I-Fluorescence quantitative PCR (eyaziwa nangokuthi i-TaqMan PCR, kamuva ebizwa ngokuthi i-FQ-PCR) ubuchwepheshe bobuningi be-nucleic acid obusha obuthuthukiswe yi-PE (Perkin Elmer) e-United States ngo-1995. Lobu buchwepheshe busekelwe ku-PCR evamile ngokungeza ama-probe anelebuli e-fluorescent.Uma kuqhathaniswa ne-PCR eguquguqukayo, i-FQ-PCR inezinzuzo eziningi zokubona umsebenzi wayo wobuningi.Le ndatshana ihlose ukuchaza kafushane izici, izimiso, izindlela, nokusetshenziswa kobuchwepheshe.

1 Izici

I-FQ-PCR ayinakho nje kuphela ukuzwela okuphezulu kwe-PCR evamile, kodwa futhi ngenxa yokusetshenziswa kwama-fluorescent probes, ingakwazi ukubona ngokuqondile ukuguqulwa kwesignali ye-fluorescent ngesikhathi sokukhulisa i-PCR ngohlelo lwe-photoelectric conduction ukuze ithole imiphumela yobuningi, enqoba ukushiyeka okuningi kwe-PCR evamile, ngakho futhi inokucaciswa okuphezulu kwe-DNA specturacy kanye nobuchwepheshe obuphezulu be-DNA specturacy.

Isibonelo, imikhiqizo ye-PCR evamile idinga ukubhekwa nge-agarose gel electrophoresis kanye ne-ethidium bromide staining ngokukhanya kwe-ultraviolet noma nge-polyacrylamide gel electrophoresis kanye ne-silver staining.Lokhu akudingi kuphela izinsimbi eziningi, kodwa futhi kuthatha isikhathi nomzamo.Amabala asetshenziswa i-Ethidium bromide ayingozi emzimbeni womuntu, futhi lezi zinqubo zokuhlola eziyinkimbinkimbi zinikeza amathuba okungcola kanye nemibono engamanga.Kodwa-ke, i-FQ-PCR idinga kuphela ukuvula isivalo kanye ngesikhathi sokulayisha isampula, futhi inqubo elandelayo iwukusebenza kweshubhu evaliwe ngokuphelele, okungadingi ukucutshungulwa kwangemuva kwe-PCR, ukugwema izithiyo eziningi ekusebenzeni kwe-PCR evamile.Ukuhlolwa ngokuvamile kusebenzisa i-ABI7100 PCR yomjikelezo oshisayo othuthukiswe inkampani ye-PE.

Ithuluzi linezici ezilandelayo: ① Ukusetshenziswa okubanzi: Ingasetshenziselwa i-DNA kanye ne-RNA PCR ukulinganisa umkhiqizo, ucwaningo lwesisho sofuzo, ukutholwa kwe-pathogen, kanye nokwenza kahle kwezimo ze-PCR.② Isimiso sobuningi esihlukile: Kusetshenziswa ama-probe anelebuli e-fluorescent, inani le-fluorescence lizonqwabelana ngomjikelezo we-PCR ngemva kokuvuswa kwe-laser, ukuze kufezwe inhloso yokulinganisa.③ Ukusebenza kahle okuphezulu: Ibhayisikili elishisayo elakhelwe ngaphakathi elingu-9600 PCR, ikhompuyutha elawulwa amahora angu-1 kuye kwayi-2 ukuze kuqedelwe ukukhulisa nokulinganisa amasampula angama-96 ngokuzenzakalelayo nangokuvumelanayo.④ Asikho isidingo sejeli ye-electrophoresis: Asikho isidingo sokuhlanjululwa kanye ne-electrophoresis isampula, vele usebenzise i-probe ekhethekile ukuthola ngqo kushubhu yokusabela.⑤Akukho ukungcola epayipini: Ishubhu lokusabela elivalwe ngokugcwele elivalwe ngokugcwele kanye nesistimu ye-photoelectric conduction iyamukelwa, ngakho-ke asikho isidingo sokukhathazeka ngokungcola.⑥Imiphumela iyakhiqizeka kabusha: ububanzi obuguquguqukayo bomthamo bufika kuma-oda amahlanu obukhulu.Ngakho-ke, njengoba lobu buchwepheshe buthuthukiswe ngempumelelo, buye baziswa abacwaningi besayensi abaningi futhi sebusetshenziswe emikhakheni eminingi.

2 Izimiso nezindlela

Umgomo wokusebenza we-FQ-PCR ukusebenzisa umsebenzi we-5′→3′ we-exonuclease we-enzyme ye-Taq ukwengeza uphenyo olunelebula olukhanyayo ohlelweni lokusabela lwe-PCR.I-probe ingakwazi ukuhlanganisa ngokukhethekile isifanekiso se-DNA esiqukethwe ngokulandelana kokuqala.I-5′end yophenyo ifakwe ilebula yofuzo olukhipha i-fluorescence emission FAM (6-carboxyfluorescein, i-fluorescence emission peak in 518nm), kanti i-3′end ibhalwe ngokuthi Iqembu le-fluorescence quenching TAMRA (6-carboxytetramethylrhodamine, i-fluorescence emission 518nm) i-phosphorylated ukuvimbela uphenyo ukuthi lunganwetshwa ngesikhathi sokukhulisa i-PCR.Lapho uphenyo luhlala lunjalo, iqembu le-quencher licindezela ukukhishwa kwe-fluorescence kweqembu elikhiphayo.Uma iqembu elikhiphayo selihlukanisiwe neqembu lokucisha, ukuvimbela kuyasuswa, futhi ukuminyana kokubona ku-518nm kuyanda futhi kutholwa uhlelo lokubona i-fluorescence.Esigabeni sokuguqulwa kabusha, uphenyo luhlanganiswa ne-DNA yesifanekiso, futhi i-enzyme ye-Taq esigabeni sokunwetshwa ihamba eduze kwesandiso se-DNA isifanekiso.Lapho i-probe inqanyuliwe, umphumela wokucisha uyakhululwa futhi isignali ye-fluorescent iyakhululwa.Ngaso sonke isikhathi uma isifanekiso sikopishwa, i-probe iyanqanyulwa, ihambisane nokukhishwa kwesignali ye-fluorescent.Njengoba kunobudlelwano obubodwa phakathi kwenani le-fluorophore ekhishwe kanye nenani lemikhiqizo ye-PCR, le nqubo ingasetshenziswa ukulinganisa ngokunembile isifanekiso.Ithuluzi lokuhlola ngokuvamile lisebenzisa umjikelezo oshisayo we-ABI7100 PCR othuthukiswe inkampani ye-PE, namanye amabhayisikili ashisayo angasetshenziswa.Uma isistimu yokusabela yohlobo lwe-ABI7700 isetshenziselwa ukuhlola, ngemva kokuqedwa kokusabela, imiphumela yobuningi inganikezwa ngokuqondile ngokuhlaziywa kwekhompyutha.Uma usebenzisa amanye amabhayisikili ashisayo, udinga ukusebenzisa umtshina we-fluorescence ukukala isignali ye-fluorescence kushubhu yokusabela ngesikhathi esifanayo ukuze ubale i-RQ+, RQ-, △RQ.I-RQ+ imele isilinganiso sokushuba kwe-luminescence kweqembu elikhipha i-fluorescent leshubhu lesampula kumfutho we-luminescence weqembu lokucisha, i-RQ- imele isilinganiso salokhu okubili eshubhuni elingenalutho, △RQ (△RQ=RQ+ -RQ-) imelela inani lemiphumela yesignali ye-fluorescence etholwe ngesikhathi sokuguqulwa kwedatha ye-PCquaRntita.Ngenxa yokwethulwa kwama-fluorescent probes, ukucaciswa kokuhlolwa kuthuthukiswa kakhulu.Idizayini ye-probe kufanele ihlangabezane nalezi zimo ezilandelayo: ①Ubude be-probe kufanele bube yizisekelo ezingaba ngu-20-40 ukuze kuqinisekiswe ukucaciswa kokubophezela.②Okuqukethwe kwezisekelo ze-GC kuphakathi kuka-40% no-60% ukuze kugwenywe impinda yokulandelana kwe-nucleotide eyodwa.③ Gwema ukuxutshwa noma ukugqagqana neziqalo.④ Ukuzinza kokubophezela phakathi kwe-probe nesifanekiso kukhulu kunokuzinza kokubopha phakathi kwe-primer nesifanekiso, ngakho-ke inani le-Tm lophenyo kufanele okungenani libe ngu-5°C ngaphezu kwevelu ye-Tm ye-primer.Ukwengeza, ukugxila kophenyo, i-homology phakathi kwe-probe nokulandelana kwesifanekiso, nebanga phakathi kwe-probe ne-primer konke kunomthelela emiphumeleni yokuhlola.

Imikhiqizo ehlobene:

I-China Lnc-RT Heroᵀᴹ I(With gDNase)(Super Premix for first-strand cDNA synthesis from lncRNA) Umkhiqizi Nomphakeli |I-Foregene (foreivd.com)

I-China Real Time PCR Easyᵀᴹ-Taqman Umkhiqizi Nomhlinzeki |I-Foregene (foreivd.com)