Imyandikire gakondo ntishobora kwihanganira ubushyuhe bwo hejuru (ubushyuhe bwiza kubikorwa bya MMLV ni 37-50 ° C, na AMV ni 42-60 ° C).Virusi irushijeho gukomera RNA ntishobora guhindurwa neza muri cDNA mubushyuhe buke, bikavamo gukora neza Kugabanuka.Gakondo RT-qPCR isaba uruhare rwa enzymes ebyiri zingenzi (reverse transcriptase na ADN polymerase), bigatuma bidashoboka koroshya imikorere ya sisitemu yo kubyitwaramo no kugabanya ikiguzi.Hano tuzamenyekanisha ibice bibiri byo hejuru-ubushyuhe-birwanya revers transcriptase, TtH na RevTaq.Iyi misemburo yombi ifite kandi imikorere ya ADN polymerase, bityo bita enzymes zifatika.

TtH ADN polymerase

Ugomba kuba warumvise ibya TtH, ikomoka kuri bacteri ya thermophilique Thermus thermophilus HB8.Imbere ya cations zingana nka Mg2 +, ifite ibikorwa bya ADN polymerase.Irakoreshwa cyane mubitekerezo bya PCR nka enzyme ya Taq, ariko ifite ubushyuhe burenze ubwinshi bwa Taq enzyme, bityo ikagira n'ingaruka nziza kuri PCR hamwe nibishusho byinshi bya GC.

· Iyi misemburo ntisanzwe ifite 3 ′ → 5 ′ ibikorwa bya exonuclease hamwe na 5 ′ → 3 ′ ibikorwa bya exonuclease, bityo irashobora no gukoreshwa mugukurikirana dideoxy.

· Iyi misemburo ifite ibikorwa bya RTase.Imbere ya Mn2 +, ibikorwa bya RTase bizamurwa.Ukoresheje iyi mikorere, irashobora gukoreshwa mugukora reaction ya reaction ya reaction na PCR reaction muri tube imwe, ni ukuvuga intambwe imwe RT-PCR.Ariko, imbere ya Mn2 +, ukuri kwa RT-PCR ntabwo ari hejuru.Ibikorwa bya RT ntaho bihuriye nibikorwa bya rnaase H.

· Ibikorwa byiyongera bya polymerase ya Tth-ADN (pH9, optimiz + 55 ℃ ～ + 70 ℃, ntarengwa + 95 ℃) itsinze ibibazo biterwa nuburyo bwa kabiri bwa RNA.CDNA yavuyemo irashobora kongererwa imbaraga na PCR hamwe na enzyme imwe imbere ya Mg2 + ion.

· Ubushobozi bwa Tth-ADN polymerase yo gukora transcription transcription hamwe no kongera ADN mubushyuhe bwinshi bituma iyi misemburo igira akamaro mukigereranyo cya RT-PCR, cloni, hamwe nisesengura ryerekana gene ya RNA na selile.
· Tth-ADN polymerase ikoreshwa kuri RT-PCR kugirango yongere RNA kugera kuri 1kb.

Ibiranga ibyiza

Tth polymerase ya ADN:

• Menya neza ko polymerase ikora neza (PCR) ingano y'ibicuruzwa, byibuze 1000 bp muri reaction ya RT-PCR

• Emera deoxyribonucleoside triphosphate yahinduwe nka substrate

• Ntabwo bifitanye isano nibikorwa bya RNase H.

• Ifite ubushyuhe bwinshi bwo gukemura ibibazo, mubisanzwe bijyanye nuburyo bwo hejuru bwisumbuye buboneka muri RNA

RevTaq RT - PCR polymerase

ADN polymerase irwanya ubushyuhe hamwe nibikorwa bya transcriptase

RevTaq-RT-PCR-ADN polymerase ni injeniyeri, irwanya ubushyuhe bwinshi, enzyme ikora ibiri hamwe na transcript transcriptase hamwe nibikorwa bya ADN polymerase byabonetse binyuze mubwihindurize.

· Igice cya kabiri cyubuzima bwa RevTaq RT-PCR ADN polymerase kuri 95 ° C irenze iminota 40.

· RevTaq RT-PCR ADN polymerase ituma ubushyuhe bwo hejuru busubirana biturutse ku cyitegererezo cya RNA, kandi intambwe yo kwandukura irashobora gusubirwamo inshuro nyinshi kugirango habeho inyandikorugero nyinshi za cDNA.

· RevTaq RT-PCR ADN polymerase yemerera "zeru-ntambwe" RT-PCR (nta ntambwe ya isothermal reverse transcription), kubera ko intambwe yo kwaguka ya PCR, kwandikirana no kwongera ADN bibera icyarimwe.Ibi kandi biteza imbere reaction ya reaction ya reaction yubushyuhe bwo hejuru, bityo bikagabanya ibibazo byahuye nabyo mugushonga imiterere ikomeye ya kabiri muri RNA mubushyuhe bwinshi.

· Bitewe na aptamer ishingiye kuri hot-itangira, RevTaq RT-PCR ADN polymerase izatanga umusaruro mwiza mugihe ubushyuhe bwa annealing no kwaguka burenze 57 ° C.

· Kubera ko enzyme idashobora kwihanganira ubushyuhe, birasabwa gukora primers na probe zifite ingingo ndende cyane (> 60 ° C).

· Birasabwa guhindura ubushyuhe bwa intambwe ya annealing / kwaguka binyuze mubushyuhe bwa gradient mugihe cyo gushiraho reaction.

· Ubushyuhe buri hejuru, niko umwihariko wa PCR.Inzira yo kwandukura isanzwe ikorwa ku bushyuhe buri hejuru ya PCR, kubera ko ADN Primer: RNA Template hybridisation isanzwe ifite aho ishonga kuruta ADN Primer: cDNA Template duplex.

· RevTaq RT-PCR ADN polymerase yakozwe mubwoko bwa genetike kandi itezimbere, kandi ubunini bwa amplicon buri hagati ya 60-300 bp.

RevTaq RT-PCR ADN ya polymerase yo kumenya igera kuri kopi 4 /

Sisitemu yogukora neza (gushiraho hejuru yo gushonga ingingo yibanze) irerekanwa mumashusho.Rev.

Ibyiza byinshi:

Imikorere yo gutangira byihuse step Intambwe yambere yo gutandukanya amashyuza irashobora gusimbuka.

Gushyushya-gutangira aptamer formula → Tanga ibikorwa bya enzyme 100% ako kanya kandi wirinde amplification idasanzwe kubushyuhe buke (<57 ° C).

Imikorere ya Cleavage step Intambwe yo gukuramo RNA irasibwe, kubera ko polymerase ya RevTaq RT-PCR ADN ishobora kandi gutunganya ibyitegererezo bitagaragara.Irashobora guhita isenya uturemangingo twa eukaryote, bagiteri na virusi mugihe gishyushye cya RT-PCR.

IVD ibikoresho fatizo standard ubuziranenge bwo hejuru nibiciro byapiganwa cyane