Fluorescence quantitative PCR (yomwe imadziwikanso kuti TaqMan PCR, yomwe pambuyo pake imatchedwa FQ-PCR) ndiukadaulo watsopano wa nucleic acid quantitative wopangidwa ndi PE (Perkin Elmer) ku United States mu 1995. Ukadaulowu umachokera pa PCR wamba powonjezera ma probe olembedwa ndi fulorosenti.Poyerekeza ndi PCR yosinthika, FQ-PCR ili ndi zabwino zambiri kuti izindikire kuchuluka kwake.Nkhaniyi ikufuna kufotokoza mwachidule mawonekedwe, mfundo, njira, ndi kagwiritsidwe ntchito kaukadaulo.
1 Zinthu
FQ-PCR sikuti imakhala ndi chidwi chachikulu cha PCR wamba, komanso chifukwa chogwiritsa ntchito ma probes a fulorosenti, imatha kuzindikira mwachindunji kusintha kwa siginecha ya fulorosenti panthawi ya kukulitsa kwa PCR kudzera mu dongosolo la photoelectric conduction kuti ipeze zotsatira zochulukirapo, zomwe zimapambana zophophonya zambiri za PCR wamba, motero ilinso ndi kutsimikizika kwakukulu kwa kusakanizidwa kwa DNA ndiukadaulo wapamwamba kwambiri.
Mwachitsanzo, zinthu zonse za PCR ziyenera kuwonedwa ndi agarose gel electrophoresis ndi ethidium bromide deined ndi kuwala kwa ultraviolet kapena polyacrylamide gel electrophoresis ndi siliva.Izi sizimangofunika zida zingapo, komanso zimatengera nthawi ndi khama.Madontho omwe amagwiritsidwa ntchito Ethidium bromide ndi owopsa m'thupi la munthu, ndipo njira zovuta zoyeserazi zimapereka mpata woipitsa ndi zolakwika zabodza.Komabe, FQ-PCR imangofunika kutsegula chivindikiro kamodzi panthawi yotsegula zitsanzo, ndipo ndondomeko yotsatirayi imakhala yotsekedwa kwathunthu, yomwe sikutanthauza kuti PCR ikasinthidwe, kupeŵa zovuta zambiri pazochitika za PCR.Kuyeserako nthawi zambiri kumagwiritsa ntchito ABI7100 PCR matenthedwe cycler yopangidwa ndi kampani ya PE.
Chidachi chili ndi izi: ① Ntchito yayikulu: Itha kugwiritsidwa ntchito powerengera kuchuluka kwazinthu za DNA ndi RNA PCR, kafukufuku wama gene, kuzindikira tizilombo toyambitsa matenda, komanso kukhathamiritsa kwa PCR.② Mfundo yapadera yochulukira: Pogwiritsa ntchito ma probe olembedwa ndi fulorosenti, kuchuluka kwa fluorescence kudzaunjikana ndi kuzungulira kwa PCR pambuyo pa chisangalalo cha laser, kuti mukwaniritse cholinga cha kuchuluka.③ Kuchita bwino kwambiri: Kumangidwa mu 9600 PCR yoyendetsa matenthedwe, makompyuta amawongoleredwa maola 1 mpaka 2 kuti amalize kukulitsa ndi kuchuluka kwa zitsanzo 96 zokha komanso mogwirizana.④ Palibe chifukwa cha gel electrophoresis: Palibe chifukwa chochepetsera ndi electrophoresis chitsanzo, ingogwiritsani ntchito kafukufuku wapadera kuti muzindikire mwachindunji mu chubu.⑤Palibe kuipitsidwa kwapaipi: Njira yapadera yotsekeredwa yotsekeredwa ndi ma photoelectric conduction system amatengera, kotero palibe chifukwa chodera nkhawa za kuipitsa.⑥Zotsatira zake ndi zobwezeredwa: kuchuluka kwa dynamic range mpaka maoda asanu a ukulu wake.Choncho, popeza lusoli linapangidwa bwino, layamikiridwa ndi akatswiri ambiri a sayansi ndipo lagwiritsidwa ntchito m'madera ambiri.
2 Mfundo ndi njira
Mfundo yogwira ntchito ya FQ-PCR ndikugwiritsa ntchito 5′→3′ exonuclease zochita za Taq enzyme kuwonjezera kafukufuku wolembedwa ndi fluorescent ku PCR reaction system.Kafukufukuyu amatha kusakanikirana ndi template ya DNA yomwe ili muzoyambira.5'mapeto a kafukufukuyo amalembedwa ndi jini ya fluorescence emission FAM (6-carboxyfluorescein, fluorescence emission peak peak pa 518nm), ndipo 3'end imalembedwa ndi The fluorescence quenching gulu TAMRA (6-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, fluorescence emission 518nm), phosphorylated kuteteza kafukufukuyu kuti asapitirire pakukulitsa kwa PCR.Pamene kafukufukuyo amakhalabe, gulu la quencher limapondereza kutuluka kwa fulorosenti ya gulu lotulutsa.Gulu lotulutsa likasiyanitsidwa ndi gulu lozimitsa, choletsacho chimakwezedwa, ndipo kuchuluka kwa kuwala kwa 518nm kumawonjezeka ndipo kumadziwika ndi njira yodziwikiratu ya fluorescence.Pamene kafukufukuyo wadulidwa, mphamvu yozimitsa imatulutsidwa ndipo chizindikiro cha fulorosenti chimatulutsidwa.Nthawi zonse template ikakopera, kafukufuku amadulidwa, limodzi ndi kutulutsidwa kwa chizindikiro cha fulorosenti.Popeza pali mgwirizano pakati pa chiwerengero cha fluorophores otulutsidwa ndi chiwerengero cha mankhwala a PCR, njirayi ingagwiritsidwe ntchito poyesa template molondola.Chida choyesera nthawi zambiri chimagwiritsa ntchito ABI7100 PCR matenthedwe cycler yopangidwa ndi kampani ya PE, ndi ma cyclers ena otentha amathanso kugwiritsidwa ntchito.Ngati mawonekedwe amtundu wa ABI7700 agwiritsidwa ntchito poyeserera, zomwe zimachitikazo zikamalizidwa, zotsatira zochulukirapo zitha kuperekedwa mwachindunji kudzera pakuwunika kwamakompyuta.Ngati mugwiritsa ntchito ma cyclers ena otenthetsera, muyenera kugwiritsa ntchito chowunikira chowunikira kuti muyese chizindikiro cha fulorosenti mu chubu chochitira nthawi yomweyo kuti muwerenge RQ+, RQ-, △RQ.RQ + imayimira chiŵerengero cha mphamvu ya luminescence ya gulu lotulutsa fulorosenti la chubu lachitsanzo ku mphamvu ya luminescence ya gulu lozimitsa, RQ- imayimira chiŵerengero cha awiriwo mu chubu chopanda kanthu, △RQ (△RQ = RQ + -RQ-) imayimira kuchuluka kwa kusintha kwa data pa PCquaRntita.Chifukwa cha kuyambitsidwa kwa ma probes a fulorosenti, kutsimikizika kwa kuyesako kumakhala bwino kwambiri.Mapangidwe a probe amayenera kukwaniritsa izi: ①Kutalika kwa probe kuyenera kukhala pafupifupi 20-40 maziko kuti muwonetsetse kuti kumangirira kumatsimikizika.②Zomwe zili m'mabasi a GC zili pakati pa 40% ndi 60% kuti mupewe kubwereza kwa ma nucleotide amodzi.③ Pewani kusakanizidwa kapena kuphatikizika ndi zoyambira.④ Kukhazikika kwakumangirira pakati pa kafukufuku ndi template ndikokulirapo kuposa kukhazikika kwa kumangiriza pakati pa choyambira ndi template, kotero mtengo wa Tm wa kafukufukuyo uyenera kukhala osachepera 5 ° C kuposa mtengo wa Tm woyambira.Kuphatikiza apo, kuchuluka kwa kafukufukuyo, homology pakati pa kafukufuku ndi ma template otsatizana, komanso mtunda pakati pa kafukufukuyo ndi choyambira zonse zimakhudza zotsatira zoyeserera.
Zogwirizana nazo:
China Real Time PCR Easyᵀᴹ-Taqman Wopanga ndi Wopereka |Foregene (foreivd.com)
Nthawi yotumiza: Oct-15-2021
