Qoraalada dhaqameed ee rogaal celiska ah uma dulqaadan karaan heerkulka sare (heerkulka ugu fiican ee dhaqdhaqaaqa MMLV waa 37-50C, iyo AMV waa 42-60C).Fayraska aadka u adag ee RNA si wax ku ool ah looguma rogi karo cDNA heerkul hoose, taasoo keentay in la ogaado waxtarka Hoos u dhaca.RT-qPCR dhaqameed guud ahaan waxay u baahan tahay ka qaybgalka laba enzymes oo muhiim ah (dib-u-dejinta transcriptase iyo DNA polymerase), taas oo ka dhigaysa mid aan suurtagal ahayn in la fududeeyo hawlgalka nidaamka falcelinta oo la yareeyo kharashka.Halkan waxaan ku soo bandhigi doonaa laba qoraal oo heerkul sare u adkaysta, TtH iyo RevTaq.Labadan ensaymes sidoo kale waxay leeyihiin shaqada DNA polymerase, sidaas darteed waxaa loo yaqaannaa enzymes bifunctional.

Tth DNA polymerase

Waa inaad maqashay wax ku saabsan TtH, taasoo ka timid bakteeriyada thermophilic Thermus thermophilus HB8.Marka ay joogaan cations kala duwan sida Mg2+, waxay leedahay firfircoonida polymerase DNA.Waxaa si weyn loogu isticmaalaa falcelinta PCR sida Taq enzyme, laakiin waxay leedahay iska caabin kuleyl ka sarreeya marka loo eego Taq enzyme, sidaas darteed waxay sidoo kale saameyn wanaagsan ku leedahay PCR oo leh qaabab sare oo GC ah.

Insaymiskani asal ahaan ma laha 3′→5′ firfircoonida exonuclease iyo 5′→3′ firfircoonida exonuclease,sidoo kale waxa loo isticmaali karaa isku xigxiga dideoxy.

Insaymiskani waxa uu leeyahay firfircooni RTase.Joogitaanka Mn2+, waxqabadka RTase waa la horumarin doonaa.Isticmaalka sifadan, waxa loo istcimaali karaa in lagu sameeyo falcelinta qoraal-celinta iyo falcelinta PCR ee isla tuubada, taas oo ah, hal-tallaabo RT-PCR.Si kastaba ha ahaatee, joogitaanka Mn2+, saxnaanta RT-PCR ma sarreeyso.Dhaqdhaqaaqa RT shaqo kuma laha dhaqdhaqaaqa rnaase H.

Dhaqdhaqaaqa kordhay ee Tth-DNA polymerase (pH9, ugu fiican +55℃CDNA-da ka soo baxda waxa lagu kordhin karaa PCR oo leh isla enzym marka ay joogaan Mg2+ ions.

Awoodda Tth-DNA polymerase si ay u sameyso qoraal-ku-noqosho iyo kordhinta DNA heerkul sare waxay ka dhigaysaa ensaymkan faa'iido u leh tirada RT-PCR, cloning, iyo falanqaynta muujinta hiddaha ee unugyada iyo fayraska RNA.
polymerase Tth-DNA waxaa loo isticmaalaa RT-PCR si loo kordhiyo RNA ilaa 1kb.

Tilmaamaha iyo faa'iidooyinka

polymerase DNA-da:

Hubi cabirka falcelinta silsilada polymerase ee la hagaajiyay (PCR), ugu yaraan 1000 bp falcelinta RT-PCR

• Aqbal triphosphate deoxyribonucleoside la beddelay substrate ahaan

• Aan la xidhiidhin waxqabadka RNase H

• Leh xasilloonida kulaylka sare si uu uga gudbo dhibaatooyinka, badiyaa la xidhiidha qaab dhismeedka sare ee RNA

RevTaq RT-PCR DNA polymerase

Polymerase DNA-da kulaylka u adkeysata leh oo leh hawlo transcriptase ah

RevTaq-RT-PCR-DNA polymerase waa injineer, aad u kulul u adkaysta, enzyme dual-functional leh transcriptase rogan iyo hawlaha DNA polymerase lagu helay iyada oo loo marayo horumar toosan iyo macmal ah.

· Nolosha nuska ah ee RevTaq RT-PCR DNA polymerase ee 95°C waxay ka badan tahay 40 daqiiqo.

RevTaq RT-PCR DNA polymerase waxa ay ogolaataa in heerkul sare dib loogu noqdo si toos ah template RNA, iyo tillaabada qoraalka dib loogu celin karaa dhowr jeer si loo dhaliyo qaabab badan oo cDNA ah.

RevTaq RT-PCR DNA polymerase waxay u ogolaataa "eber-tallaabo" RT-PCR (ma jiro tillaabada qoraalka isbedbeddelka isothermal), sababtoo ah tallaabada kordhinta PCR ee wareegtada ah, qoraal-ku-celinta iyo kordhinta DNA-da ayaa isku mar dhaca.Tani waxay sidoo kale kor u qaadaysaa falcelinta qoraal-celinta ee heerkulka sare, taas oo yaraynaysa dhibaatooyinka la kulmay dhalaalidda qaabdhismeedka sare ee xooggan ee RNA heerkul sarreeya.

· Qaabka bilowga kulul ee ku salaysan aptamer-ka awgeed, RevTaq RT-PCR DNA polymerase waxa ay soo saari doontaa natiijooyin ka wanaagsan marka ciribtirka iyo heerkulka fidinta uu ka sarreeyo 57°C.

Mar haddii ensaymku yahay mid kulaylka adkaysi u leh, waxa lagu talinayaa in la qaabeeyo asal-raacyo iyo baarayaal leh dhibco dhalaalid aad u sarreeya (>60°C).

Waxaa lagu talinayaa in la hagaajiyo heerkulka tallaabada annealing/kordhinta iyada oo loo marayo heerkulbeeg inta lagu jiro habka habaynta falcelinta.

• Markasta oo heerkulku sarreeyo, ayaa sare u kaca gaar ahaanta PCR.Wareegga qoraalka gadaasha waxaa badanaa lagu sameeyaa heerkul ka sarreeya wareegga PCR, sababtoo ah DNA Primer: RNA Template hybridization badanaa waxay leedahay meel dhalaalaysa oo ka sareysa DNA Primer: cDNA Template duplex.

RevTaq RT-PCR DNA polymerase si hidde ahaan waa loo farsameeyay waana la hagaajiyay, cabirka amplicon-kuna wuxuu u dhexeeyaa 60-300 bp.

Xadka ogaanshaha RevTaq RT-PCR DNA polymerase wuxuu gaarayaa 4 nuqul/

Habka falcelinta ee la hagaajiyay (samaynta aasaasayaasha sare ee dhalaalka) ayaa lagu muujiyay shaxanka.RevTaq RT-PCR DNA polymerase-driven RT-PCR waxay muujineysaa dareen ka wanaagsan TaqPath 1-talaabo RT-qPCR isku dhafka sare, iyo ogaanshaha hoose ee Tusaalaha milanka.

Faa'iidooyin badan:

Shaqada bilawga degdega ah → Tallaabada denaturation kulaylka bilowga ah waa laga boodi karaa.

Kuleelka qaacadaha abtamer-ka-bilaabashada → Bixi 100% dhaqdhaqaaqa enzyme isla markaaba kana ilaali kor uqaadid aan gaar ahayn heerkulka hooseeya (<57°C).

Shaqada nadiifinta → Talaabada soo saarista RNA waa laga tagay, sababtoo ah RevTaq RT-PCR DNA polymerase waxa kale oo ay farsamayn kartaa muunado falcelin cayriin ah.Waxay isla markiiba burburin kartaa xuubka unugyada eukaryotes, bakteeriyada iyo fayraska ee wareegga RT-PCR ee kulul.

Heerka alaabta ceeriin IVD → heerar tayo sare leh iyo qiimo aad u tartan badan