• PCR wata hanya ce da ake amfani da ita don haɓaka DNA daga ƙaramin adadin samfurin DNA.RT-PCR tana amfani da juzu'i don samar da samfurin DNA daga tushen RNA wanda za'a iya haɓakawa.
• PCR da RT-PCR yawanci halayen ƙarshe ne, yayin da qPCR da RT-qPCR suna amfani da ƙididdiga na ƙimar haɗin samfur yayin amsawar PCR don ƙididdige adadin samfurin da ake ciki.
• Sabbin hanyoyin, irin su PCR na dijital, suna ba da cikakkiyar ƙididdige ƙididdige samfurin DNA na farko, yayin da hanyoyin kamar PCR isothermal suna rage buƙatar kayan aiki masu tsada don samar da ingantaccen sakamako.

Maganin sarkar polymerase (PCR) dabara ce mai sauƙi kuma da amfani da ko'ina don haɓakawa da gano jerin DNA da RNA.Idan aka kwatanta da hanyoyin gargajiya na cloning DNA da haɓakawa, waɗanda galibi kan ɗauki kwanaki, PCR na buƙatar sa'o'i kaɗan kawai.PCR yana da matukar kulawa kuma yana buƙatar ƙaramin samfuri don ganowa da haɓaka takamaiman jeri.Hanyoyin PCR na asali sun ci gaba daga gano DNA da RNA mai sauƙi.A ƙasa, mun ba da bayyani na hanyoyin PCR daban-daban da masu sakewa da muke samarwa a Enzo Life Sciences don buƙatun bincikenku.Muna nufin taimaka wa masana kimiyya da sauri samun damar PCR reagents don amfani da su a cikin aikin bincike na gaba!

PCR

Don daidaitaccen PCR, duk abin da kuke buƙata shine polymerase na DNA, magnesium, nucleotides, masu haɓakawa, samfurin DNA don haɓakawa, da na'ura mai zafi.Tsarin PCR yana da sauƙi kamar manufarsa: 1) DNA mai ɗaure biyu (dsDNA) zafi ne mai ƙima, 2) na'urori masu auna siginar DNA guda ɗaya, kuma 3) ana ƙaddamar da na'urar ta hanyar DNA polymerase, wanda ya haifar da kwafi biyu na asalin DNA strand.The denaturation, annealing, da elongation tsari a kan jerin yanayin zafi da kuma lokuta da aka sani da daya sake zagayowar na ƙarawa (Fig. 1).

Kowane mataki na zagayowar ya kamata a inganta shi don samfuri da saitin fidda da aka yi amfani da su.Ana maimaita wannan sake zagayowar kusan sau 20-40, sannan za'a iya bincikar haɓakar samfurin, yawanci ta hanyar gel agarose (Fig. 2).

Kamar yadda PCR hanya ce mai mahimmanci kuma ana buƙatar ƙananan ƙididdiga don halayen guda ɗaya, ana ba da shawarar shirye-shiryen haɗin gwaninta don halayen da yawa.Dole ne a haɗa haɗin maigidan da kyau sannan a raba ta adadin halayen, tabbatar da cewa kowane amsa zai ƙunshi adadin enzyme iri ɗaya, dNTPs, da masu farawa.Yawancin masu samar da kayayyaki, irin su Enzo Life Sciences, suma suna ba da gaurayawan PCR waɗanda suka riga sun ƙunshi komai ban da firamare da samfurin DNA.

Yankunan Guanine/Cytosine-rich (GC-rich) suna wakiltar ƙalubale a daidaitattun dabarun PCR.Jerin wadatattun GC sun fi karɓuwa fiye da jeri tare da ƙananan abun ciki na GC.Bugu da ƙari, jeri-arziƙi na GC yakan haifar da sifofi na biyu, kamar madaukai na gashi.Sakamakon haka, igiyoyi biyu masu wadatar GC suna da wahalar rabuwa gaba ɗaya yayin lokacin denaturation.Saboda haka, DNA polymerase ba zai iya haɗa sabon igiyoyin ba tare da shamaki ba.Mafi girman zafin jiki na iya inganta wannan, kuma gyare-gyare zuwa yanayin zafi mai girma da gajeriyar lokacin raɗaɗi zai iya hana ƙayyadaddun daurin abubuwan da ke da wadatar GC.Ƙarin reagents na iya haɓaka haɓakar jerin wadatattun GC.DMSO, glycerol, da betaine suna taimakawa wajen rushe tsarin na biyu wanda ke haifar da hulɗar GC kuma ta haka yana sauƙaƙe rarrabuwar igiyoyi biyu.

Hot Start PCR

Ƙwaƙwalwar da ba ta da takamaiman matsala matsala ce da za ta iya faruwa yayin PCR.Yawancin polymerases na DNA waɗanda ake amfani da su don PCR suna aiki mafi kyau a yanayin zafi a kusa da 68 ° C zuwa 72 ° C.Enzyme na iya, duk da haka, yana aiki a ƙananan yanayin zafi, kodayake zuwa ƙananan digiri.A yanayin zafi mai nisa da ke ƙasa da zafin jiki, na'urori na iya ɗaure ba musamman ba kuma suna haifar da haɓakawa mara takamaiman, koda kuwa an saita matakin akan kankara.Ana iya hana wannan ta yin amfani da masu hana polymerase waɗanda ke rabu da DNA polymerase kawai da zarar an kai wani zazzabi, don haka kalmar fara PCR mai zafi.Mai hanawa na iya zama antibody wanda ke ɗaure polymerase da denatures a farkon yawan zafin jiki (95°C yawanci).

Babban Fidelity Polymerase

Yayin da polymerases na DNA suna haɓaka daidai daidai zuwa jerin samfurin asali, kurakurai a cikin daidaitawar nucleotide na iya faruwa.Rashin daidaituwa a cikin aikace-aikace kamar cloning na iya haifar da tarkace rubuce-rubucen, da kuskuren fassara ko furotin da ba su aiki a ƙasa.Don guje wa waɗannan rashin daidaituwa, an gano polymerases tare da aikin "tabbatacce" kuma an haɗa su cikin aikin aiki.Polymerase na farko proofreading, Pfu, an gano shi a cikin 1991 a Pyrococcus furiosus.Wannan enzyme na Pfu yana da 3' zuwa 5' aikin exonuclease.Yayin da DNA ke ƙaruwa, exonuclease yana cire nucleotides da basu dace ba a ƙarshen 3' na madaidaicin.Ana maye gurbin madaidaicin nucleotide, kuma haɗin DNA ya ci gaba.Gano jerin nucleotide da ba daidai ba yana dogara ne akan alaƙar ɗaure don daidaitaccen nucleoside triphosphate tare da enzyme, inda ɗaurin da ba ta dace ba yana rage haɗuwa kuma yana ba da damar maye gurbin daidai.Ayyukan tantancewa na Pfu polymerase yana haifar da ƴan kurakurai a jere na ƙarshe idan aka kwatanta da Taq DNA polymerase.A cikin 'yan shekarun nan, an gano wasu enzymes na tantancewa, kuma an yi gyare-gyare na asali na Pfu enzyme don ƙara rage yawan kuskure yayin haɓaka DNA.

RT-PCR

Juya kwafin PCR, ko RT-PCR, yana ba da damar amfani da RNA azaman samfuri.Ƙarin mataki yana ba da damar ganowa da haɓaka RNA.Ana juyar da RNA zuwa DNA na ƙarin (cDNA), ta amfani da juzu'i.Inganci da tsarkin samfurin RNA suna da mahimmanci don nasarar RT-PCR.Mataki na farko na RT-PCR shine haɗin haɗin DNA/RNA.Reverse transcriptase shima yana da aikin RNase H, wanda ke lalata sashin RNA na matasan.Kwayoyin DNA mai ɗaure guda ɗaya sannan ana kammala ta aikin DNA polymerase mai dogaro da DNA na juyar da rubutun zuwa cDNA.Ingantacciyar amsawar-marar farko na iya shafar tsarin haɓakawa.Daga nan gaba, ana amfani da daidaitaccen tsarin PCR don haɓaka cDNA.Yiwuwar mayar da RNA zuwa cDNA ta RT-PCR yana da fa'idodi da yawa, kuma ana amfani da shi da farko don nazarin maganganun kwayoyin halitta.RNA mai ɗaci ɗaya ce kuma mara ƙarfi sosai, wanda ke sa ya zama ƙalubale don yin aiki da shi.Yawanci yana aiki azaman mataki na farko a qPCR, wanda ke ƙididdige kwafin RNA a cikin samfurin halitta.

qPCR da RT-qPCR

Ana amfani da PCR mai ƙididdigewa (qPCR) don ganowa, ƙididdigewa da ƙididdige acid nucleic don aikace-aikace masu yawa.A cikin RT-qPCR, ana ƙididdige kwafin RNA sau da yawa ta hanyar juya su zuwa cDNA da farko, kamar yadda aka bayyana a sama, sannan qPCR ana aiwatar da shi daga baya.Kamar yadda yake a daidaitaccen PCR, ana ƙara DNA ta matakai uku masu maimaitawa: denaturation, annealing, da elongation.Koyaya, a cikin qPCR, alamar kyalli yana ba da damar tattara bayanai yayin da PCR ke ci gaba.Wannan dabarar tana da fa'idodi da yawa saboda kewayon hanyoyin da sinadarai da ake da su.

A cikin qPCR na tushen rini (yawanci kore), lakabin kyalli yana ba da damar ƙididdige adadin ƙwayoyin DNA da aka haɓaka ta yin amfani da rini mai ɗaure dsDNA.A yayin kowane zagayowar, ana auna hasken haske.Siginar kyalli yana ƙaruwa daidai gwargwado ga adadin DNA da aka kwafi.Saboda haka, ana ƙididdige DNA a cikin "ainihin lokaci" (Fig. 3).Lalacewar qPCR na tushen rini shine cewa manufa ɗaya kawai za a iya bincika a lokaci guda kuma cewa rini za ta ɗaure ga kowane ds-DNA da ke cikin samfurin.

A cikin qPCR na tushen bincike, ana iya gano maƙasudi da yawa a lokaci guda a cikin kowane samfuri, amma wannan yana buƙatar haɓakawa da ƙira na takamaiman bincike (s) da aka yi amfani da shi ban da firamare.Akwai nau'ikan ƙirar bincike da yawa, amma nau'in da aka fi sani shine binciken hydrolysis, wanda ya haɗa da fluorophore da quencher.Canja wurin wutar lantarki mai haske (FRET) yana hana fitar da fluorophore ta hanyar mai kashe wuta yayin binciken yana nan.Koyaya, yayin amsawar PCR, binciken yana hydrolyzed yayin haɓakawa na farko da haɓaka takamaiman jerin da aka ɗaure zuwa.Ragewar binciken ya raba fluorophore daga mai kashewa kuma yana haifar da haɓakar haɓakawar haɓakawa a cikin haske (Fig. 4).Don haka, siginar kyalli daga amsawar qPCR na tushen bincike yayi daidai da adadin jerin maƙasudin binciken da ke cikin samfurin.Saboda tushen binciken qPCR ya fi ƙayyadaddun ƙayyadaddun qPCR na tushen rini, galibi fasaha ce da ake amfani da ita a cikin ƙididdigar tushen qPCR.

Ƙaddamar da Isothermal

Dabarun na PCR da aka ambata a sama suna buƙatar kayan aikin zafi masu tsada don daidaita yanayin zafi sama da ƙasa don ƙwanƙwasa, cirewa, da matakan tsawaitawa.An ƙirƙiri wasu fasahohin da ba sa buƙatar irin waɗannan na'urori masu mahimmanci kuma ana iya yin su a cikin wanka mai sauƙi na ruwa ko ma a cikin sel masu sha'awa.Waɗannan fasahohin ana kiran su tare da haɓaka haɓakar isothermal da aiki bisa ga haɓakawa, madaidaiciya, ko haɓakar cascade.

Mafi sanannun nau'in haɓakawar isothermal shine haɓakawar isothermal na madauki, ko LAMP.LAMP yana amfani da ƙarawa mai ƙarfi a 65⁰C don haɓaka samfurin DNA ko RNA.Lokacin yin LAMP, ana amfani da firam ɗin huɗu zuwa shida waɗanda ke dacewa da yankuna na DNA da aka yi niyya tare da DNA polymerase don haɗa sabon DNA.Biyu daga cikin waɗannan firamare suna da jeri na kyauta waɗanda ke gane jeri a cikin sauran firamare kuma su ɗaure su, suna ba da damar tsarin “madauki” ya samar a cikin sabuwar DNA ɗin da aka haɗe wanda sannan yana taimakawa haɓakar matakin ƙarawa a zagaye na gaba na ƙarawa.Ana iya ganin fitilun ta hanyoyi da yawa, gami da haske, agarose gel electrophoresis, ko colorimetry.Sauƙin gani da gano kasancewar ko rashi samfurin ta hanyar launi da ƙarancin kayan aiki masu tsada da ake buƙata sun sanya LAMP zaɓi mai dacewa don gwajin SARS-CoV-2 a wuraren da ba a samun gwajin gwajin asibiti cikin sauri, ko adanawa da jigilar samfuran. ba zai yiwu ba, ko a cikin dakunan gwaje-gwaje waɗanda ba su da kayan aikin zafi na PCR.