• PCR ke mokhoa o sebelisoang ho holisa DNA ho tsoa palo e nyane ea template ea DNA.RT-PCR e sebelisa transcript ho hlahisa template ea DNA ho tsoa mohloling oa RNA o ka holisoang.
• PCR le RT-PCR hangata ke karabelo ea pheletso, ha qPCR le RT-qPCR li sebelisa kinetics ea sekhahla sa motsoako oa sehlahisoa nakong ea karabelo ea PCR ho lekanya palo ea template e teng.
• Mekhoa e mecha, joalo ka PCR ea dijithale, e fana ka palo e felletseng ea template ea pele ea DNA, athe mekhoa e kang isothermal PCR e fokotsa tlhoko ea lisebelisoa tse theko e boima ho fana ka liphetho tse tšepahalang.

Polymerase chain reaction (PCR) ke mokhoa o bonolo le o sebelisoang haholo oa baeloji ea limolek'hule ho holisa le ho lemoha tatelano ea DNA le RNA.Ha ho bapisoa le mekhoa e tloaelehileng ea DNA cloning le amplification, eo hangata e ka nkang matsatsi, PCR e hloka lihora tse seng kae feela.PCR e hlokolosi haholo 'me e hloka thempleite e fokolang bakeng sa ho lemoha le ho holisa tatellano e itseng.Mekhoa ea mantlha ea PCR e tsoetse pele ho tloha ho DNA e bonolo le ho lemoha RNA.Ka tlase, re fane ka kakaretso ea mekhoa e fapaneng ea PCR le li-reagents tseo re fanang ka tsona ho Enzo Life Sciences bakeng sa litlhoko tsa hau tsa lipatlisiso.Re ikemiselitse ho thusa bo-ramahlale ho fihlella li-reagents tsa PCR ka potlako ho li sebelisa morerong oa bona o latelang oa lipatlisiso!

PCR

Bakeng sa PCR e tloaelehileng, sohle seo u se hlokang ke DNA polymerase, magnesium, nucleotides, primers, template ea DNA e lokelang ho holisoa, le thermocycler.Mochini oa PCR o bonolo joalo ka morero oa ona: 1) DNA e nang le likhoele tse peli (dsDNA) e kentse mocheso, 2) li-primers li tsamaisana le likhoele tse le 'ngoe tsa DNA, le 3) li-primers li atolosoa ke DNA polymerase, e leng se hlahisang likopi tse peli tsa DNA strand ea pele.The denaturation, annealing, and elongation process over the series of the temperatures and times is known as one cycle of amplification (sa feiga. 1).

Mohato o mong le o mong oa potoloho o lokela ho ntlafatsoa bakeng sa template le sete ea primer e sebelisitsoeng.Potoloho ena e phetoa ka makhetlo a ka bang 20-40, 'me sehlahisoa se matlafalitsoeng se ka hlahlojoa, hangata ka gel ea agarose (setšoantšo sa 2).

Kaha PCR ke mokhoa o hlokolosi haholo 'me ho hlokahala li-volume tse nyane haholo bakeng sa karabelo e le 'ngoe, ho khothaletsoa ho lokisoa ha motsoako o matla bakeng sa likarabelo tse ngata.Motsoako o moholo o tlameha ho tsoakoa hantle ebe o aroloa ka palo ea liketso, ho etsa bonnete ba hore karabelo ka 'ngoe e tla ba le palo e lekanang ea enzyme, dNTPs le li-primers.Barekisi ba bangata, joalo ka Enzo Life Sciences, le bona ba fana ka metsoako ea PCR e seng e na le ntho e 'ngoe le e' ngoe ntle le li-primers le template ea DNA.

Libaka tsa Guanine/Cytosine-rich (GC-rich) li emela phephetso mekhoeng e tloaelehileng ea PCR.Tatelano e ruileng ka GC e tsitsitse ho feta tatelano e nang le litaba tse tlase tsa GC.Ho feta moo, tatellano e ruileng ea GC e na le ho theha meaho ea bobeli, joalo ka loops ea hairpin.Ka lebaka leo, likhoele tse habeli tse ruileng tsa GC ho thata ho arohana ka botlalo nakong ea mokhahlelo oa denaturation.Ka lebaka leo, DNA polymerase ha e khone ho kopanya khoele e ncha ntle le tšitiso.Thempereichara e phahameng ea denaturation e ka ntlafatsa sena, 'me litokiso tse lebisang mochesong o phahameng oa mocheso le nako e khuts'oane ea ho koala li ka thibela ho tlamahanngoa ho sa tobang ha li-primers tse ruileng tsa GC.Li-reagents tse ling li ka ntlafatsa tatellano ea GC-rich.DMSO, glycerol, le betaine li thusa ho senya mehaho ea bobeli e bakoang ke ho sebelisana ha GC 'me ka hona ho nolofalletsa ho arohana ha likhoele tse peli.

Hot Start PCR

Ho holisa ka mokhoa o sa tobang ke bothata bo ka bang teng nakong ea PCR.Boholo ba lipolymerase tsa DNA tse sebelisoang bakeng sa PCR li sebetsa hantle haholo mochesong o ka bang 68°C ho isa ho 72°C.Leha ho le joalo, enzyme e ka boela ea sebetsa maemong a mocheso a tlaase, le hoja e le ka tekanyo e tlaase.Lithempereicharang tse ka tlase ho mocheso o kenang, li-primers li ka tlama ka mokhoa o sa tobang 'me tsa lebisa ho matlafatso e sa tobang, le haeba karabelo e behiloe holim'a leqhoa.Sena se ka thibeloa ka ho sebelisa li-polymerase inhibitors tse ikarohanyang le DNA polymerase hang feela ha mocheso o itseng o fihletsoe, ke ka hona poleloana e reng hot start PCR.Inhibitor e ka ba lesole la 'mele le tlamang polymerase le li-denature ho mocheso oa pele oa denaturation (95°C ka tloaelo).

High Fidelity Polymerase

Le ha DNA polymerase e holisa ka nepo ho latela tatellano ea template ea mantlha, liphoso tsa ho bapisa ha nucleotide li ka etsahala.Ho se lumellane lits'ebetsong tse joalo ka cloning ho ka fella ka lingoloa tse khutsufalitsoeng, le liprotheine tse fetoletsoeng hampe kapa tse sa sebetseng tlase ho metsi.Ho qoba liphoso tsena, li-polymerase tse nang le ts'ebetso ea "proofreading" li khethiloe mme li kenyellelitsoe tšebetsong ea mosebetsi.Polymerase ea pele ea ho hlahloba liphoso, Pfu, e ile ea fumanoa ka 1991 Pyrococcus furiosus.Enzyme ena ea Pfu e na le ts'ebetso ea 3' ho isa ho 5' exonuclease.Ha DNA e ntse e eketseha, exonuclease e tlosa li-nucleotide tse sa tšoaneng qetellong ea 3' ea khoele.Joale nucleotide e nepahetseng e nkeloa sebaka, 'me DNA synthesis e tsoela pele.Khethollo ea tatellano e fosahetseng ea li-nucleotide e ipapisitse le kamano e tlamang bakeng sa nucleoside triphosphate e nepahetseng le enzyme, moo tlamo e sa sebetseng e liehisang motsoako le ho lumella phetoho e nepahetseng.Mosebetsi oa ho hlahloba liphoso oa Pfu polymerase o fella ka liphoso tse fokolang ka tatellano ea ho qetela ha e bapisoa le Taq DNA polymerase.Lilemong tsa morao tjena, ho ile ha fumanoa li-enzyme tse ling tsa ho hlahloba liphoso, 'me ho entsoe liphetoho tsa enzyme ea pele ea Pfu ho fokotsa sekhahla sa liphoso nakong ea ho holisa DNA.

RT-PCR

Reverse transcription PCR, kapa RT-PCR, e lumella tšebeliso ea RNA e le template.Mohato o mong o lumella ho lemoha le ho holisa RNA.RNA e fetoletsoe ho DNA e tlatsanang (cDNA), ho sebelisoa reverse transcriptase.Boleng le bohloeki ba template ea RNA li bohlokoa bakeng sa katleho ea RT-PCR.Mohato oa pele oa RT-PCR ke motsoako oa motsoako oa DNA/RNA.Reverse transcriptase e boetse e na le tšebetso ea RNase H, e theolang karolo ea RNA ea lebasetere.Molek'hule oa DNA e nang le khoele e le 'ngoe e phethoa ke tšebetso ea DNA polymerase e itšetlehileng ka DNA ea reverse transcriptase ho cDNA.Ts'ebetso ea karabelo ea strand ea pele e ka ama ts'ebetso ea amplification.Ho tloha mona ho ea pele, mokhoa o tloaelehileng oa PCR o sebelisoa ho holisa cDNA.Monyetla oa ho khutlisetsa RNA ho cDNA ka RT-PCR e na le melemo e mengata, 'me e sebelisoa haholo-holo bakeng sa tlhahlobo ea liphatsa tsa lefutso.RNA e na le ntlha e le 'ngoe ebile ha e tsitsa, e leng se etsang hore ho be thata ho sebetsa le eona.Hangata e sebetsa joalo ka mohato oa pele ho qPCR, e lekanyang lingoloa tsa RNA sampole ea baeloji.

qPCR le RT-qPCR

Quantitative PCR (qPCR) e sebelisetsoa ho lemoha, ho khetholla le ho lekanya li-nucleic acid bakeng sa lits'ebetso tse ngata.Ho RT-qPCR, lingoloa tsa RNA hangata li hlalosoa ka ho li fetolela ho cDNA pele, joalo ka ha ho hlalositsoe kaholimo, ebe qPCR e etsoa kamora moo.Joalo ka PCR e tloaelehileng, DNA e holisoa ka mehato e meraro e pheta-phetoang: denaturation, annealing, le elongation.Leha ho le joalo, ho qPCR, ho ngola ka fluorescent ho nolofalletsa ho bokella lintlha ha PCR e ntse e tsoela pele.Mokhoa ona o na le melemo e mengata ka lebaka la mefuta e mengata ea mekhoa le lik'hemik'hale tse teng.

Ho qPCR e thehiloeng ho dae (hangata e tala), ho ngola ka fluorescent ho lumella palo ea limolek'hule tsa DNA tse holisitsoeng ka ho sebelisa dae e tlamang ea dsDNA.Nakong ea potoloho ka 'ngoe, fluorescence e lekanngoa.Letšoao la fluorescence le eketseha ka ho lekana le palo ea DNA e phetoang.Kahoo, DNA e lekantsoe ka "nako ea sebele" (setšoantšo sa 3).Mefokolo ea qPCR e thehiloeng ho dae ke hore sepheo se le seng feela se ka hlahlojoang ka nako le hore dae e tla tlama ho ds-DNA efe kapa efe e teng sampoleng.

Ho qPCR e thehiloeng ho lipatlisiso, lipheo tse ngata li ka bonoa ka nako e le 'ngoe sampoleng e' ngoe le e 'ngoe, empa sena se hloka ntlafatso le moralo oa li-probe tse ikhethileng tse sebelisoang ho kenyelletsa li-primers.Ho na le mefuta e mengata ea liqapi tsa lipatlisiso, empa mofuta o tloaelehileng haholo ke hydrolysis probe, e kenyelletsang fluorophore le quencher.Fluorescence resonance energy transfer (FRET) e thibela ho tsoa ha fluorophore ka quencher ha probe e ntse e tiile.Leha ho le joalo, nakong ea karabelo ea PCR, probe e entsoe ka hydrolyzed nakong ea katoloso ea primer le ho holisa tatellano e ikhethileng eo e tlamehang ho e etsa.Ho phunyeha ha probe ho arola fluorophore ho tloha ho tima 'me ho fella ka keketseho e itšetlehileng ka matla a fluorescence (setšoantšo sa 4).Kahoo, lets'oao la fluorescence le tsoang ho probe-based qPCR reaction le lekana le palo ea tatellano ea sepheo sa probe e teng sampoleng.Hobane qPCR e thehiloeng ho probe e totobetse ho feta qPCR e thehiloeng ho dae, hangata ke theknoloji e sebelisoang litlhahlobong tsa tlhahlobo tse thehiloeng ho qPCR.

Matlafatso ea Isothermal

PCR e boletsoeng ka holimo e hloka lisebelisoa tse theko e boima tsa thermocycling ho nyolla le ho theosa mocheso oa kamore bakeng sa mehato ea denaturation, annealing, le katoloso.Ho entsoe mekhoa e mengata e sa hlokeng lisebelisoa tse nepahetseng joalo 'me e ka etsoa ka ho hlapa metsi ka mokhoa o bonolo kapa esita le ka har'a lisele tsa thahasello.Mekhoa ena ka kakaretso e bitsoa isothermal amplification le mosebetsi o ipapisitse le exponential, linear, kapa cascade amplification.

Mofuta o tsebahalang haholo oa amplification ea isothermal ke loop-mediated isothermal amplification, kapa LAMP.LAMP e sebelisa exponential amplification ho 65⁰C ho holisa template ea DNA kapa RNA.Ha ho etsoa LAMP, li-primers tse 'nè ho isa ho tse tšeletseng tse tlatselletsang libakeng tsa DNA e reretsoeng li sebelisoa ka DNA polymerase ho kopanya DNA e ncha.Tse peli tsa li-primers tsena li na le tatellano e ntle e hlokomelang tatellano ho li-primers tse ling ebe e li tlama, e lumellang hore sebopeho sa "loop" se thehoe ho DNA e sa tsoa etsoa, ​​​​e ntan'o thusa ho kenella ka har'a mekhahlelo e latelang ea amplification.LAMP e ka bonoa ka mekhoa e mengata, ho kenyelletsa fluorescence, agarose gel electrophoresis, kapa colorimetry.Boiketlo ba ho bona le ho bona boteng kapa ho ba sieo ha sehlahisoa ka colorimetry le khaello ea lisebelisoa tse theko e phahameng tse hlokahalang li entse hore LAMP e be khetho e nepahetseng bakeng sa tlhahlobo ea SARS-CoV-2 libakeng tseo tlhahlobo ea laboratori ea bongaka e neng e sa fumanehe habonolo, kapa polokelo le ho tsamaisa lisampole. e ne e sa khonehe, kapa li-lab tse neng li se na lisebelisoa tsa thermocycling tsa PCR pele.