Fluorescence quantitative PCR (wanda kuma aka sani da TaqMan PCR, daga baya ake magana da shi a matsayin FQ-PCR) sabuwar fasaha ce ta ƙididdige adadin acid nucleic da PE (Perkin Elmer) ta haɓaka a Amurka a cikin 1995. Wannan fasaha ta dogara ne akan PCR na al'ada ta hanyar ƙara abubuwan bincike mai suna fluorescent.Idan aka kwatanta da PCR mai sassauƙa, FQ-PCR yana da fa'idodi da yawa don gane yawan aikin sa.Wannan labarin yana nufin a taƙaice bayyana halaye, ƙa'idodi, hanyoyin, da aikace-aikacen fasaha.
1 Features
FQ-PCR ba wai kawai yana da babban ji na PCR na yau da kullun ba, amma kuma saboda aikace-aikacen bincike na kyalli, yana iya gano canjin siginar kyalli kai tsaye yayin haɓakawar PCR ta hanyar tsarin sarrafa hoto don samun sakamako mai ƙima, wanda ke shawo kan ƙarancin ƙarancin PCR na al'ada, don haka yana da babban ƙayyadaddun ƙayyadaddun DNA hybridization da fasaha mai girma na spectroscopy.
Misali, samfuran PCR na gaba ɗaya suna buƙatar a lura da su ta hanyar agarose gel electrophoresis da ethidium bromide tabo tare da hasken ultraviolet ko ta polyacrylamide gel electrophoresis da tabon azurfa.Wannan ba kawai yana buƙatar kayan aiki da yawa ba, amma kuma yana ɗaukar lokaci da ƙoƙari.Tabon da aka yi amfani da su na Ethidium bromide yana da illa ga jikin ɗan adam, kuma waɗannan rikitattun hanyoyin gwaji suna ba da damammaki ga gurɓata yanayi da haɓakar ƙarya.Koyaya, FQ-PCR kawai yana buƙatar buɗe murfi sau ɗaya yayin ɗaukar samfuri, kuma tsarin na gaba yana aiki ne gaba ɗaya rufe-tube, wanda baya buƙatar aiwatar da PCR, yana guje wa raguwa da yawa a cikin ayyukan PCR na al'ada.Gwajin gabaɗaya yana amfani da ABI7100 PCR thermal cycler wanda kamfanin PE ya haɓaka.
Kayan aiki yana da halaye masu zuwa: ① Faɗin aikace-aikacen: Ana iya amfani da shi don ƙididdige samfuran samfuran DNA da RNA PCR, binciken maganganun kwayoyin halitta, gano ƙwayoyin cuta, da haɓaka yanayin PCR.② Ƙa'idar ƙididdiga ta musamman: Yin amfani da bincike mai alamar haske, adadin haske zai tara tare da zagayowar PCR bayan tashin hankali na laser, don cimma manufar ƙididdigewa.③ Babban ingancin aiki: Gina 9600 PCR thermal cycler, kwamfuta sarrafa 1 zuwa 2 hours don kammala haɓakawa da ƙididdige samfuran 96 ta atomatik da aiki tare.④ Babu buƙatar gel electrophoresis: Babu buƙatar tsarma da electrophoresis samfurin, kawai amfani da bincike na musamman don gano kai tsaye a cikin bututun amsawa.⑤Babu gurɓataccen abu a cikin bututun: An karɓi bututun amsawa na musamman da tsarin gudanarwa na hoto, don haka babu buƙatar damuwa game da gurɓatawa.⑥ Sakamako suna iya sakewa: madaidaicin kewayo mai ƙarfi ya kai umarni biyar na girma.Don haka, tun lokacin da aka samu nasarar samar da wannan fasaha, masana kimiyya da dama sun yi amfani da ita a fannoni da dama.
2 Ka'idoji da hanyoyin
Ka'idar aiki na FQ-PCR shine yin amfani da 5'→3' ayyukan exonuclease na Taq enzyme don ƙara bincike mai alamar haske zuwa tsarin amsawar PCR.Binciken na iya haɗawa musamman tare da samfurin DNA wanda ke ƙunshe a cikin jeri na farko.Ƙarshen 5'arshen binciken ana lakafta shi tare da ƙwayar fluorescence emission gene FAM (6-carboxyfluorescein, fluorescence emission peak a 518nm), kuma 3'end an lakafta shi tare da The Fluorescence quenching group TAMRA (6-carboxytetramethylrhodamine, peakginning da 3) phosphorylated don hana ƙaddamar da binciken yayin haɓaka PCR.Lokacin da binciken ya kasance lafiyayye, ƙungiyar masu kashe wutar lantarki tana hana fitar da hayaƙi na ƙungiyar masu fitarwa.Da zarar an rabu da ƙungiyar emitting daga ƙungiyar masu kashewa, an ɗaga hanawa, kuma ƙimar gani a 518nm yana ƙaruwa kuma ana gano shi ta tsarin gano haske.Lokacin da aka yanke binciken, ana fitar da tasirin quenching kuma ana sakin siginar kyalli.Duk lokacin da aka kwafi samfurin, ana yanke bincike, tare da sakin siginar kyalli.Tun da akwai alaƙa ɗaya zuwa ɗaya tsakanin adadin da aka fitar da fluorophores da adadin samfuran PCR, ana iya amfani da wannan dabara don ƙididdige samfurin daidai.Kayan aikin gwajin gabaɗaya yana amfani da ABI7100 PCR thermal cycler wanda kamfanin PE ya haɓaka, kuma ana iya amfani da sauran masu kekuna masu zafi.Idan an yi amfani da tsarin amsawa na nau'in amsawa na ABI7700 don gwaji, bayan an gama amsawa, ana iya ba da sakamakon ƙididdiga kai tsaye ta hanyar nazarin kwamfuta.Idan kuna amfani da sauran masu kekuna na thermal, kuna buƙatar amfani da na'urar gano haske don auna siginar kyalli a cikin bututun dauki lokaci guda don lissafin RQ+, RQ-, △RQ.RQ + wakiltar da rabo daga cikin luminescence tsanani na kyalli watsi kungiyar na samfurin tube zuwa luminescence tsanani na quenching kungiyar, RQ- wakiltar da rabo daga cikin biyu a cikin blank tube, △ RQ (△ RQ = RQ + -RQ-) wakiltar adadin fluorescence sakamakon canji na iya zama da yawa sakamakon canji na PC.Saboda gabatarwar bincike na fluorescent, ƙayyadaddun gwajin yana inganta sosai.Tsarin binciken ya kamata gabaɗaya ya cika waɗannan sharuɗɗan: ① Tsawon binciken ya kamata ya zama kusan tushe 20-40 don tabbatar da ƙayyadaddun ɗauri.② Abubuwan da ke cikin tushen GC yana tsakanin 40% da 60% don guje wa kwafi na jerin nucleotide guda ɗaya.③ Guji haɗawa ko haɗawa tare da firamare.④ Tsayayyar dauri tsakanin bincike da samfuri ya fi girma fiye da kwanciyar hankali na ɗaure tsakanin firam da samfuri, don haka ƙimar Tm na binciken ya kamata ya zama aƙalla 5 ° C sama da ƙimar Tm na firam.Bugu da ƙari, ƙaddamar da bincike, homology tsakanin bincike da jerin samfuri, da nisa tsakanin binciken da na farko duk suna da tasiri akan sakamakon gwaji.
Samfura masu alaƙa:
China Real Time PCR Easyᵀᴹ-Taqman Manufacturer and Supplier |Foregene (foreivd.com)
Lokacin aikawa: Oktoba-15-2021
