ʻAʻole hiki i nā transcriptases hoʻohuli kuʻuna ke ʻae i nā wela kiʻekiʻe (ʻo ka mahana maikaʻi loa no ka hana MMLV ʻo 37-50 ° C, a ʻo AMV ʻo 42-60 ° C).ʻAʻole hiki ke hoʻohuli maikaʻi ʻia ka RNA viral paʻakikī i ka cDNA i nā haʻahaʻa haʻahaʻa, e hopena i ka ʻike pono ʻana.Pono ka RT-qPCR kuʻuna i ke komo ʻana o ʻelua mau enzymes nui (reverse transcriptase a me DNA polymerase), ʻaʻole hiki ke hoʻomaʻamaʻa i ka hana o ka ʻōnaehana pane a hoʻemi i ke kumukūʻai.Ma ʻaneʻi e hoʻolauna mākou i ʻelua transcriptases hoʻohuli i ka wela kiʻekiʻe, TtH a me RevTaq.Loaʻa i kēia mau enzyme ʻelua ka hana o ka DNA polymerase, no laila ua kapa ʻia lākou he enzyme bifunctional.

TtH DNA polymerase

Ua lohe paha ʻoe e pili ana i ka TtH, ka mea i loaʻa mai ka thermophilic bacterium Thermus thermophilus HB8.Ma ke alo o nā cations divalent e like me Mg2+, loaʻa iā ia ka hana DNA polymerase.Hoʻohana nui ʻia ia i nā hopena PCR e like me Taq enzyme, akā ʻoi aku ka ʻoi aku o ke kūpaʻa wela ma mua o ka Taq enzyme, no laila ʻoi aku ka maikaʻi o ka hopena ma PCR me nā kiʻi maʻiʻo GC kiʻekiʻe.

· ʻAʻohe hana exonuclease 3′→5′ a me ka hana exonuclease 5′→3′ no kēia enzyme, no laila hiki ke hoʻohana ʻia no ke kaʻina hana dideoxy.

· He hana RTase kēia enzyme.Ma ke alo o Mn2+, e hoʻonui ʻia ka hana RTase.Me ka hoʻohana ʻana i kēia hiʻohiʻona, hiki ke hoʻohana ʻia e hana i ka hoʻohuli ʻana i ka unuhi ʻana a me ka hopena PCR i ka paipu hoʻokahi, ʻo ia hoʻi, hoʻokahi ʻanuʻu RT-PCR.Eia naʻe, ma ke alo o Mn2 +, ʻaʻole kiʻekiʻe ka pololei o ka RT-PCR.ʻAʻole pili ka hana RT me ka hana rnaase H.

· ʻO ka hoʻonui ʻana o ka hana o Tth-DNA polymerase (pH9, ʻoi loa + 55 ℃～ + 70 ℃, kiʻekiʻe + 95 ℃) lanakila i nā pilikia i hoʻokumu ʻia e ka RNA lua.Hiki ke hoʻonui ʻia ka cDNA e ka PCR me ka enzyme like i mua o nā ion Mg2+.

· ʻO ka hiki o Tth-DNA polymerase ke hana i ka hoʻololi ʻana a me ka hoʻonui ʻana i ka DNA ma nā wela kiʻekiʻe e pono ai kēia enzyme no ka quantitative RT-PCR, cloning, a me ka kānana hōʻike gene o RNA cellular a viral.
· Hoʻohana ʻia ka Tth-DNA polymerase no RT-PCR e hoʻonui i ka RNA a hiki i 1kb.

Nā hiʻohiʻona a me nā pono

Tth DNA polymerase:

• E hōʻoia i ka nui o ka huahana polymerase chain reaction (PCR), ma kahi o 1000 bp ma ka hopena RT-PCR.

• E ʻae i ka deoxyribonucleoside triphosphate i hoʻololi ʻia ma ke ʻano he pani

• 'A'ole pili i ka hana RNase H

• Loaʻa i ke kūpaʻa wela kiʻekiʻe no ka hoʻoponopono ʻana i nā pilikia, pili maʻamau i ka hale kiʻekiʻe kiʻekiʻe i loaʻa i ka RNA

RevTaq RT-PCR DNA polymerase

He DNA polymerase pale wela me ka hana transcriptase hoʻohuli

ʻO RevTaq-RT-PCR-DNA polymerase kahi enzyme i hana ʻia, wela loa, ʻelua hana me ka reverse transcriptase a me nā hana DNA polymerase i loaʻa ma o ka hoʻomohala kuhikuhi a me ka hoʻomohala ʻana.

· ʻOi aku ka hapalua o ke ola o RevTaq RT-PCR DNA polymerase ma 95°C ma mua o 40 mau minuke.

· ʻO RevTaq RT-PCR DNA polymerase hiki ke hoʻololi pololei i ka hoʻololi ʻana i ka wela kiʻekiʻe mai ka RNA template, a hiki ke hana hou ʻia ke kaʻina hana hoʻohuli i nā manawa he nui no ka hoʻonui ʻana i nā mamana cDNA.

· RevTaq RT-PCR DNA polymerase hiki i ka “zero-step” RT-PCR (ʻaʻohe isothermal reverse transcription step), no ka mea, i ka cyclic PCR extension step, hoʻohuli ʻia ka transcription a me ka hoʻonui DNA i ka manawa like.Hoʻoikaika pū kēia i ka hoʻihoʻi ʻana i ka unuhi ʻana i nā wela kiʻekiʻe, a laila e hōʻemi ana i nā pilikia i loaʻa i ka hoʻoheheʻe ʻana i ke kūkulu lua ikaika ma RNA i nā wela kiʻekiʻe.

· Ma muli o ke kumu hoʻomaka wela e pili ana i ka aptamer, RevTaq RT-PCR DNA polymerase e hoʻopuka i nā hopena ʻoi aku ka maikaʻi ke kiʻekiʻe ke kiʻekiʻe o ka annealing a me ka hoʻonui ʻana ma mua o 57°C.

· No ka mea he mea wela ka enzyme, pono ia e hoʻolālā i nā kumu mua a me nā ʻimi me nā helu heheʻe kiʻekiʻe loa (>60°C).

· Manaʻo ʻia e hoʻomaikaʻi i ka mahana o ka annealing/extension step ma waena o kahi gradient wela i ka wā o ke kaʻina hana hoʻonohonoho.

· ʻO ke kiʻekiʻe o ka mahana, ʻoi aku ka kiʻekiʻe o ka kikoʻī o PCR.Hana ʻia ka pōʻaiapili hoʻohuli ʻana ma kahi wela kiʻekiʻe ma mua o ka pōʻaiapuni PCR, no ka mea, ʻoi aku ka nui o ka helu heheʻe o ka DNA Primer: RNA Template hybridization ma mua o ka DNA Primer: cDNA Template duplex.

· RevTaq RT-PCR DNA polymerase he genetically engineered a optimized, a aia ka nui amplicon ma waena o 60-300 bp.

ʻO RevTaq RT-PCR DNA polymerase palena palena hiki i 4 kope/

Hōʻike ʻia ka ʻōnaehana hopena maikaʻi (ka hoʻokumu ʻana i nā kumu hoʻoheheʻe kiʻekiʻe) ma ke kiʻi.Hōʻike ʻo RevTaq RT-PCR DNA polymerase-driven RT-PCR i ka ʻoi aku ka maikaʻi ma mua o TaqPath 1-step RT-qPCR master mix, a me ka ʻike haʻahaʻa.

Nā mea maikaʻi hou aku:

Hana hoʻomaka wikiwiki → Hiki ke hoʻokuʻu ʻia ke kaʻina denaturation thermal mua.

Hoʻomaka wela aptamer formula → Hāʻawi koke i ka hana enzyme 100% a pale i ka hoʻonui ʻole kikoʻī ma nā wela haʻahaʻa (<57°C).

Hana ʻia ʻo Cleavage → Hoʻopau ʻia ka ʻanuʻu ʻoki ʻana o RNA, no ka mea, hiki nō iā RevTaq RT-PCR DNA polymerase ke hoʻoponopono i nā laʻana.Hiki iā ia ke hoʻopau koke i nā membrane cell o nā eukaryotes, bacteria a me nā maʻi i loko o kahi pōʻai wela RT-PCR.

IVD raw material level → nā kūlana kiʻekiʻe a me nā kumukūʻai hoʻokūkū loa