Ukusungulwa okuningana okuguquguqukayo emlandweni wokuthola ubuchwepheshe emqondweni wami ubuchwepheshe be-immunolabeling obusekelwe kumgomo wokubopha okukhethekile kwe-antigen-antibody, ubuchwepheshe be-PCR kanye nobuchwepheshe bokulandelana.Namuhla sizokhuluma ngobuchwepheshe be-PCR.Ngokuvumelana nokuvela kobuchwepheshe be-PCR, abantu bavamise ukuhlukanisa ubuchwepheshe be-PCR babe izizukulwane ezintathu: ubuchwepheshe obujwayelekile be-PCR, ubuchwepheshe besikhathi sangempela be-fluorescent quantitative PCR kanye nobuchwepheshe be-PCR yedijithali.
Cinqubo ye-PCR ejwayelekile
UKARY MULLIS (1944.12.28-2019.8.7)
U-Kary Mullis wasungula i-polymerase chain reaction (i-polymerase chain reaction, i-PCR) ngo-1983. Kuthiwa lapho eshayela intombi yakhe, ngokuzumayo waba ne-flash of ugqozi futhi wacabanga ngesimiso se-PCR (ngezinzuzo zokushayela).UKary Mullis wanikezwa uMklomelo KaNobel Kwezekhemikhali ngo-1993. I-New York Times yaphawula: “Eyangempela kakhulu futhi ephawulekayo, ecishe ihlukanise isayensi yezinto eziphilayo zibe inkathi yangaphambi kwe-PCR neyangemva kwe-PCR.
Umgomo we-PCR: Ngaphansi kwe-catalysis ye-DNA polymerase, i-DNA yomucu kamama isetshenziswa njengesifanekiso, futhi i-primer ethize isetshenziswa njengendawo yokuqala yokunwetshwa, futhi i-DNA yendodakazi yomucu ehambisana ne-DNA yesifanekiso somucu kamama ikopishwa ku-vitro ngokusebenzisa i-denaturation, annealing, extension kanye nezinye izinyathelo.Iwubuchwepheshe be-DNA synthesis amplification in vitro, engakhulisa ngokushesha futhi ngokuqondile noma iyiphi i-DNA in vitro eqondiwe.
Izinzuzo ze-PCR ezejwayelekile
1.Indlela yakudala, amazinga aphelele wamazwe ngamazwe nawasekhaya
2.Izindleko eziphansi zama-reagents wensimbi
3.Imikhiqizo ye-PCR ingabuyiselwa kwezinye izivivinyo zebhayoloji yamangqamuzana
Umshini we-Foregene PCR onconyiwe: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Imikhiqizo ehlobene: https://www.foreivd.com/pcr-herotm-with-dye-product/
Ukungalungi kwe-PCR evamile
1.kulula ukungcolisa
2.ukusebenza kanzima
3.ukuhlaziywa kwekhwalithi kuphela
4.Ukuzwela okumaphakathi
5.Kukhona ukukhulisa okungaqondile, futhi uma ibhendi engacacisiwe ilingana nebhendi eqondiwe, ayikwazi ukuhlukaniswa.
Ci-apillary electrophoresis-based PCR
Ukuphendula ukushiyeka kwe-PCR evamile, abanye abakhiqizi baye bethula izinsimbi ezisekelwe kumgomo we-capillary electrophoresis.Isinyathelo se-electrophoresis ngemva kokuqedwa kokukhulisa i-PCR ku-capillary.Ukuzwela kuphakeme, futhi umehluko wezisekelo ezimbalwa ungahlukaniswa futhi ukukhulisa kungabalwa nge-MAERKER.okuqukethwe komkhiqizo.Okubi ukuthi umkhiqizo we-PCR usadinga ukuvulwa futhi ufakwe ensimbini, futhi kusenengozi enkulu yokungcola.
Ci-apillaryEi-lectrophoresis
2. Ubuchwepheshe besikhathi sangempela se-fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR)I-Fluorescent quantitative PCR, ebizwa nangokuthi i-Real-Time PCR, ubuchwepheshe obusha be-nucleic acid quantitative obuthuthukiswe yi-PE (Perkin Elmer) ngo-1995. Umlando wokuthuthuka we-fluorescent quantitative PCR umlando wokuzabalaza okushukumisa umphefumulo wamadondoshiya afana ne-ABI, i-Roche, ne-Bio-Rad.Uma unentshisekelo, ungayihlola.Le nqubo okwamanje iyindlela ye-PCR evuthwe kakhulu futhi esetshenziswa kabanzi ye-semi-quantitative.
Umshini we-qPCR onconyiwe:https://www.foreivd.com/mini-real-time-pcr-system-forequant-sf2sf4-product/
Indlela yokudaya ye-Fluorescent (SYBR Green I):I-SYBR Green I wudayi ovame ukusetshenziswa kakhulu we-DNA we-PCR yomthamo, obophezela ngokungaqondile ku-DNA enemicu ekabili.Esimeni esikhululekile, i-SYBR Green ikhipha i-fluorescence ebuthakathaka, kodwa uma iboshelwe ku-DNA enemicu ekabili, i-fluorescence yayo inyuka ngokuphindwe ka-1000.Ngakho-ke, isignali ye-fluorescent ephelele ekhishwa ukusabela ilingana nenani le-DNA enemicu ekabili ekhona futhi izokhula ngokwanda komkhiqizo okhulisiwe.Njengoba udayi ubophezela ngokungaqondile ku-DNA enemicu ekabili, kungase kukhiqizwe imiphumela emihle engamanga.
Imikhiqizo ehlobene: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/
Indlela ye-Fluorescent probe (ubuchwepheshe be-Taqman): NgesikhathiUkukhulisa i-PCR, i-probe ethize ye-fluorescent yengezwa ngesikhathi esifanayo njengepheya lezinto zokuqala.I-probe iyi-oligonucleotide eqondile, eneqembu lezintatheli ze-fluorescent kanye neqembu le-fluorescent quencher ngokulandelanayo elibhalwe kuzo zombili iziphetho.Uma i-probe iphelele, isignali ye-fluorescent ekhishwa yiqembu lentatheli imuncwa iqembu le-quencher, futhi ukutholwa Ayikho isignali ye-fluorescent;ngesikhathi sokukhulisa i-PCR (esigabeni sokunwetshwa), umsebenzi we-5'-3' Dicer we-enzyme ye-Taq uzogaya futhi wehlise i-probe, ukuze iqembu lentatheli ye-fluorescent kanye neqembu le-quencher fluorescent lihlukaniswe, ukuze uhlelo lokuqapha lwe-fluorescence Isignali ye-fluorescent ingatholwa, okungukuthi, ngaso sonke isikhathi lapho i-molecule ye-DNA ibona uchungechunge lwe-fluorescent oluphelele, oluyi-syndrome e-fluorescent. ukuqoqwa kwezimpawu ze-fluorescent kanye nokwakhiwa kwemikhiqizo ye-PCR.Indlela ye-Taqman probe iyindlela evame ukusetshenziswa kakhulu yokuthola ukutholwa komtholampilo.
Imikhiqizo ehlobene: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-real-time-pcr-kit-taqman-product/
Izinzuzo ze-qPCR
1.Indlela isivuthiwe futhi okokusebenza okusekelayo nama-reagents sekuqediwe
2.Izindleko ezimaphakathi zama-reagents
3.kulula ukuyisebenzisa
4.Ukuzwela okuphezulu kokutholwa kanye nokucacisa
Ukungalungi kwe-qPCR
Ukuguqulwa kofuzo oluqondiwe kuholela ekugejisweni kokutholwa.
Umphumela wokutholwa kwesifanekiso sokugxila kancane awukwazi ukunqunywa.
Kunephutha elikhulu uma kusetshenziswa ijika elijwayelekile ukuthola ubuningi.
3. I-Digital PCR (i-PCR yedijithali, i-dPCR) ubuchwepheshe
I-Digital PCR iyindlela yokwenza inani eliphelele lama-nucleic acid molecule.Uma kuqhathaniswa ne-qPCR, i-PCR yedijithali ingafunda ngokuqondile inani lama-molecule e-DNA/RNA, okuwumthamo ophelele wama-nucleic acid kusampula yokuqala.Ngo-1999, u-Bert Vogelstein kanye no-Kenneth W. Kin-zler bahlongoza ngokusemthethweni umqondo we-dPCR.
Ngo-2006, i-Fluidigm yaba ngeyokuqala ukukhiqiza ithuluzi le-dPCR elisekelwe ku-chip.Ngo-2009, i-Life Technologies yethula izinhlelo ze-OpenArray kanye ne-QuantStudio 12K Flex dPCR.Ngo-2013, i-Life Technologies yethula uhlelo lwe-QuantStudio 3DdPCR, olusebenzisa ubuchwepheshe be-chip ye-nanoscale microfluidic ephezulu ukuze kusabalalise ngokulinganayo amasampula kumaseli angama-20,000 ngamanye.ekuphenduleni kahle.
Ngo-2011, i-Bio-Rad yethula ithuluzi elisekelwe ngamaconsi le-QX100 dPCR, esebenzisa ubuchwepheshe bamanzi ku-oyela ukuze isabalalise ngokulinganayo isampula kuwoyela wamaconsi angama-20,000, futhi isebenzisa i-droplet analyzer ukuze ihlaziye amaconsi.Ngo-2012, i-RainDance yethula ithuluzi le-RainDrop dPCR, eliqhutshwa igesi enomfutho ophezulu, ukuze ihlukanise isistimu yokusabela evamile ibe i-emulsion yokusabela equkethe amaconsi amancane e-picoliter-level eyizigidi ezingu-1 kuya kweziyi-10.
Kuze kube manje, i-PCR yedijithali isisungule izingxenye ezimbili ezinkulu, uhlobo lwe-chip kanye nohlobo lwamaconsi.Akunandaba ukuthi hlobo luni lwe-PCR yedijithali, izimiso zayo eziyinhloko zikhawulela ukuhlanjululwa, i-endpoint PCR kanye nokusabalalisa kwe-Poisson.Isistimu yokusabela ye-PCR evamile equkethe izifanekiso ze-nucleic acid ihlukaniswe ngokulinganayo ibe amashumi ezinkulungwane zokusabela kwe-PCR, ezisatshalaliswa kuma-chips noma ama-microdroplets, ukuze ukusabela ngakunye kuqukethe okuningi ngangokunokwenzeka i-molecule yesifanekiso, kanye nokusabela kwe-PCR yesifanekiso se-molecule eyodwa kwenziwa.Ngokufunda i-fluorescence Ukuba khona noma ukungabikho kwesignali kuyabalwa, futhi ukulinganisa okuphelele kwenziwa ngemva kokulinganiswa kokusabalalisa kwezibalo ze-Poisson.
Okulandelayo yizici zezinkundla ezimbalwa zedijithali ze-PCR engizisebenzisile:
1. I-Bio-Rad QX200 droplet digital PCR Bio-RadI-QX200 iyinkundla ye-PCR yedijithali yakudala kakhulu, inqubo yokutholwa eyisisekelo: amasampuli angu-20,000 akhiqizwa umshini wokuphehla amaconsi Amaconsi amancane afakwe kuwoyela akhuliswa emshinini ojwayelekile we-PCR, futhi ekugcineni isignali ye-fluorescence yethonsi ngalinye elincane lifundwa ngumfundi we-micro-droplet.Ukusebenza kuyinkimbinkimbi, futhi ingozi yokungcola iphakathi.
I-Xinyi TD1 micro-droplet digital PCRI-Xinyi TD1 iyinkundla ye-PCR yedijithali yasekhaya, inqubo eyisisekelo yokuthola: khiqiza amaconsi kawoyela angama-30,000-50,000 ngejeneretha yamaconsi, akhulise ethuluzini elivamile le-PCR, futhi ekugcineni adlule I-droplet reader ifunda isignali ye-fluorescent yethonsi ngalinye.Kokubili ukukhiqizwa kwamaconsi nokufunda kule nkundla kwenziwa nge-chip ezinikele enengozi ephansi yokungcola.
STILLA Naica micro-droplet chip digital PCRI-STILLA Naica iyinkundla entsha ye-PCR yedijithali.Inqubo yokutholwa eyisisekelo yilena: engeza isisombululo sokusabela ku-chip, faka i-chip ku-micro-droplet generation kanye nohlelo lokukhulisa, bese ukhiqiza amaconsi amancane angu-30,000.Sabalalisa ku-chip, futhi ukukhulisa i-PCR kuqediwe ku-chip.Khona-ke i-chip ekhulisiwe idluliselwa ohlelweni lokuhlaziya ukufundwa kwe-micro-droplet, futhi isignali ye-fluorescent ifundwa ngokuthatha izithombe.Njengoba yonke inqubo yenzeka ku-chip evaliwe, ingozi yokungcola iphansi.
4. I-ThermoFisher QuantStudio 3D chip digital PCR
I-ThermoFisher QuantStudio 3D ingenye inkundla ye-PCR yedijithali esekwe ku-chip.Inqubo yayo eyisisekelo yokuthola ukuthi: engeza isixazululo sokusabela kusipredishithi, bese usakaza isisombululo sokusabela ngokulinganayo ku-chip ngama-microwells angu-20,000 ngokusebenzisa isisakazeli., faka i-chip emshinini we-PCR ukuze ukhulise, futhi ekugcineni ufake i-chip kumfundi bese uthatha isithombe ukuze ufunde isignali ye-fluorescent.Ukusebenza kuyinkimbinkimbi, futhi yonke inqubo yenziwa ku-chip evaliwe, futhi ingozi yokungcola iphansi.
5. I-JN MEDSYS Clarity chip digital PCR
I-JN MEDSYS Clarity iyinkundla entsha ye-PCR yedijithali entsha uma kuqhathaniswa.Inqubo yayo eyisisekelo yokuthola ukuthi: engeza isixazululo sokusabela kumfakisicelo, futhi usabalalise isixazululo sokusabela ngokulinganayo kumashubhu e-PCR ayi-10,000 ahlanganiswe eshubhu ye-PCR ngesifakeli.Ku-chip ye-microporous, isisombululo sokusabela singena ku-chip ngokusebenzisa isenzo se-capillary, futhi ishubhu le-PCR eline-chip lifakwa emshinini we-PCR ukuze likhuliswe, futhi ekugcineni i-chip ifakwa kumfundi ukuze afunde isignali ye-fluorescent ngokuthatha isithombe.Ukusebenza kuyinkimbinkimbi kakhulu.Ingozi yokutheleleka iphansi.
Amapharamitha weplathifomu ngayinye yedijithali ye-PCR afinyezwa kanje:
Izinkomba zokuhlola zeplathifomu ye-PCR yedijithali yilezi: inani lamayunithi ahlukanisiwe, inani lamashaneli e-fluorescent, inkimbinkimbi yokusebenza kanye nengozi yokungcola.Kodwa okubaluleke kakhulu ukunemba kokutholwa.Enye indlela yokuhlola izinkundla ze-PCR yedijithali ukusebenzisa izinkundla eziningi ze-PCR zedijithali ukuze uqinisekisena, futhi enye indlela iwukusebenzisa izinto ezijwayelekile ezinamavelu anembile.
Izinzuzo ze-dPCR
1.Ukuthola ukulinganisa okuphelele
2.Ukuzwela okuphezulu nokucacisa
3.Ingathola amasampula amakhophi aphansi
Ukungalungi kwe-dPCR1. Imishini ebizayo nama-reagents 2. Ukusebenza okuyinkimbinkimbi nesikhathi eside sokutholwa 3. Ibanga lokutholwa elincane
Njengamanje, izizukulwane ezintathu zobuchwepheshe be-PCR zinezinzuzo zazo kanye nokubi, futhi ngayinye inezinkambu zayo zokufaka isicelo, futhi akubona ubuhlobo ukuthi isizukulwane esisodwa sithatha indawo yesinye.Ukuthuthuka okuqhubekayo kobuchwepheshe kufake ubungqabavu obusha kubuchwepheshe be-PCR, obuyenza ikwazi ukuvula indlela yohlelo lokusebenza ngemva kwenye, okwenza ukutholwa kwe-nucleic acid kube lula futhi kunembe kakhudlwana.
Umthombo: UDkt. Yuan uyakuthatha ayohlolwa
Imikhiqizo Enconyiwe:
Isikhathi sokuthumela: Nov-18-2022
