PCR, da yawa PCR, A cikin PCR, Farashin PCR, RT-PCR, qPCR (1)-PCR

Za mu warware ra'ayoyi, matakai da cikakkun bayanai na PCR daban-daban

Ⅰ. PCR

Polymerase Chain Reaction, wanda ake magana da shi azaman PCR, fasaha ce ta kwayoyin halitta wacce ake amfani da ita don haɓaka takamaiman gutsuttsuran DNA.Ana iya ɗaukarsa azaman kwafi na musamman na DNA a cikin vitro.An gano DNA polymerase (DNA Polymerase I) a farkon 1955, kuma Klenow Fragment na E. Coli, wanda ke da ƙimar gwaji da kuma amfani, Dr. H. Klenow ya gano a farkon shekarun 1970, amma saboda wannan enzyme ba ya jure wa zafin jiki, yawan zafin jiki na iya lalata shi, don haka bai dace da sarkar degeneration na polymerase ba.Enzymes da ake amfani da su a yau (wanda ake kira Taq polymerase), an ware su daga Thermus aquaticus, kwayar cutar zafi mai zafi a 1976. Halinsa shine zai iya tsayayya da zafi mai zafi kuma shine ingantaccen enzyme, amma ana amfani dashi sosai bayan 1980s.Ma'anar asali na asali na asali na asali na PCR yana kama da gyaran kwayoyin halitta da kwafi, wanda Dokta KJell Kleppe ya ba da shawara a cikin 1971. Ya buga kwafin kwayoyin halitta na farko mai sauƙi da gajeren lokaci (mai kama da na farko biyu na sake zagayowar PCR).Dokta Kary B. Mullis ya haɓaka PCR a yau a cikin 1983. Dr. Mullis ya yi hidima ga kamfanonin PE a waccan shekarar, don haka PE yana da matsayi na musamman a cikin masana'antar PCR.Dokta Mullis a hukumance ya buga takarda mai alaƙa ta farko tare da Saiki da sauransu a cikin 1985. Tun daga wannan lokacin, amfani da PCR yana da dubban mil a rana, kuma ana iya faɗi ingancin takaddun da ke da alaƙa da sauran hanyoyin bincike da yawa.Bayan haka, ana amfani da fasahar PCR sosai a cikin binciken kimiyyar halittu da aikace-aikacen asibiti, ta zama mafi mahimmancin fasaha na binciken ilimin halitta.Mullis kuma ya lashe kyautar Nobel a fannin ilmin sinadarai a shekarar 1993.

PCRKa'ida

Asalin ka'idar fasahar PCR tayi kama da tsarin kwafi na dabi'a na DNA, kuma ƙayyadaddun sa ya dogara da madaidaicin oligonucleotide wanda ke dacewa da ƙarshen jeri biyu.PCR ya ƙunshi degeneration-annealing-extending uku na asali halayen matakai: ①Degeneration of template DNA: Bayan da samfurin DNA da aka mai tsanani zuwa game da 93°C na wani wani lokaci, da dual DNA bayani ga dual-sarkar DNA kafa ta PCR amplification na DNA Leaving, sanya shi a shirya guda sarkar domin da zagaye na gaba dauki.②Annealing (compound) na samfuri na DNA da firamare: Bayan da samfurin DNA ya yi zafi kuma ya lalace zuwa sarka ɗaya, zafin jiki yana raguwa zuwa kusan 55°C.Madaidaicin jeri na farko da samfurin DNA guda sarkar.③ Tsawaita na farko: Samfurin DNA-haɗin dauri na farko ya dogara ne akan aikin TaqDNA polymerase, tare da dNTP azaman ɗanyen abu.Ci gaba da ka'idar kwafi, haɗa sabon sarkar kwafin da aka tanada wanda ya dace da sarkar DNA ɗin samfuri, da maimaita sake zagayowar lalacewa-annealing-tsawo matakai uku na iya samun ƙarin "sarkar kwafin da aka tanadar", kuma wannan sabuwar sarkar tana nan sake zama samfuri don zagayowar gaba.Yana ɗaukar mintuna 2-4 don kammala madauki, ana iya haɓaka ƙwayar da aka yi niyya sau miliyan da yawa a cikin sa'o'i 2-3.

DaidaitawaPCRTsarin martani

Abubuwa biyar na amsawar PCR

Akwai nau'ikan abubuwa guda biyar da ke da hannu a cikin amsawar PCR, wato primer, enzyme, dNTP, template da buffer (ana buƙatar Mg2+).[Tsarin PCR]

Daidaitaccen tsari na PCR ya kasu kashi uku

1. DNA degeneration (90 ° C-96 ° C): Dual-sarkar DNA samfuri a karkashin thermal mataki, hydrogen bond karya, forming DNA guda sarkar.

2. Annealing (25 ℃ -65 ℃): An rage yawan zafin jiki na tsarin, ana haɗe firamare tare da samfurin DNA don samar da sarkar dual-chain na gida.

3. Tsawaita (70 ℃ -75 ℃): A karkashin aikin Taq enzyme (kimanin 72 ° C, mafi kyawun aiki), ana amfani da dNTP azaman albarkatun ƙasa, ƙarawa daga ƙarshen 5' na farkon → 3' ƙarshen, haɓakawa da samfuri suna haɗawa da sarkar DNA.

Kowane zagayowar an cire shi, an share shi kuma yana tsawaita, yana ninka abun cikin DNA.A halin yanzu, saboda gajeriyar wurin haɓakawa, ana iya maimaita wasu PCR cikin ɗan ƙanƙanin lokaci ko da aikin Taq enzyme bai fi kyau ba, don haka ana iya canza shi zuwa matakai biyu, wato, ana iya aiwatar da annealing da tsawo a 60 ° C-65 ° C a lokaci guda.Don rage tsarin ɗagawa da sanyaya da inganta saurin amsawa.

Fasalolin amsawar PCR

● Ƙayyadaddun Ƙira

Takaitattun dalilai masu mahimmanci na amsawar PCR sune: ①Takamammen haɗe-haɗe na farko da DNA samfuri.②Ka'idar haɗin ginin tushe.③ Amincewa da amsawar TaqDNA polymerase.④ Ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun kwayoyin halitta.

Daidaitaccen haɗe-haɗe na firamare da samfuri shine maɓalli.Ƙimar ƙaddamarwa da samfuri da kuma tsawo na sarkar farko sun dogara ne akan ka'idar daidaitawar tushen alkaline.Amincewa da halayen haɗin gwiwar polymerase da babban juriya na zafin jiki na Taq DNA polymerase don yin ɗaurin (haɗin) na samfuri da firamare a cikin abin da za a iya yi a mafi girman zafin jiki.Ƙayyadaddun haɗin haɗin yana ƙaruwa sosai.Hoton na iya kiyaye babban matakin daidai.Ta zaɓar yankin da aka yi niyya tare da babban ra'ayin mazan jiya da babban ra'ayin mazan jiya, ƙayyadaddun sa ya fi girma.

● Babban Hankali

Ana haɓaka ƙarar samar da samfuran PCR ta index, wanda zai iya faɗaɗa samfurin farawa na Picker (PG=10-12) don ƙara matakin microcontroller zuwa matakin micrograms (μg= -6).Ana iya gano ƙwayoyin da aka yi niyya daga sel miliyan 1;a cikin gano ƙwayoyin cuta, hankalin PCR zai iya kaiwa 3 RFUs (rabo mara kyau da aka kafa);Mafi ƙarancin ganowa a cikin ilimin ƙwayoyin cuta shine ƙwayoyin cuta 3.

● Sauƙi da sauri

Tunanin PCR yana amfani da Taq DNA polymerase mai zafi mai zafi, wanda ke ƙara maganin amsawa a lokaci ɗaya, wato, haɓakar haɓaka-anneal-tsawo akan maganin ƙarawar DNA da tukunyar wanka na ruwa.Gabaɗaya, ana kammala aikin haɓakawa a cikin sa'o'i 2 zuwa 4.Abubuwan da aka haɓaka gabaɗaya ana nazarin su ta takobin lantarki, kuma ba dole ba ne su yi amfani da isotopes, babu gurɓataccen rediyo, da haɓakawa cikin sauƙi.

● Tsaftar samfurin yana da ƙasa

Babu buƙatar raba ƙwayoyin cuta ko ƙwayoyin cuta da ƙwayoyin al'adu.Za'a iya amfani da samfuran ɗanyen DNA da RNA azaman amplifiers.Ana iya amfani da gano ƙarar DNA kai tsaye ta amfani da samfuran asibiti kamar jini, ruwan jiki, ruwan wanke tari, gashi, sel, da nama mai rai.

PCRmatsalolin gama gari

● Ƙarya mara kyau, babu ƙararrakin makada

Mahimmin matakai na amsawar PCR sun haɗa da: ① shirye-shiryen samfuri na nucleic acid, ② inganci da ƙayyadaddun abubuwan farko, ③ ingancin enzymes ④ yanayin sake zagayowar PCR.Nemo dalilin kuma yakamata a bincika kuma a yi nazari don hanyoyin haɗin da ke sama.

Samfura: ① Samfurin ya ƙunshi furotin daban-daban, ② Samfurin yana ƙunshe da mai hana enzyme Taq, ③ Ba a kawar da furotin da ke cikin samfuri, musamman furotin na rukuni a cikin chromosome.⑤ Deminer nucleic acid degeneration ba cikakke ba ne.Lokacin da ingancin enzymes da furotin suna da kyau, babu ƙararrawa band, wanda shine mafi kusantar maganin narkewar samfuran.Akwai wani abu da ba daidai ba tare da tsarin hakar samfurin nucleic acid, don haka don shirya ingantaccen maganin narkewar abinci mai inganci, ya kamata a gyara tsarin sa kuma ba a canza shi ba.

Rashin kunnawar Enzyme: sabon enzyme ko duka tsofaffi da sabbin enzymes yakamata a yi amfani dasu tare don tantance ko aikin enzyme ya ɓace ko bai isa ba, yana haifar da rashin ƙarfi na ƙarya.Ya kamata a lura cewa Taq enzyme ko ethidium bromide wani lokaci ana mantawa da shi.

Mahimmanci: ingancin na'ura mai mahimmanci, ƙaddamar da ƙaddamarwa, da kuma ko ƙaddamar da ƙaddamarwa guda biyu yana da daidaituwa.Yana da dalili na gama gari na gazawar PCR ko ƙarar band bai dace ba kuma yana da saurin yaduwa.Akwai matsaloli tare da ingancin firamare na wasu lambobi.Abubuwan da aka tsara guda biyu suna da babban taro da ƙananan ƙaddamarwa, suna haifar da ƙananan haɓakaccen haɓakaccen haɓakawa na asymmetric.Matakan magance su sune: ① Zaɓan fidda kai mai kyau don haɗa raka'a.② Ƙaddamar da ƙaddamarwa ba kawai ya dogara da ƙimar OD ba, amma kuma yana kula da asalin ruwa na asali don yin agar sugar gel electrophoresis.Dole ne a sami yankin tsiri na firamare, kuma hasken firamare biyu ya kamata ya kasance daidai.Belt, PCR na iya gazawa a wannan lokacin, kuma yakamata a warware shi tare da naúrar haɗakarwa.Idan firamare yana da girma, haske yana da ƙasa, kuma dole ne a daidaita maida hankali lokacin da aka diluted.③ Yakamata a biya firamare kuma a adana shi a babban taro don hana daskarewa da yawa ko ɓangarorin firji na dogon lokaci, wanda zai sa na'urar ta lalace kuma ta lalace.④ Tsarin ƙira ba shi da ma'ana, irin su tsawon lokacin da ba a isa ba, kuma an kafa cluster a tsakanin masu farawa.

Mg2 + maida hankali: Mg2 + ion maida hankali yana da babban tasiri akan ingantaccen haɓaka PCR.Yawan maida hankali zai iya rage kishiyar jima'i na haɓaka PCR.Idan maida hankali ya yi ƙasa da ƙasa, fitarwar haɓakawa na PCR har ma zai haifar da gazawar haɓakar PCR ba tare da band ɗin faɗaɗa ba.

Canjin ƙarar amsawa: Ƙarar da aka yi amfani da ita a cikin haɓakawa na PCR shine 20ul, 30ul, da 50ul ko 100uL, babban adadin aikace-aikacen don haɓaka PCR an saita shi bisa dalilai daban-daban na binciken kimiyya da gwajin asibiti.Bayan yin ƙananan ƙananan kamar 20ul, wajibi ne don yin yanayin igiya lokacin yin girman, in ba haka ba zai kasa.

Dalilai na jiki: Canji yana da mahimmanci don haɓaka PCR.Idan yawan zafin jiki na raguwa ya ragu, lokacin raguwa ya kasance takaice, yana yiwuwa ya faru a cikin rashin kuskure;ƙananan zafin jiki na annealing zai iya haifar da haɓakawa maras takamaiman kuma yana rage ƙayyadaddun ingantaccen haɓakawa.Yi tasiri sosai akan haɗar firamare da samfura don rage haɓakar haɓakawa na PCR.Wani lokaci ya zama dole don amfani da ma'aunin ma'aunin zafi da sanyio don gano bambance-bambancen, annealing da tsawaita zafin jiki a cikin tsawo ko tukunyar ruwa mai narkewa, wanda shine ɗayan dalilan gazawar PCR.

Bambance-bambancen jeri na manufa: Idan jerin abubuwan da aka yi niyya sun faru, maye gurbi ko gogewa, an haɗa haɗin samfuri da samfuri, ko kuma saboda rashin tsarin maƙasudi, na farko da samfuri za su rasa jerin ƙarin, kuma haɓakawar PCR ɗinsa ba zai yi nasara ba.

● Ƙarya mai kyau

Ƙungiyar haɓakawa ta PCR tana bayyana daidai da ƙungiyar jerin abubuwan da aka yi niyya, kuma wani lokacin ƙungiyar sa tana da kyau kuma mafi girma.

Zane na farko bai dace ba: jerin ƙarawa da aka zaɓa da jerin ƙarawa marasa manufa suna da kamanceceniya, don haka lokacin haɓaka PCR, samfuran PCR da aka haɓaka ba jerin ma'ana ba ne.Jerin abubuwan da aka yi niyya ya yi gajeru sosai ko kuma na farko ya yi gajere sosai, kuma yana da saurin samun tabbataccen ƙarya.Bukatar sake fasalin

Rashin gurɓatawar jerin abubuwan da aka yi niyya ko haɓakawa: Akwai dalilai guda biyu na wannan gurɓacewar: Na farko, giciye - gurɓatawar dukkanin kwayoyin halitta ko manyan sassan, wanda ke haifar da tabbataccen ƙarya.Ana iya magance wannan nau'in tabbataccen ƙarya ta hanyoyi masu zuwa: Yi hankali da tausasawa yayin aiki don hana jerin abubuwan da aka yi niyya shakar su cikin bindigar samfurin ko fantsama daga bututun tsakiya.Ban da enzymes da abubuwan da ba za su iya jure yanayin zafi ba, duk reagents ko kayan aiki ya kamata a lalata su da babban matsa lamba.Ya kamata a yi amfani da bututun centrifugal da samfurori a lokaci ɗaya.Lokacin da ya cancanta, kafin ƙara samfurori, bututun amsawa da reagent ana fallasa su zuwa hasken ultraviolet don lalata acid nucleic da ke akwai.Na biyu, ƙananan gutsuttsura a cikin gurɓataccen iska.Waɗannan ƙananan gutsuttsura sun fi jerin abubuwan da aka yi niyya gajarta, amma suna da ƙayyadaddun ƙayyadaddun ƙa'idodi.Ana iya raba shi da juna.Bayan kammala abubuwan farko, ana iya fadada samfurin PCR, wanda zai haifar da samar da ingantaccen inganci.Ana iya amfani dashi don rage ko kawar da hanyar PCR gida.

● Bayyana bandejin ƙarawa da ba takamaiman ba

Ƙungiyoyin da suka bayyana bayan haɓakawa na PCR ba su dace da girman da ake tsammani ba, ko babba ko ƙarami, ko a lokaci guda, ko a lokaci guda, ƙayyadaddun makada na ƙarawa da ƙananan ƙararrawa marasa takamaiman.Fitowar makada da ba takamaiman ba shine: Na farko, ginshiƙan ba su cika cika madaidaicin jerin abubuwan da aka yi niyya ba, ko kuma polymerization na firamare don samar da gungu.Na biyu shi ne cewa maida hankali na MG2+ ions ya yi yawa, yawan zafin jiki na annealing ya yi ƙasa sosai, kuma adadin hawan PCR yana da alaƙa.Abu na biyu, inganci da adadin enzymes.Sau da yawa, enzymes na wasu kafofin suna da haɗari ga ƙungiyoyin da ba na musamman ba kuma enzymes na wani tushe ba su faru ba.Wasu lokuta ba takamaiman haɓakar enzymes kuma suna faruwa.Matakan magance su sune: sake tsara abubuwan ban sha'awa idan ya cancanta.Rage adadin enzyme ko maye gurbin enzyme na wani tushe.Rage adadin firamare, ƙara adadin samfuri yadda ya kamata, da rage adadin zagayowar.Ƙara yawan zafin jiki da ya dace ko amfani da hanyar yanayin zafin jiki guda biyu (93 ° C degeneration, annealing da tsawo a kimanin 65 ° C).

● Ya bayyana ja ko shafa tef

Ƙwaƙwalwar PCR wani lokaci yana bayyana ana amfani da shi ko harsashi ko bel mai kama da kafet.Don dalili, saboda yawan adadin enzymes ko rashin ingancin enzyme, ƙaddamarwar dNTP ya yi yawa, ƙaddamarwar Mg2+ ya yi yawa, yawan zafin jiki na annealing yana da yawa, kuma yawan hawan keke yana da yawa.Ma'auni shine: ①Rage adadin enzymes, ko canza enzyme na wani tushe.②Rage taro na dNTP ③A rage yawan taro na Mg2+ daidai.④ Ƙara yawan samfuri kuma rage adadin zagayowar.

Samfura masu dangantaka

Jarumin PCR (Tare da Dye)

◮ Babban aminci: sau 6 na talakawan Taq enzyme;

◮ Saurin haɓakawa

◮ Ƙarin daidaitawar samfuri

◮ Ingantaccen haɓakawa mafi girma

◮ Haƙurin muhalli ya fi ƙarfi: sanya shi a 37 ° C na mako guda, yana riƙe da fiye da 90% aiki;

Yana da 5'→3' DNA polymerase aiki da 5'→3' aikin exonuclease, ba tare da 3'→5' aikin exonuclease ba.

PCR Easy ᴹ (Tare da Dye)

Tsarin amsawa na musamman da ingantaccen ingantaccen Taq DNA Polymerase yana sa amsawar PCR ta sami ingantaccen haɓakawa, ƙayyadaddun da hankali.

RT-qPCR Easyᵀᴹ (Mataki Daya) -SYBR Green I

◮ Kit ɗin mataki ɗaya yana yin jujjuya rubutu da qPCR halayen biyu a cikin bututu ɗaya, kawai buƙatar ƙara samfuri RNA, takamaiman abubuwan PCR da RNase-Free ddH₂O.

◮ Kit ɗin na iya yin nazari da sauri da inganci cikin ƙididdigewa na RNA na hoto ko kuma gano RNA.

◮ Kit ɗin yana amfani da na musamman na Foregene reverse reagent reagent da Foregene HotStar Taq DNA Polymerase haɗe tare da na'urar amsawa ta musamman don haɓaka ingantaccen haɓakawa da ƙayyadaddun halayen.

◮ Ingantattun tsarin amsawa yana sa abin ya sami mafi girman ganewar ganewa, kwanciyar hankali mai ƙarfi, da mafi kyawun haƙuri.

◮ RT-qPCR Mai Sauƙi^TM(Mataki Daya) -SYBR Green I kit ya zo tare da rini na ciki na ROX, wanda za'a iya amfani dashi don kawar da bayanan sigina da kurakuran sigina tsakanin rijiyoyi, wanda ya dace da abokan ciniki don amfani da su a cikin nau'ikan kayan aikin PCR masu yawa.

RT Sauƙi^TMII (Master Premix don farko-strand cDNA kira donReal Time PCR)

-Ingantacciyar ikon cire gDNA, wanda zai iya cire gDNA a cikin samfuri a cikin mintuna 2.

-Ingantaccen tsarin jujjuya rubutu, yana ɗaukar mintuna 15 kawai don kammala haɗin layin farko na cDNA.

-Complex samfuri: samfura tare da babban abun ciki na GC da hadaddun tsarin sakandare kuma ana iya juyawa tare da ingantaccen inganci.

-Tsarin jujjuya juzu'i mai girma, samfuran pg-matakin kuma na iya samun cDNA mai inganci.

-Tsarin rubutun baya yana da babban kwanciyar hankali na thermal, mafi kyawun zafin jiki shine 42 ℃, kuma har yanzu yana da kyakkyawan aikin juzu'i a 50 ℃.