Ƙirƙirar juyin-juya-hali da yawa a cikin tarihin fasahar ganowa a cikin raina fasahar rigakafin rigakafi ne bisa ƙa'idar ƙayyadaddun dauri na antigen-antibody, fasaha na PCR da fasahar jerin abubuwa.Yau za mu yi magana game da fasahar PCR.Bisa ga juyin halittar fasahar PCR, mutane sun saba raba fasahar PCR zuwa tsararraki uku: fasahar PCR ta yau da kullun, fasahar PCR mai kyalli na gaske da fasahar PCR na dijital.
Common PCR fasaha
KARY MULLIS (1944.12.28-2019.8.7)
Kary Mullis ya ƙirƙira nau'in sarkar polymerase (polymerase chain reaction, PCR) a cikin 1983. An ce lokacin da yake tuki budurwarsa, ba zato ba tsammani ya sami haske na wahayi kuma yayi tunanin ka'idar PCR (a kan amfanin tuki).An bai wa Kary Mullis lambar yabo ta Nobel a ilmin sinadarai a shekarar 1993. Jaridar New York Times ta yi sharhi: “Mai girma na asali kuma mai mahimmanci, kusan rarraba ilmin halitta zuwa zamanin pre-PCR da bayan PCR.
Ka'idar PCR: A ƙarƙashin catalysis na DNA polymerase, ana amfani da DNA strand na uwar a matsayin samfuri, kuma ana amfani da takamaiman madaidaicin a matsayin wurin farawa na tsawo, kuma ɗiyar ɗiyar DNA ta dace da samfurin ƙirar uwar strand DNA ana kwafi a cikin vitro ta hanyar denaturation, annealing, tsawo da sauran matakai.Fasaha ce ta haɓaka haɓakawar DNA a cikin vitro, wacce za ta iya haɓaka da sauri da kuma haɓaka duk wani DNA da aka yi niyya a cikin vitro.
Amfanin PCR na yau da kullun
1.Hanyar gargajiya, cikakkiyar ma'auni na ƙasa da ƙasa
2.Ƙananan farashin kayan aikin reagents
3.Ana iya dawo da samfuran PCR don wasu gwaje-gwajen ilimin halitta
Nasihar Foregene PCR inji: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Samfura masu alaƙa: https://www.foreivd.com/pcr-herotm-with-dye-product/
Hasara na talakawa PCR
1.mai sauki ga gurbatawa
2.m aiki
3.nazari mai inganci kawai
4.Matsakaicin hankali
5.Akwai ƙarawa mara ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙidayar, kuma lokacin da ƙungiyar da ba ta dace ba ta kasance girman daidai da rukunin da aka yi niyya, ba za a iya bambanta ta ba.
Capillary electrophoresis na tushen PCR
Dangane da gazawar PCR na yau da kullun, wasu masana'antun sun gabatar da kayan aikin bisa ka'idar electrophoresis capillary.Matakan electrophoresis bayan an kammala haɓaka PCR a cikin capillary.Hankali ya fi girma, kuma ana iya bambanta bambancin tushe da yawa kuma ana iya ƙididdige haɓakawa ta MAERKER.abun ciki na samfur.Rashin hasara shine cewa samfurin PCR har yanzu yana buƙatar buɗewa kuma a saka shi cikin kayan aiki, kuma har yanzu akwai babban haɗarin gurɓata.
CapillaryElectrophoresis
2. Fasahar qPCR mai kyalli na ainihi (Quantitative Real-time PCR, qPCR) fasahaFluorescent quantitative PCR, wanda kuma ake kira Real-Time PCR, sabuwar fasaha ce ta nucleic acid mai ƙididdigewa ta PE (Perkin Elmer) a cikin 1995. Tarihin ci gaba na PCR mai ƙyalƙyali shine tarihin gwagwarmayar ruhi na ƙattai irin su ABI, Roche, da Bio-Rad.Idan kuna sha'awar, kuna iya duba shi.Wannan dabarar a halin yanzu ita ce mafi girma kuma mafi girman dabarar PCR da ake amfani da ita.
Na'urar qPCR da aka ba da shawarar:https://www.foreivd.com/mini-real-time-pcr-system-forequant-sf2sf4-product/
Hanyar rini mai walƙiya (SYBR Green I):SYBR Green I shine rini mai ɗaure DNA da aka fi amfani dashi don ƙididdige PCR, wanda ke ɗaure ba musamman ga DNA mai ɗauri biyu ba.A cikin jihar kyauta, SYBR Green yana fitar da haske mai rauni, amma da zarar an ɗaure shi zuwa DNA mai ɗaure biyu, hasken sa yana ƙaruwa sau 1000.Sabili da haka, jimlar siginar kyalli da aka fitar ta hanyar amsawa yayi daidai da adadin DNA mai ɗauri biyu da ke akwai kuma zai ƙaru tare da haɓakar samfur.Tun da rini ya ɗaure ba musamman ga DNA mai ɗaure biyu ba, ana iya haifar da kyakkyawan sakamako na ƙarya.
Abubuwan da ke da alaƙa: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/
Hanyar binciken fluorescent (fasaha ta Taqman): LokacinƘwaƙwalwar PCR, ana ƙara takamaiman bincike mai kyalli a lokaci guda kamar nau'i-nau'i.Binciken shine oligonucleotide na layi mai layi, tare da ƙungiyar masu ba da rahoto mai kyalli da kuma ƙungiyar masu ba da haske a bi da bi a ƙarshen duka.Lokacin da binciken ya kasance cikakke, siginar mai kyalli da ƙungiyar masu ba da rahoto ke fitarwa tana ɗaukar ƙungiyar masu kashewa, kuma ganowa Babu siginar kyalli;a lokacin haɓakawa na PCR (a cikin matakin haɓaka), 5'-3' Dicer aiki na Taq enzyme zai narke kuma ya ƙasƙantar da bincike, don haka ƙungiyar masu ba da rahoto da ƙungiyar masu ba da haske sun rabu, don haka tsarin kula da hasken wuta Ana iya karɓar sigina mai kyalli, wato, duk lokacin da aka haɓaka sarkar DNA, cikakkiyar siginar da ke haifar da siginar siginar moleculation, wanda ke haifar da cikakkiyar siginar haɓakawa. s da samuwar samfuran PCR.Hanyar binciken Taqman ita ce hanyar gano da aka fi amfani da ita wajen gano asibiti.
Samfura masu dangantaka: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-real-time-pcr-kit-taqman-product/
Abubuwan da aka bayar na qPCR
1.Hanyar ya balaga kuma kayan aikin tallafi da reagents sun cika
2.Matsakaicin farashin reagents
3.sauki don amfani
4.Babban ganewar ganewa da ƙayyadaddun bayanai
Lalacewar qPCR
Sauyewar kwayar halittar da aka yi niyya tana kaiwa ga ganowar da aka rasa.
Ba za a iya tantance sakamakon gano ƙarancin hankali ba.
Akwai babban kuskure lokacin amfani da daidaitaccen lanƙwasa don gano ƙididdiga.
3. Digital PCR (dijital PCR, dPCR) fasaha
PCR na dijital wata dabara ce don cikakkiyar ƙididdige ƙwayoyin ƙwayoyin acid nucleic.Idan aka kwatanta da qPCR, PCR na dijital na iya karanta adadin ƙwayoyin DNA/RNA kai tsaye, wanda shine cikakken ƙididdige ƙwayoyin ƙwayoyin acid nucleic a cikin samfurin farawa.A cikin 1999, Bert Vogelstein da Kenneth W. Kin-zler sun ba da shawarar manufar dPCR.
A cikin 2006, Fluidigm shine farkon wanda ya samar da kayan aikin dPCR na tushen guntu na kasuwanci.A cikin 2009, Life Technologies sun ƙaddamar da tsarin OpenArray da QuantStudio 12K Flex dPCR.A cikin 2013, Life Technologies ta ƙaddamar da tsarin QuantStudio 3DdPCR, wanda ke amfani da fasahar guntu mai girma na nanoscale microfluidic don rarraba samfuran daidai gwargwado zuwa sel guda 20,000.a cikin dauki da kyau.
A cikin 2011, Bio-Rad ya ƙaddamar da kayan aikin QX100 dPCR na tushen droplet, wanda ke amfani da fasahar ruwa a cikin mai don rarraba samfurin daidai zuwa 20.A cikin 2012, RainDance ya ƙaddamar da kayan aikin RainDrop dPCR, wanda iskar gas mai ƙarfi ke motsawa, don rarraba kowane daidaitaccen tsarin amsawa cikin emulsion mai ɗauke da ɗigon ɗigon ɗigon picoliter miliyan 1 zuwa miliyan 10.
Ya zuwa yanzu, PCR na dijital ya kafa manyan ƙungiyoyi biyu, nau'in guntu da nau'in droplet.Komai irin nau'in PCR na dijital, ainihin ƙa'idodin sa suna iyakance dilution, PCR na ƙarshe da rarraba Poisson.Daidaitaccen tsarin amsawar PCR mai ɗauke da samfuran nucleic acid an raba shi daidai zuwa dubun dubatar halayen PCR, waɗanda ake rarraba su zuwa guntu ko microdroplets, ta yadda kowane amsa ya ƙunshi adadin samfurin samfuri gwargwadon yuwuwar, kuma ana aiwatar da samfurin PCR guda ɗaya.Ta hanyar karanta haske Ana ƙidaya kasancewar ko rashin siginar, kuma ana yin cikakken ƙididdigewa bayan daidaita rarraba Poisson na ƙididdiga.
Waɗannan su ne halayen dandamali na PCR na dijital da yawa da na yi amfani da su:
1. Bio-Rad QX200 droplet dijital PCR Bio-RadQX200 shine dandamalin PCR na dijital na yau da kullun, ainihin tsarin ganowa: samfuran 20,000 ana samar da su ta hanyar janareta na ɗigon ruwa Ana ƙara ƙaramar na'ura na PCR na yau da kullun, kuma a ƙarshe siginar kyalli na kowane micro-droplet mai karatu ne ya karanta.Aikin ya fi rikitarwa, kuma haɗarin gurɓata yana da matsakaici.
Xinyi TD1 micro-drolet dijital PCRXinyi TD1 dandamali ne na PCR na dijital na cikin gida, tsarin gano asali: yana samar da ɗigon ruwa na 30,000-50,000 ta hanyar janareta na ɗigon ruwa, ƙarawa akan kayan aikin PCR na yau da kullun, sannan ya wuce Mai karanta droplet yana karanta siginar kyalli na kowane digo.Dukansu tsararrun ɗigo da karatu a cikin wannan dandali ana yin su ne a cikin keɓaɓɓen guntu tare da ƙananan haɗarin kamuwa da cuta.
STILLA Naica micro-drolet guntu na dijital PCRSTILLA Naica sabon dandali ne na PCR na dijital.Babban tsarin ganowa shine: ƙara maganin amsawa ga guntu, saka guntu a cikin tsarin haɓakar ƙarami da haɓakawa, da samar da ƙananan ɗigon 30,000.Yada akan guntu, kuma an kammala haɓaka PCR akan guntu.Sa'an nan kuma ƙarar guntu an canza shi zuwa tsarin nazarin karatun micro-droplet, kuma ana karanta siginar mai kyalli ta hanyar ɗaukar hotuna.Tun da dukan tsari yana faruwa a cikin rufaffiyar guntu, haɗarin kamuwa da cuta yana da ƙasa.
4. ThermoFisher QuantStudio 3D guntu PCR dijital
ThermoFisher QuantStudio 3D wani babban dandamali ne na PCR dijital na tushen guntu.Tsarin gano ainihin sa shine: ƙara maganin amsawa a cikin mai watsawa, kuma yada maganin amsa daidai akan guntu tare da microwells 20,000 ta hanyar mai watsawa., sanya guntu akan na'urar PCR don haɓakawa, sannan a ƙarshe sanya guntu a cikin mai karatu kuma ɗaukar hoto don karanta siginar kyalli.Ayyukan yana da rikitarwa, kuma ana aiwatar da dukkan tsari a cikin guntu mai rufaffiyar, kuma haɗarin kamuwa da cuta yana da ƙasa.
5. JN MEDSYS Clarity chip dijital PCR
JN MEDSYS Clarity sabon dandamali ne na PCR dijital na nau'in guntu.Babban tsarin gano shi shine: ƙara maganin amsawa a cikin applicator, kuma yada maganin amsa daidai a kan bututun PCR 10,000 da aka gyara a cikin bututun PCR ta hanyar applicator.A kan guntuwar microporous, maganin amsawa yana shiga guntu ta hanyar aikin capillary, kuma ana sanya bututun PCR tare da guntu akan injin PCR don haɓakawa, kuma a ƙarshe an saka guntu a cikin mai karatu don karanta siginar kyalli ta hanyar ɗaukar hoto.Aikin ya fi rikitarwa.Haɗarin kamuwa da cuta yana da ƙasa.
An taƙaita ma'auni na kowane dandamali na PCR na dijital kamar haka:
Alamun kimantawa na dandamali na PCR na dijital sune: adadin raka'a raka'a, adadin tashoshi mai kyalli, rikitarwar aiki da haɗarin gurɓatawa.Amma abu mafi mahimmanci shine daidaiton ganowa.Hanya ɗaya don kimanta dandamali na PCR na dijital shine amfani da dandamali na PCR na dijital da yawa don tantance juna, wata hanya kuma ita ce amfani da daidaitattun abubuwa tare da ingantattun ƙima.
Abubuwan da aka bayar na dPCR
1.Samun cikakken ƙididdigewa
2.Mafi girman hankali da ƙayyadaddun bayanai
3.Za a iya gano ƙananan samfuran kwafi
Lalacewar dPCR1. Kayan aiki masu tsada da reagents 2. Rikicin aiki da lokacin ganowa mai tsayi 3. kunkuntar gano kewayon
A halin yanzu, tsararraki uku na fasahar PCR suna da nasu fa'ida da rashin amfani, kuma kowanne yana da nasa filayen aikace-aikacen, kuma ba dangantaka ce wani zamani ke maye gurbin ɗayan ba.Ci gaba da ci gaban fasaha ya shigar da sabon kuzari a cikin fasahar PCR, yana ba ta damar buɗe hanyar aikace-aikacen ɗaya bayan ɗaya, yana sa gano nucleic acid ya fi dacewa kuma daidai.
Source: Dr. Yuan ya kai ku don gwaji
Abubuwan da aka Shawarta:
Lokacin aikawa: Nuwamba 18-2022
