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 Fluorescence quantitative PCR (inozivikanwawo seTaqMan PCR, inozonzi FQ-PCR) inyanzvi itsva yenucleic acid quantitative tekinoroji yakagadziridzwa nePE (Perkin Elmer) muUnited States muna 1995. Tekinoroji iyi inobva paPCR yakajairika nekuwedzera maprobes ane fluorescent akanyorwa.Kuenzaniswa nePCR inochinjika, FQ-PCR ine zvakawanda zvakanakira kuona huwandu hwayo basa.Ichi chinyorwa chinotarisira kutsanangura muchidimbu maitiro, misimboti, nzira, uye mashandisirwo etekinoroji.

1 Zvinoumba

FQ-PCR haingove nekunzwa kwakanyanya kwePCR yakajairwa, asiwo nekuda kwekushandiswa kwefluorescent probes, inogona kuona zvakananga shanduko yefluorescent chiratidzo panguva yePCR kukwidziridzwa kuburikidza neiyo photoelectric conduction system kuti iwane huwandu hwemhedzisiro, iyo inokunda kukanganisa kwakawanda kweyakajairwa PCR, saka inewo yakakwira hunhu hweiyo DNA hybridization uye highoscopy tekinoroji.

Semuyenzaniso, zvakajairwa PCR zvigadzirwa zvinoda kutariswa neagarose gel electrophoresis uye ethidium bromide staining ne ultraviolet light kana ne polyacrylamide gel electrophoresis uye sirivheri stain.Izvi hazvingodi midziyo yakawanda chete, asiwo zvinotora nguva uye kushanda nesimba.Mavara anoshandiswa Ethidium bromide anokuvadza kumuviri wemunhu, uye aya akaomarara ekuedza maitiro anopa mikana yekusvibiswa uye manyepo.Nekudaro, FQ-PCR inongoda kuvhura chivharo kamwe chete panguva yekurodha sampuli, uye iyo inotevera nzira yakavharwa-chubhu oparesheni, iyo isingade PCR post-processing, ichidzivirira zvakawanda zvinokanganisa mune yakajairika PCR mashandiro.Kuedza kunowanzo shandisa iyo ABI7100 PCR inopisa cycler yakagadziriswa nekambani yePE.

Chiridzwa chine zvinotevera hunhu: ① Wide application: Inogona kushandiswa kuDNA uye RNA PCR chigadzirwa quantification, gene expression tsvakiridzo, pathogen kuona, uye optimization yePCR mamiriro.② Yakasarudzika kuwanda musimboti: Uchishandisa fluorescent yakanyorwa probes, huwandu hwefluorescence huchaungana pamwe nePCR kutenderera mushure meiyo laser excitation, kuitira kuti uwane chinangwa chehuwandu.③ Kushanda kwakanyanya kwekushanda: Yakavakwa-mu9600 PCR yekupisa cycler, komputa inodzorwa 1 kusvika 2 maawa kuti ipedze kukwidziridzwa uye kuwanda kwemasample makumi mapfumbamwe nematanhatu otomatiki uye synchronously.④ Hapana chikonzero che gel electrophoresis: Hapana chikonzero chekudzikisa uye electrophoresis sampuli, ingoshandisa yakakosha probe kuona zvakananga muhubhu yekuita.⑤Hapana kusvibiswa mupombi: Iyo yakasarudzika yakavharika yakazara reaction chubhu uye photoelectric conduction system inogamuchirwa, saka hapana chikonzero chekunetseka nezve kusvibiswa.⑥ Mhedzisiro yacho inogadzirwazve: huwandu hwesimba renji rinosvika pamaodha mashanu ehukuru.Naizvozvo, sezvo tekinoroji iyi yakagadziridzwa zvakabudirira, yakakosheswa nevazhinji vaongorori vesainzi uye yakashandiswa munzvimbo dzakawanda.

2 Nheyo uye nzira

Nheyo yekushanda yeFQ-PCR ndeye kushandisa iyo 5′→3′ exonuclease chiitiko cheTaq enzyme kuwedzera fluorescent yakanyorwa probe kuPCR reaction system.Iyo probe inogona kunyanya kusanganiswa neiyo DNA template iri mukutanga kutevedzana.Iyo 5'kuguma kweiyo probe yakanyorwa neiyo fluorescence emission gene FAM (6-carboxyfluorescein, fluorescence emission peak pa518nm), uye iyo 3'end yakanyorwa neThe fluorescence quenching boka TAMRA (6-carboxytetramethylrhodamine, 3-carboxytetramethylrhodamine, fluorescence emission peak8 phosphorylated kudzivirira iyo probe kuti isawedzerwe panguva yePCR amplification.Kana iyo probe ikaramba yakasimba, boka re quencher rinodzvinyirira kubuda kwefluorescence yeboka rinobudisa.Kana boka rekubudisa rikaparadzaniswa kubva kuboka rekudzima, inhibition inosimudzwa, uye kupenya kwemaziso pa518nm kunowedzera uye kunoonekwa nefluorescence yekuongorora system.Muchikamu chekudzorerazve, probe inosanganiswa ne template DNA, uye enzyme yeTaq muchikamu chekuwedzera inofamba pamwe nekuwedzera kweDNA template ine template.Kana iyo probe yakagurwa, iyo yekudzima inoburitswa uye chiratidzo chefluorescent chinoburitswa.Nguva imwe neimwe iyo template inokopwa, probe inogurwa, inoperekedzwa nekuburitswa kwechiratidzo chefluorescent.Sezvo paine hukama hweumwe-kune-umwe pakati pehuwandu hweakaburitswa fluorophores uye nhamba yePCR zvigadzirwa, iyi nzira inogona kushandiswa kunyatso kuenzanisa template.Chimbo chekuyedza chinowanzo shandisa iyo ABI7100 PCR yekupisa cycler yakagadziriswa nekambani yePE, uye mamwe mabhasikoro ekupisa anogona kushandiswa zvakare.Kana iyo ABI7700 reaction type reaction system ikashandiswa kuyedza, mushure mekuita kwapera, mhedzisiro yehuwandu inogona kupihwa zvakananga kuburikidza nekuongorora komputa.Kana iwe ukashandisa mamwe mabhasikoro ekupisa, iwe unofanirwa kushandisa fluorescence detector kuyera iyo fluorescence siginecha mune rekuita chubhu panguva imwe chete kuverenga RQ+, RQ-, △RQ.RQ + inomiririra chiyero chekupenya kwechiedza che fluorescent emission group yechubhu yemuenzaniso kune luminescence intensity yeboka rekudzima, RQ- inomiririra chiyero chevaviri muchubhu isina chinhu, △RQ (△RQ = RQ + -RQ-) inomiririra huwandu hweiyo fluorescence yekushandurwa kwe data.Nekuda kwekuiswa kwefluorescent probes, iyo chaiyo yekuyedza inovandudzwa zvakanyanya.Iyo probe dhizaini inofanirwa kusangana neanotevera mamiriro: ①Kureba kweprobe kunofanirwa kunge kuri makumi maviri-40 mabhesi kuti ive nechokwadi chekusunga.②Zviri mukati meGC zvigadziko zviri pakati pe40% ne60% kudzivirira kudzokorora kwekamwechete nucleotide sequences.③ Dzivisa kusanganiswa kana kupindirana nemaprimers.④ Kugadzikana kwekusungirirwa pakati peprobe uye template yakakura kudarika kugadzikana kwekusungirirwa pakati pekutanga uye template, saka kukosha kweTm kweprobe kunofanira kunge kune 5 ° C yakakwirira kudarika kukosha kweTm kwekutanga.Pamusoro pezvo, kusanganisirwa kweprobe, iyo homology pakati peprobe uye template kutevedzana, uye chinhambwe pakati peprobe neprimer zvese zvine chekuita pamhedzisiro yekuyedza.

Zvigadzirwa zvinoenderana:

China Lnc-RT Heroᵀᴹ I(Ne gDNase)(Super Premix yekutanga-strand cDNA synthesis kubva kulncRNA) Mugadziri uye Mutengesi |Foregene (foreivd.com)

China Real Time PCR Easyᵀᴹ-Taqman Manufacturer uye Supplier |Foregene (foreivd.com)


Nguva yekutumira: Oct-15-2021