• I-PCR yindlela esetyenziselwa ukukhulisa i-DNA kwixabiso elincinci le-template ye-DNA.I-RT-PCR isebenzisa i-reverse transcription ukuvelisa itemplate ye-DNA kumthombo we-RNA onokunyuswa.
• I-PCR kunye ne-RT-PCR ngokuqhelekileyo ziimpendulo ze-endpoint, ngelixa i-qPCR kunye ne-RT-qPCR zisebenzisa i-kinetics yezinga lokwenziwa kwemveliso ngexesha lokusabela kwe-PCR ukulinganisa inani lethempleyithi ekhoyo.
• Iindlela ezintsha, ezifana ne-PCR yedijithali, zibonelela ngobungakanani obupheleleyo be-template ye-DNA yokuqala, ngelixa iindlela ezifana ne-PCR ye-isothermal zinciphisa imfuno yezixhobo ezibizayo ukunika iziphumo ezithembekileyo.

I-Polymerase chain reaction (PCR) yindlela elula nesetyenziswa ngokubanzi kwimolekyuli yebhayoloji ukukhulisa kunye nokufumanisa ulandelelwano lwe-DNA kunye ne-RNA.Xa kuthelekiswa neendlela zemveli ze-DNA cloning kunye nokukhulisa, ezinokudla ngokuthatha iintsuku, i-PCR ifuna iiyure ezimbalwa kuphela.I-PCR inovakalelo oluphezulu kwaye ifuna ithempleyithi encinci yokuchongwa kunye nokukhulisa ulandelelwano oluthile.Iindlela ze-PCR ezisisiseko ziye zahambela phambili ngakumbi ukusuka kwi-DNA elula kunye nobhaqo lwe-RNA.Apha ngezantsi, sinikeze isishwankathelo seendlela ezahlukeneyo ze-PCR kunye nee-reagents esizibonelela kwi-Enzo Life Sciences kwiimfuno zakho zophando.Sijolise ekuncedeni izazinzulu zifikelele ngokukhawuleza kwii-reagents ze-PCR ukuze zizisebenzise kwiprojekthi yazo yophando elandelayo!

I-PCR

Kwi-PCR eqhelekileyo, yonke into oyifunayo yi-polymerase ye-DNA, i-magnesium, i-nucleotides, i-primers, i-template ye-DNA ukuba ikhuliswe, kunye ne-thermocycler.Indlela ye-PCR ilula njengenjongo yayo: 1) i-DNA enemisonto ephindwe kabini (dsDNA) ibubushushu, i-2) i-primers ilungelelaniswa kwimicu ye-DNA enye, kunye ne-3) iiprimers zandiswa nge-DNA polymerase, okukhokelela kwiikopi ezimbini ze-DNA. umcu wokuqala weDNA.I-denaturation, i-annealing, kunye nenkqubo yokwandisa phezu koluhlu lwamaqondo obushushu kunye namaxesha aziwa njengomjikelo omnye wokukhulisa (umzobo 1).

Isinyathelo ngasinye somjikelo kufuneka siphuculwe kwi-template kunye nesethi ye-primer esetyenzisiweyo.Lo mjikelo uphindwe malunga namaxesha angama-20-40, kwaye imveliso ekhulisiwe ingahlalutywa ke, ngokuqhelekileyo ngejel ye-agarose (umzobo 2).

Njengoko i-PCR iyindlela enovakalelo oluphezulu kunye nemithamo emincinci kakhulu iyafuneka kwimpendulo enye, ukulungiswa komxube obalaseleyo kwiimpendulo ezininzi kuyacetyiswa.I-master mix kufuneka ixutywe kakuhle kwaye emva koko yahlulwe ngenani leempendulo, ukuqinisekisa ukuba impendulo nganye iya kuba nenani elifanayo le-enzyme, i-dNTPs, kunye ne-primers.Ababoneleli abaninzi, njenge-Enzo Life Sciences, banika kwakhona imixube ye-PCR esele iqulethe yonke into ngaphandle kwe-primers kunye ne-template ye-DNA.

Imimandla yaseGuanine/Cytosine-rich (GC-rich) ibonisa umngeni kwiindlela eziqhelekileyo ze-PCR.Ulandelelwano olutyebileyo lwe-GC luzinzile ngakumbi kunolandelelwano olunomxholo ophantsi we-GC.Ngaphaya koko, ulandelelwano olutyebileyo lwe-GC ludla ngokwenza izakhiwo zesibini, ezinje nge-hairpin loops.Ngenxa yoko, imisonto ephindwe kabini ye-GC kunzima ukuyohlula ngokupheleleyo ngexesha lesigaba sokudinwa.Ngenxa yoko, i-DNA polymerase ayikwazi ukwenza umtya omtsha ngaphandle kwesithintelo.Iqondo lobushushu eliphakamileyo le-denaturation linokuphucula oku, kwaye uhlengahlengiso olubhekiselele kubushushu obuphezulu be-annealing kunye nexesha elifutshane lokuvalela kunokuthintela ukubophelela okungacacanga kweeprimers ezityebileyo ze-GC.Ii-reagents ezongezelelweyo zinokuphucula ukukhulisa ulandelelwano olutyebileyo lwe-GC.I-DMSO, i-glycerol, kunye ne-betaine inceda ukuphazamisa izakhiwo zesibini ezibangelwa ukusebenzisana kwe-GC kwaye ngaloo ndlela ziququzelele ukuhlukana kweentambo ezimbini.

I-Hot Start PCR

Ukwandisa okungaqhelekanga yingxaki enokuthi yenzeke ngexesha le-PCR.Uninzi lweepolymerasi ze-DNA ezisetyenziswa kwi-PCR zisebenza ngcono kumaqondo obushushu amalunga nama-68°C ukuya kuma-72°C.I-enzyme inokuthi, nangona kunjalo, isebenze kumaqondo obushushu aphantsi, nangona ukuya kwinqanaba elisezantsi.Kumaqondo obushushu angaphantsi kakhulu kweqondo lobushushu le-annealing, iiprimers zinokubophelela ngokungathe ngqo kwaye zikhokelele kulwandiso olungangqalanga, nokuba ukusabela kusetiwe emkhenkceni.Oku kunokuthintelwa ngokusebenzisa ii-polymerase inhibitors ezahlukana ne-DNA polymerase kanye nje ukuba ubushushu obuthile bufikelelwe, kungoko igama elithi hot start PCR.I-inhibitor ingaba yi-antibody ebopha i-polymerase kunye ne-denatures kwiqondo lokushisa lokuqala le-denaturation (95 ° C ngokuqhelekileyo).

Ukuthembeka okuphezulu kwePolymerase

Ngelixa i-DNA polymerases ikhulisa ngokuchanekileyo ngokuchanekileyo kulandelelwano lwetemplate yasekuqaleni, iimpazamo kwi-nucleotide yokulinganisa zinokwenzeka.Ukungafani kwizicelo ezifana ne-cloning kunokubangela ukukhutshelwa okuncinciweyo, kunye neeprotheni eziguqulelwe kakubi okanye ezingasebenziyo ezantsi komsinga.Ukunqanda oku kungafaniyo, iipolymerases ezinomsebenzi "wokuhlola ubungqina" ziye zachongwa zaza zadityaniswa nokuhamba komsebenzi.Ipolymerase yokuqala yokuhlola uvavanyo, iPfu, yachongwa ngo-1991 kwiPyrococcus furiosus.Le enzyme ye-Pfu inomsebenzi we-3' ukuya kwi-5' exonuclease.Njengoko i-DNA ikhulisiwe, i-exonuclease isusa i-nucleotides engafanelekanga ekupheleni kwe-3 'ye-strand.Emva koko i-nucleotide echanekileyo iyatshintshwa, kwaye i-DNA synthesis iyaqhubeka.Ukuchongwa kolandelelwano lwe-nucleotide olungachanekanga lusekelwe kwi-affinity ebophezelayo ye-nucleoside triphosphate echanekileyo kunye ne-enzyme, apho ukubopha okungahambi kakuhle kunciphisa i-synthesis kwaye kuvumela ukutshintshwa okuchanekileyo.Umsebenzi wokuhlola i-Pfu polymerase uphumela kwiimpazamo ezimbalwa kulandelelwano lokugqibela xa kuthelekiswa ne-Taq DNA polymerase.Kwiminyaka yakutshanje, ezinye ii-enzymes zokuhlola zichongiwe, kwaye uhlengahlengiso lwe-enzyme yokuqala ye-Pfu luye lwenziwa ukuze kuncitshiswe ngakumbi izinga lempazamo ngexesha lokukhulisa i-DNA.

I-RT-PCR

Reverse transcription PCR, okanye RT-PCR, ivumela ukusetyenziswa RNA njenge template.Inyathelo elongezelelweyo livumela ukuchongwa kunye nokukhulisa i-RNA.I-RNA iguqulelwa umva kwi-DNA ehambelanayo (cDNA), kusetyenziswa i-reverse transcriptase.Umgangatho kunye nokucoceka kwe-template ye-RNA kubalulekile kwimpumelelo ye-RT-PCR.Isinyathelo sokuqala se-RT-PCR yi-synthesis ye-DNA / RNA hybrid.I-Reverse transcriptase inomsebenzi we-RNase H, othoba isahlulo se-RNA somxube.I-molecule ye-DNA enemisonto enye igqityezelwa yi-DNA exhomekeke kwi-DNA polymerase umsebenzi we-reverse transcriptase kwi-cDNA.Ukusebenza kwe-reaction ye-strand yokuqala kunokuchaphazela inkqubo yokukhulisa.Ukusuka apha ukuya phambili, inkqubo ye-PCR eqhelekileyo isetyenziselwa ukukhulisa i-cDNA.Ithuba lokubuyisela i-RNA kwi-cDNA nge-RT-PCR ineengenelo ezininzi, kwaye isetyenziselwa ikakhulu uhlalutyo lwembonakalo yemfuza.I-RNA inentambo enye kwaye ayizinzi kakhulu, nto leyo eyenza kube nzima ukusebenza nayo.Idla ngokusebenza njengenyathelo lokuqala kwi-qPCR, ebonisa ubungakanani bemibhalo ye-RNA kwisampulu yebhayoloji.

qPCR kunye ne-RT-qPCR

I-PCR yobungakanani (qPCR) isetyenziselwa ukufumanisa, ukulinganisa kunye nokulinganisa i-nucleic acids kwizicelo ezininzi.Kwi-RT-qPCR, imibhalo ye-RNA isoloko ibalwa ngokuyiguqulela umva ibe yi-cDNA kuqala, njengoko kuchaziwe ngasentla, kwaye emva koko i-qPCR iye yenziwa.Njengakwi-PCR eqhelekileyo, i-DNA ikhuliswa ngamanyathelo amathathu aphindaphindayo: i-denaturation, i-annealing, kunye nokwandisa.Nangona kunjalo, kwi-qPCR, ukuleyibheli kwefluorescent kwenza ukuqokelelwa kwedatha njengoko i-PCR iqhubela phambili.Obu buchule buneenzuzo ezininzi ngenxa yoluhlu lweendlela kunye neekhemistri ezikhoyo.

Kwidayi esekwe kwi-qPCR (ngokuqhelekileyo eluhlaza), ilebhile yefluorescent ivumela ukubalwa kwemolekyuli ye-DNA eyandisiweyo ngokusebenzisa ukusetyenziswa kwedayi ebophelelayo ye-dsDNA.Ngethuba lomjikelo ngamnye, i-fluorescence ilinganiswa.Isignali ye-fluorescence inyuka ngokulinganayo kwisixa se-DNA ephindwe kabini.Ngenxa yoko, i-DNA ibalwa "kwixesha langempela" (umzobo 3).Ukungalungi kwi-qPCR esekwe kwidayi kukuba enye kuphela ekujoliswe kuyo inokuvavanywa ngexesha kwaye idayi iya kubophelela kuyo nayiphi na i-ds-DNA ekhoyo kwisampulu.

Kwi-probe-based qPCR, iithagethi ezininzi zinokubonwa ngaxeshanye kwisampulu nganye, kodwa oku kufuna ukuphuculwa kunye noyilo lweprobe (s) ekujoliswe kuyo esetyenziswa ukongezelela kwiiprimers.Iindidi ezininzi zoyilo lweprobe ziyafumaneka, kodwa olona hlobo luqhelekileyo yi-hydrolysis probe, ebandakanya i-fluorophore kunye ne-quencher.I-Fluorescence resonance energy transfer (FRET) inqanda ukukhutshwa kwe-fluorophore nge-quencher ngelixa i-probe ingaguquki.Nangona kunjalo, ngexesha lokuphendula kwe-PCR, i-probe i-hydrolyzed ngexesha lokwandiswa kwe-primer kunye nokukhulisa ulandelelwano oluthile olubophelelweyo.I-cleavage ye-probe ihlukanisa i-fluorophore kwi-quencher kwaye ibangele ukwanda okuxhomekeke kwi-amplification kwi-fluorescence (Umfanekiso 4).Ke, isiginali ye-fluorescence evela kwi-probe-based qPCR reaction is proporing to the amount of the probe target sequence ekhoyo kwisampulu.Ngenxa yokuba i-qPCR esekwe kwiprobe icace ngakumbi kune-qPCR esekwe kwidayi, ihlala ibuchwephesha obusetyenziswa kwiimvavanyo zokuxilonga ezisekwe kwi-qPCR.

I-Isothermal Amplification

I-PCR ekhankanywe ngasentla ifuna izixhobo zethermocycling ezibizayo ukuze zinyuse kwaye zihle ngokuchanekileyo amaqondo obushushu egumbi kuhlengahlengiso, ukunyathela, kunye namanyathelo okwandisa.Kuye kwaphuhliswa iindlela ezininzi ezingadingi zixhobo zichanekileyo kwaye zinokwenziwa kwindawo yokuhlambela amanzi okanye ngaphakathi kweeseli ezinomdla.Obu buchule bubizwa ngokuba yi-isothermal amplification kunye nomsebenzi osekwe kwi-exponential, linear, okanye i-cascade amplification.

Olona hlobo lwaziwa kakhulu lwe-isothermal amplification yi-loop-mediated isothermal amplification, okanye i-LAMP.I-LAMP isebenzisa i-exponential amplification kwi-65⁰C ukukhulisa i-template yeDNA okanye i-RNA.Xa kusenziwa i-LAMP, iiprimers ezine ukuya kwezintandathu ezincedisana nemimandla ekujoliswe kuyo i-DNA zisetyenziswa kunye ne-DNA polymerase ukudibanisa iDNA entsha.Ezimbini kwezi ziqalo zinolandelelwano oluncomekayo oluqaphela ulandelelwano kwezinye iiprimers kwaye luzibophe, luvumela ulwakhiwo lwe-"loop" ukuba lwenze kwi-DNA esanda kwenziwa enceda ukufakwa kwe-primer annealing kwimijikelo elandelayo yokwandisa.I-LAMP inokubonwa ngeendlela ezininzi, kuquka i-fluorescence, i-agarose gel electrophoresis, okanye i-colorimetry.Ubulula bokubona kunye nokufumanisa ubukho okanye ukungabikho kwemveliso ngemibala kunye nokunqongophala kwezixhobo ezibiza kakhulu ezifunekayo kwenza i-LAMP ibe yinto efanelekileyo yovavanyo lwe-SARS-CoV-2 kwiindawo apho uvavanyo lwelebhu yeklinikhi lwalungafumaneki lula, okanye ukugcinwa kunye nokuthuthwa kweesampulu. yayingenzeki, okanye kwiilebhu ezazingenazo ngaphambili izixhobo ze-PCR ze-thermocycling.