Fluorescence quantitative PCR (eo hape e tsejoang ka hore ke TaqMan PCR, eo hamorao e bitsoang FQ-PCR) ke theknoloji e ncha ea nucleic acid quantitative e entsoeng ke PE (Perkin Elmer) United States ka 1995. Theknoloji ena e itšetlehile ka PCR e tloaelehileng ka ho eketsa lisebelisoa tse ngotsoeng ka fluorescent.Ha ho bapisoa le PCR e tenyetsehang, FQ-PCR e na le melemo e mengata ea ho hlokomela tšebetso ea eona ea bongata.Sengoliloeng sena se ikemiselitse ho hlalosa ka bokhutšoanyane litšobotsi, melao-motheo, mekhoa le ts'ebeliso ea theknoloji.
1 Likarolo
FQ-PCR ha e na kutlo e phahameng feela ea PCR e tloaelehileng, empa hape ka lebaka la ts'ebeliso ea li-fluorescent probes, e khona ho bona ka kotloloho phetoho ea lets'oao la fluorescent nakong ea ntlafatso ea PCR ka sistimi ea photoelectric conduction ho fumana liphetho tse ngata, tse hlolang mefokolo e mengata ea PCR e tloaelehileng, ka hona e boetse e na le boits'oaro bo phahameng ba DNA hybrid hybridization.
Mohlala, lihlahisoa tse akaretsang tsa PCR li hloka ho bonoa ke agarose gel electrophoresis le ethidium bromide staining e nang le khanya ea ultraviolet kapa ka polyacrylamide gel electrophoresis le matheba a silevera.Sena ha se hloke lisebelisoa tse ngata feela, empa hape se hloka nako le boiteko.Matheba a sebelisitsoeng Ethidium bromide a kotsi 'meleng oa motho,' me mekhoa ena e rarahaneng ea liteko e fana ka menyetla ea tšilafalo le maikutlo a bohata.Leha ho le joalo, FQ-PCR e hloka feela ho bula sekoahelo hanngoe nakong ea ho kenya sampuli, 'me ts'ebetso e latelang ke ts'ebetso ea tube e koetsoeng ka ho feletseng, e sa hlokeng PCR post-processing, ho qoba litšitiso tse ngata ts'ebetsong e tloaelehileng ea PCR.Teko ka kakaretso e sebelisa ABI7100 PCR sebaesekele se futhumatsang se entsoeng ke k'hamphani ea PE.
Sesebelisoa se na le litšobotsi tse latelang: ① Ts'ebeliso e pharalletseng: E ka sebelisoa bakeng sa palo ea lihlahisoa tsa DNA le RNA PCR, lipatlisiso tsa liphatsa tsa lefutso, ho lemoha pathogen, le ho ntlafatsa maemo a PCR.② Molao-motheo o ikhethileng oa palo: Ho sebelisa li-probes tse ngotsoeng ka fluorescent, palo ea fluorescence e tla bokellana le potoloho ea PCR kamora ho hlasimoloha ha laser, e le ho fihlela sepheo sa quantification.③ Ts'ebetso e phahameng ea ts'ebetso: E hahelletsoeng ka har'a 9600 PCR cycler e futhumatsang, komporo e laoloa 1 ho lihora tsa 2 ho phethela katoloso le palo ea lisampole tse 96 ka bo eona le ka mokhoa o lumellanang.④ Ha ho na tlhoko ea gel electrophoresis: Ha ho na tlhoko ea ho hlapolla le electrophoresis sampole, sebelisa feela probe e khethehileng ho bona ka kotloloho mothapong oa karabelo.⑤Ha ho na tšilafalo liphaepheng: Ho amoheloa ka mokhoa o ikhethileng oa karabelo tube le photoelectric conduction system, kahoo ha ho na lebaka la ho tšoenyeha ka tšilafalo.⑥Liphetho li ka ikatisetsa hape: palo e matla ea mefuta e fihlang ho liodara tse hlano tsa boholo.Ka hona, kaha theknoloji ena e ile ea ntlafatsoa ka katleho, e ’nile ea nkoa e le ea bohlokoa ke bafuputsi ba bangata ba saense ’me e ’nile ea sebelisoa mafapheng a mangata.
2 Melao-motheo le mekhoa
Molao-motheo oa ts'ebetso oa FQ-PCR ke ho sebelisa ts'ebetso ea 5′→3′ exonuclease ea enzyme ea Taq ho eketsa probe e ngotsoeng ka fluorescent ho sistimi ea karabelo ea PCR.Probe e ka kopanngoa ka ho khetheha le template ea DNA e fumanehang ka tatellano ea pele.Qetello ea 5 ea probe e ngotsoe ka mofuta oa fluorescence emission FAM (6-carboxyfluorescein, tlhoro ea tlhahiso ea fluorescence ho 518nm), 'me 3'end e ngotsoe ka The fluorescence quenching sehlopha TAMRA (6-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, 2orescence emission 518nm) phosphorylated ho thibela probe ho atolosoa nakong ea ntlafatso ea PCR.Ha probe e lula e tiile, sehlopha sa quencher se hatella tlhahiso ea fluorescence ea sehlopha se ntšang.Hang ha sehlopha se emitting se arohane le sehlopha sa quenching, thibelo ea phahamisoa, 'me boholo ba optical ho 518nm bo eketseha' me bo bonoa ke mokhoa oa ho lemoha ha fluorescence.Ha probe e khaotsoe, phello ea ho tima e lokolloa 'me letšoao la fluorescent le lokolloe.Nako le nako ha template e kopitsoa, probe e khaoloa, e tsamaisana le ho lokolloa ha lets'oao la fluorescent.Kaha ho na le kamano e le 'ngoe pakeng tsa palo ea fluorophores e lokollotsoeng le palo ea lihlahisoa tsa PCR, mokhoa ona o ka sebelisoa ho lekanya template ka nepo.Sesebelisoa sa liteko hangata se sebelisa ABI7100 PCR sebaesekele se futhumatsang se entsoeng ke k'hamphani ea PE, 'me libaesekele tse ling tsa mocheso le tsona li ka sebelisoa.Haeba mokhoa oa karabelo oa mofuta oa ABI7700 o sebelisoa bakeng sa teko, kamora hore karabelo e phetheloe, liphetho tsa palo li ka fanoa ka kotloloho ka tlhahlobo ea komporo.Haeba u sebelisa libaesekele tse ling tse futhumatsang, u lokela ho sebelisa sesebelisoa sa fluorescence ho lekanya lets'oao la fluorescence ka har'a tube ea karabelo ka nako e ts'oanang ho bala RQ+, RQ-, △RQ.RQ+ e emela karo-karolelano ea matla a luminescence a sehlopha sa phallo ea fluorescent ea tube ea sampole ho matla a luminescence a sehlopha sa quenching, RQ- e emela karo-karolelano ea tse peli ka har'a tube e se nang letho, △RQ (△RQ = RQ + -RQ-) e emela palo ea liphetho tsa data tsa fluorescence ka mor'a hore ho fumanoe liphetoho tsa data tsa fluorescence.Ka lebaka la ho kenngoa ha lisebelisoa tsa fluorescent, ho khetheha ha teko ho ntlafala haholo.Moralo oa probe ka kakaretso o tlameha ho fihlela lipehelo tse latelang: ①Bolelele ba probe e lokela ho ba li-bases tse ka bang 20-40 ho netefatsa bonnete ba ho tlama.②Likahare tsa metheo ea GC li pakeng tsa 40% le 60% ho qoba ho pheta-pheta ha tatellano e le 'ngoe ea nucleotide.③ Qoba ho kopanya kapa ho kopana le li-primers.④ Botsitso ba tlamahano pakeng tsa probe le template e kholo ho feta botsitso ba tlamahano pakeng tsa primer le template, kahoo boleng ba Tm ba probe bo lokela ho ba bonyane 5 ° C ho feta boleng ba Tm ba primer.Ho feta moo, ho tsepama ha lipatlisiso, homology pakeng tsa probe le tatellano ea thempleite, le sebaka se pakeng tsa probe le primer kaofela li na le tšusumetso liphethong tsa liteko.
Lihlahisoa tse amanang:
China Real Time PCR Easyᵀᴹ-Taqman Manufacturer le Supplier |Foregene (foreivd.com)
Nako ea poso: Oct-15-2021
