70 ℃, pazipita +95 ℃) kumathetsa zovuta zomwe zimachitika chifukwa cha RNA yachiwiri.Zotsatira za cDNA zimatha kukulitsidwa ndi PCR ndi enzyme yomweyi pamaso pa Mg2 + ions.

Kuthekera kwa Tth-DNA polymerase kupanga reverse transcription and DNA amplification pa kutentha kwambiri kumapangitsa enzyme iyi kukhala yothandiza pakuchulukira kwa RT-PCR, cloning, ndi kusanthula kwa ma geneti a RNA ndi ma virus.
· Tth-DNA polymerase imagwiritsidwa ntchito pa RT-PCR kukulitsa RNA mpaka 1kb.

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Mbali ndi ubwino

Tth DNA polymerase:

• Onetsetsani kukula kwa malonda a polymerase chain reaction (PCR), osachepera 1000 bp pamachitidwe a RT-PCR

• Landirani deoxyribonucleoside triphosphate yosinthidwa ngati gawo lapansi

• Zosagwirizana ndi zochitika za RNase H

• Ali ndi kukhazikika kwa kutentha kwapamwamba kuti athetse mavuto, omwe nthawi zambiri amakhudzana ndi mawonekedwe apamwamba achiwiri omwe amapezeka mu RNA

RevTaq RT-PCR DNA polymerase

DNA polymerase yosamva kutentha yokhala ndi reverse transcriptase ntchito

RevTaq-RT-PCR-DNA polymerase ndi enzyme yopangidwa, yosatentha kwambiri, yogwira ntchito pawiri yokhala ndi reverse transcriptase ndi DNA polymerase ntchito zomwe zimapezeka kudzera mu chisinthiko cholunjika komanso chochita kupanga.

· Theka la moyo wa RevTaq RT-PCR DNA polymerase pa 95 ° C ndi wamkulu kuposa mphindi 40.

· RevTaq RT-PCR DNA polymerase imalola kusindikiza kwapamwamba kwambiri kuchokera pa template ya RNA, ndipo sitepe yolembera m'mbuyo imatha kubwerezedwa kangapo kuti apange ma tempulo ambiri a cDNA.

· RevTaq RT-PCR DNA polymerase imalola "zero-step" RT-PCR (palibe isothermal reverse transcript sitepe), chifukwa mu gawo la cyclic PCR yowonjezera, kumasulira mobwereza ndi kukulitsa kwa DNA kumachitika nthawi imodzi.Izi zimalimbikitsanso kusintha kwa kalembedwe kameneka pakatentha kwambiri, motero kuchepetsa mavuto omwe amakumana nawo posungunula dongosolo lachiwiri la RNA pa kutentha kwakukulu.

· Chifukwa cha mawonekedwe oyambira otentha otengera aptamer, RevTaq RT-PCR DNA polymerase ipanga zotulukapo zabwinoko pamene kutentha kwa annealing ndi kukulitsa kumakhala kopitilira 57°C.

· Popeza enzymeyi imalimbana ndi kutentha, tikulimbikitsidwa kupanga zoyambira ndi zofufuzira zokhala ndi malo osungunuka kwambiri (>60 ° C).

· Ndi bwino konza kutentha kwa annealing/wowonjezera sitepe kudzera kutentha gradient pa zimene khwekhwe ndondomeko.

· Kutentha kwapamwamba, kumapangitsa kuti PCR ikhale yeniyeni.Kuzungulira kwa reverse transcript nthawi zambiri kumachitika pa kutentha kwakukulu kuposa momwe PCR cycle, chifukwa DNA Primer:RNA Template hybridization nthawi zambiri imakhala ndi malo osungunuka kwambiri kuposa DNA Primer:cDNA Template duplex.

RevTaq RT-PCR DNA polymerase imapangidwa mwachibadwa ndikukonzedwa bwino, ndipo kukula kwa amplicon kuli pakati pa 60-300 bp.

RevTaq RT-PCR DNA polymerase kuzindikira malire amafika makope 4/Awiri (2)

Dongosolo lokonzekera bwino (kukhazikitsidwa kwa zoyambira zazikulu zosungunuka) zikuwonetsedwa pachithunzichi.RevTaq RT-PCR DNA polymerase-driven RT-PCR imawonetsa kukhudzika kwabwinoko kuposa TaqPath 1-step RT-qPCR master mix, komanso kuzindikira kutsika Zitsanzo za dilution gradient.

Ubwino winanso:

Ntchito yoyambira mwachangu → Gawo loyambira la kutentha likhoza kudumpha.

Fomula yoyambira yotentha ya aptamer → Perekani zochita za enzyme 100% nthawi yomweyo ndikupewa kukulitsa kosagwirizana ndi kutentha kochepa (<57°C).

Ntchito ya Cleavage → Sitepe yochotsa RNA yasiyidwa, chifukwa RevTaq RT-PCR DNA polymerase imathanso kukonza zitsanzo zamwano.Itha kuwononga nthawi yomweyo ma cell a eukaryotes, mabakiteriya ndi ma virus mumayendedwe otentha a RT-PCR.

IVD raw material level → miyezo yapamwamba kwambiri komanso mitengo yopikisana kwambiri


Nthawi yotumiza: Aug-12-2021