Ntụgharị ntụgharị ọdịnala enweghị ike ịnabata oke okpomọkụ (okpomọkụ kacha mma maka ọrụ MMLV bụ 37-50°C, yana AMV bụ 42-60°C).Enweghị ike ịtụgharị RNA viral dị mgbagwoju anya nke ọma ka ọ bụrụ cDNA n'obere okpomọkụ, na-ebute nrụpụta nrụpụta Mbelata ahụ.RT-qPCR omenala na-achọkarị ntinye nke enzymes isi abụọ (reverse transcriptase na DNA polymerase), nke na-eme ka ọ ghara ikwe omume ime ka ọrụ nke usoro mmeghachi omume dị mfe ma belata ọnụ ahịa.N'ebe a, anyị ga-ewebata transcriptase abụọ na-eguzogide okpomọkụ, TtH na RevTaq.Enzymes abụọ a nwekwara ọrụ nke DNA polymerase, ya mere a na-akpọ ha bifunctional enzymes.

TTH DNA polymerase

Ọ ga-abụrịrị na ị nụla gbasara TtH, nke sitere na nje bacteria Thermus thermophilus HB8.Na ọnụnọ nke divalent cations dị ka Mg2+, ọ nwere DNA polymerase ọrụ.A na-ejikarị ya eme ihe na mmeghachi omume PCR dị ka enzyme Taq, mana ọ nwere nguzogide okpomọkụ dị elu karịa Taq enzyme, ya mere ọ na-enwekwa mmetụta dị mma na PCR na nnukwu ọdịnaya GC.

· Nke a enzyme ihu ọma enweghị 3′→5′ exonuclease ọrụ na 5′→3′ exonuclease ọrụ, n'ihi ya, ọ nwekwara ike-eji maka dideoxy sequencing.

Nke a enzyme nwere ọrụ RTase.N'ebe Mn2+ nọ, a ga-akwalite ọrụ RTase.Iji njirimara a, enwere ike iji ya mee mmeghachi omume ntụgharị na PCR n'otu tube ahụ, ya bụ, otu nzọụkwụ RT-PCR.Agbanyeghị, na ọnụnọ Mn2+, izi ezi nke RT-PCR adịghị elu.Ọrụ RT enweghị ihe jikọrọ ya na ọrụ rnaase H.

Ọrụ mụbara nke Tth-DNA polymerase (pH9, kacha +55℃～+70℃, kacha +95℃) na-emeri nsogbu nke usoro nke abụọ RNA kpatara.Enwere ike ịbawanye cDNA sitere na PCR yana otu enzyme ahụ n'ihu Mg2+ ion.

Ikike nke Tth-DNA polymerase ime ntụgharị ntụgharị na mmụba DNA na oke okpomọkụ na-eme ka enzyme a bara uru maka ọnụọgụ RT-PCR, cloning, na nyocha okwu mkpụrụ ndụ nke RNA cell na viral.
A na-eji polymerase Tth-DNA maka RT-PCR iji bulie RNA ruo 1kb.

Atụmatụ na uru

DNA polymerase:

• Gbaa mbọ hụ na nha ngwaahịa polymerase chain (PCR) kachasị, opekata mpe 1000 bp na mmeghachi omume RT-PCR.

• Nabata triphosphate deoxyribonucleoside gbanwetụrụ dị ka mkpụrụ

• Enweghị njikọ na ọrụ RNase H

• Nwere nkwụsi ike nke okpomọkụ dị elu iji merie nsogbu, na-ejikọta ya na nhazi nke abụọ dị elu dị na RNA

RevTaq RT-PCR DNA polymerase

DNA polymerase na-eguzogide okpomọkụ nwere ọrụ transcriptase ntụgharị

RevTaq-RT-PCR-DNA polymerase bụ ihe arụpụtara, nke na-ekpo ọkụ nke ukwuu, enzyme na-arụ ọrụ abụọ nwere ntụgharị transcriptase na ọrụ DNA polymerase enwetara site na ntuzi aka na mgbanwe mmadụ.

· Ọkara ndụ nke RevTaq RT-PCR DNA polymerase na 95°C karịrị nkeji iri anọ.

RevTaq RT-PCR DNA polymerase na-enye ohere ntụgharị mgbanwe okpomọkụ dị elu ozugbo site na ndebiri RNA, na ntụgharị ntụgharị ntụgharị nwere ike ịmegharị ugboro ugboro iji mepụta ndebiri cDNA ọzọ.

RevTaq RT-PCR DNA polymerase na-enye ohere "efu-nzọụkwụ" RT-PCR (enweghị usoro ntụgharị ntụgharị isothermal), n'ihi na n'usoro mgbatị PCR cyclic, ntụgharị ntụgharị na mmụba DNA na-eme n'otu oge.Nke a na-akwalite mmeghachi omume ntụgharị ntụgharị na oke okpomọkụ, si otú a na-ebelata nsogbu ndị a na-enwe na agbaze ihe owuwu nke abụọ siri ike na RNA na okpomọkụ dị elu.

N'ihi usoro mmalite ọkụ na-ekpo ọkụ nke dabeere na aptamer, RevTaq RT-PCR DNA polymerase ga-arụpụta ihe dị mma mgbe nkwụsịtụ na mgbatị ahụ dị elu karịa 57 ° C.

Ebe ọ bụ na enzyme na-eguzogide okpomọkụ, a na-atụ aro ka ịmepụta primers na nyocha nwere nnukwu agbaze (> 60 ° C).

A na-atụ aro ka ebuli ọnọdụ okpomọkụ nke nkwụsịtụ / ndọtị site na gradient okpomọkụ n'oge usoro nhazi mmeghachi omume.

· Ọnụ ọgụgụ dị elu nke okpomọkụ, ihe dị elu nke PCR dị elu.A na-emekarị okirikiri ntụgharị ntụgharị n'ogo dị elu karịa okirikiri PCR, n'ihi na DNA Primer: RNA Template hybridization na-enwekarị ebe mgbaze dị elu karịa DNA Primer: cDNA Template duplex.

· RevTaq RT-PCR DNA polymerase ka emebere site na mkpụrụ ndụ ihe nketa yana kachasị, na nha amplicon dị n'etiti 60-300 bp.

Oke nchọpụta nke RevTaq RT-PCR DNA polymerase ruru nnomi 4/

A na-egosipụta usoro mmeghachi omume kachasị mma (ntọala nke ihe ọkụkụ dị elu) na ọnụ ọgụgụ ahụ.RevTaq RT-PCR DNA polymerase na-ebute RT-PCR na-egosi nghọta ka mma karịa TaqPath 1-step RT-qPCR master mix, na nchọpụta ala dị ala Sample dilution gradient.

Uru ndị ọzọ:

Ọrụ mmalite ngwa ngwa → Enwere ike ịwụpụ nzọụkwụ denaturation nke mbụ.

Usoro aptamer na-ekpo ọkụ na-ekpo ọkụ → Nye 100% ọrụ enzyme ozugbo ma gbochie mmụba na-enweghị isi na obere okpomọkụ (<57 ° C).

Ọrụ mkpochapụ → Ahapụla nzọụkwụ mmịpụta RNA, n'ihi na RevTaq RT-PCR DNA polymerase nwekwara ike hazie nlele mmeghachi omume crude.Ọ nwere ike ibibi membranes cell nke eukaryotes, nje bacteria na nje ozugbo na usoro RT-PCR na-ekpo ọkụ.

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