• PCR ndi njira yomwe imagwiritsidwa ntchito kukulitsa DNA kuchokera ku template yaying'ono ya DNA.RT-PCR imagwiritsa ntchito reverse transcript kuti ipange template ya DNA kuchokera ku RNA yomwe imatha kukulitsidwa.
• PCR ndi RT-PCR nthawi zambiri zimakhala zomaliza, pomwe qPCR ndi RT-qPCR amagwiritsa ntchito kinetics ya kuchuluka kwa kaphatikizidwe kazinthu panthawi ya PCR kuti athe kuwerengera kuchuluka kwa template yomwe ilipo.
• Njira zatsopano, monga digito PCR, zimapereka chiwerengero chokwanira cha template yoyambirira ya DNA, pamene njira monga isothermal PCR zimachepetsa kufunikira kwa zipangizo zodula kuti zipereke zotsatira zodalirika.

Polymerase chain reaction (PCR) ndi njira yosavuta komanso yogwiritsidwa ntchito kwambiri ndi ma molekyulu a biology kukulitsa ndi kuzindikira ma DNA ndi RNA.Poyerekeza ndi njira zachikhalidwe za DNA cloning ndi kukulitsa, zomwe nthawi zambiri zimatha kutenga masiku, PCR imafuna maola ochepa okha.PCR imakhudzidwa kwambiri ndipo imafuna template yocheperako kuti izindikire ndikukulitsa mndandanda wazinthu zinazake.Njira zoyambira za PCR zapita patsogolo kwambiri kuchokera ku DNA yosavuta ndi RNA kuzindikira.Pansipa, tapereka chithunzithunzi cha njira zosiyanasiyana za PCR ndi ma reagents omwe timapereka ku Enzo Life Sciences pazosowa zanu pa kafukufuku.Tikufuna kuthandiza asayansi kupeza ma reagents a PCR mwachangu kuti agwiritse ntchito pofufuza kafukufuku wawo wotsatira!

PCR

Pa PCR yokhazikika, zonse zomwe mungafune ndi DNA polymerase, magnesium, nucleotides, primers, template ya DNA kuti ikulitsidwe, ndi thermocycler.Makina a PCR ndi osavuta monga cholinga chake: 1) DNA yamitundu iwiri (dsDNA) imakhala yotentha, 2) zoyambira zimagwirizana ndi chingwe chimodzi cha DNA, ndipo 3) zoyambira zimakulitsidwa ndi DNA polymerase, zomwe zimapangitsa kuti makope awiri apangidwe. choyambirira cha DNA strand.The denaturation, annealing, ndi elongation ndondomeko pa mndandanda wa kutentha ndi nthawi amadziwika ngati mkombero umodzi wa matalikidwe (mkuyu. 1).

Gawo lirilonse la kuzungulira liyenera kukonzedwa kuti likhale template ndi zoyambira zomwe zimagwiritsidwa ntchito.Kuzungulira uku kubwerezedwa pafupifupi 20-40 nthawi, ndipo mankhwala okulitsa amatha kusanthula, makamaka ndi gel osakaniza agarose (mkuyu. 2).

Popeza PCR ndi njira yovuta kwambiri ndipo ma voliyumu ang'onoang'ono amafunikira pakuchita kamodzi, kukonzekera kaphatikizidwe kabwino ka machitidwe angapo ndikofunikira.Kusakaniza kwakukulu kuyenera kusakanizidwa bwino ndikugawidwa ndi kuchuluka kwa zomwe zimachitika, kuwonetsetsa kuti chilichonse chizikhala ndi ma enzyme, ma dNTP, ndi zoyambira.Otsatsa ambiri, monga Enzo Life Sciences, amaperekanso zosakaniza za PCR zomwe zili kale ndi chirichonse kupatula zoyambira ndi template ya DNA.

Madera a Guanine/Cytosine-rich (GC-rich) akuyimira zovuta mu njira zodziwika bwino za PCR.Mayendedwe olemera a GC amakhala okhazikika kuposa ma GC otsika.Kuphatikiza apo, kutsatizana kolemera kwa GC kumakonda kupanga zida zachiwiri, monga malupu atsitsi.Zotsatira zake, zingwe zapawiri za GC zimakhala zovuta kuzilekanitsa panthawi ya denaturation.Chifukwa chake, DNA polymerase singathe kupanga chingwe chatsopano popanda cholepheretsa.Kutentha kwapamwamba kwambiri kumatha kuwongolera izi, ndipo kusintha kopitilira kutentha kocheperako komanso nthawi yayifupi yotsekera kumatha kuletsa kumangirira mosadziwika bwino kwa zoyambira zolemera za GC.Ma reagents owonjezera amatha kukulitsa kuchulukitsitsa kwa ma GC-rich-process.DMSO, glycerol, ndi betaine zimathandiza kusokoneza zigawo zachiwiri zomwe zimayambitsidwa ndi kuyanjana kwa GC ndipo potero zimathandizira kupatukana kwa zingwe ziwiri.

Hot Start PCR

Kukulitsa mosadziwika bwino ndi vuto lomwe limatha kuchitika pa PCR.Ma DNA polymerases ambiri omwe amagwiritsidwa ntchito pa PCR amagwira ntchito bwino pa kutentha kozungulira 68°C mpaka 72°C.Enzymeyi imatha kukhalanso yogwira ntchito pamatenthedwe otsika, ngakhale pang'ono.Pakutentha kwambiri pansi pa kutentha kwa annealing, zoyambira zimatha kumangirira mosadukiza ndikupangitsa kukulitsa kosagwirizana, ngakhale zomwe zimayikidwa pa ayezi.Izi zitha kupewedwa pogwiritsa ntchito ma polymerase inhibitors omwe amasiyanitsidwa ndi DNA polymerase pokhapokha kutentha kwina kwafika, motero mawu akuti "hot start PCR".Inhibitor ikhoza kukhala antibody yomwe imamanga ma polymerase ndi ma denatures pa kutentha koyambirira kwa denaturation (95 ° C nthawi zambiri).

High Fidelity Polymerase

Ngakhale ma polymerase a DNA amakulitsa molondola kutsatana koyambirira kwa template, zolakwika pakufananitsa ma nucleotide zitha kuchitika.Zosemphana zamapulogalamu monga kupanga ma cloning zitha kupangitsa kuti zolembedwazo zikhale zocheperako, komanso mapuloteni otanthauziridwa molakwika kapena osagwira ntchito kutsika.Pofuna kupewa izi, ma polymerases okhala ndi "kuwerengera" azindikirika ndikuphatikizidwa mumayendedwe ogwirira ntchito.Polymerase yoyamba yowerengera, Pfu, idadziwika mu 1991 ku Pyrococcus furiosus.Enzyme iyi ya Pfu ili ndi ntchito ya 3' mpaka 5 'exonuclease.Pamene DNA imakulitsidwa, exonuclease imachotsa ma nucleotide osagwirizana kumapeto kwa 3 'mapeto a chingwe.Nucleotide yolondola imasinthidwa, ndipo kaphatikizidwe ka DNA kakupitilira.Kuzindikirika kwa ma nucleotide olakwika kumatengera kuyanjana kolondola kwa nucleoside triphosphate ndi enzyme, komwe kumangiriza kosakwanira kumachepetsa kaphatikizidwe ndikulola m'malo olondola.Ntchito yowunikiranso ya Pfu polymerase imabweretsa zolakwika zochepa pamndandanda womaliza poyerekeza ndi Taq DNA polymerase.M’zaka zaposachedwapa, ma enzyme ena otsimikizira zolondola azindikiridwa, ndipo kusinthidwa kwa puloteni yoyambirira ya Pfu apangidwa kuti achepetsenso kuchuluka kwa zolakwika pakukula kwa DNA.

RT-PCR

Reverse transcript PCR, kapena RT-PCR, imalola kugwiritsa ntchito RNA ngati template.Njira yowonjezera imalola kuzindikira ndi kukulitsa kwa RNA.RNA imasinthidwa kukhala DNA yowonjezera (cDNA), pogwiritsa ntchito reverse transcriptase.Ubwino ndi chiyero cha template ya RNA ndizofunikira kuti RT-PCR ikhale yabwino.Gawo loyamba la RT-PCR ndikuphatikiza kwa hybrid ya DNA/RNA.Reverse transcriptase ilinso ndi ntchito ya RNase H, yomwe imasokoneza gawo la RNA la haibridi.Molekyu ya DNA ya chingwe chimodzi imatsirizidwa ndi DNA-yodalira DNA polymerase ntchito ya reverse transcriptase mu cDNA.Kuchita bwino kwa chingwe choyamba kumatha kukhudza njira yokulitsa.Kuyambira pano, njira yokhazikika ya PCR imagwiritsidwa ntchito kukulitsa cDNA.Kuthekera kobwezeretsa RNA kukhala cDNA ndi RT-PCR kuli ndi zabwino zambiri, ndipo kumagwiritsidwa ntchito makamaka pakuwunika ma gene.RNA ndi ya chingwe chimodzi komanso yosakhazikika, zomwe zimapangitsa kuti zikhale zovuta kugwira ntchito.Nthawi zambiri imakhala ngati sitepe yoyamba mu qPCR, yomwe imawerengera zolemba za RNA mu zitsanzo zachilengedwe.

qPCR ndi RT-qPCR

Quantitative PCR (qPCR) imagwiritsidwa ntchito pozindikira, kuwonetsa ndi kuwerengera ma nucleic acid pamapulogalamu angapo.Mu RT-qPCR, zolembedwa za RNA nthawi zambiri zimawerengedwa ndikuzilembanso kukhala cDNA poyamba, monga tafotokozera pamwambapa, kenako qPCR imachitika pambuyo pake.Monga mu PCR wamba, DNA imakulitsidwa ndi masitepe atatu obwerezabwereza: denaturation, annealing, and elongation.Komabe, mu qPCR, kulemba zilembo za fulorosenti kumathandiza kusonkhanitsa deta pamene PCR ikupita patsogolo.Njirayi ili ndi maubwino ambiri chifukwa cha kuchuluka kwa njira ndi ma chemistry omwe alipo.

Mu utoto wopangidwa ndi qPCR (nthawi zambiri wobiriwira), zolemba za fulorosenti zimalola kuchulukitsidwa kwa mamolekyu a DNA pogwiritsa ntchito utoto womangira wa dsDNA.Panthawi iliyonse yozungulira, fluorescence imayesedwa.Chizindikiro cha fluorescence chimawonjezeka molingana ndi kuchuluka kwa DNA yofananizidwa.Chifukwa chake, DNA imawerengedwa mu "nthawi yeniyeni" (mkuyu 3).Kuipa kwa qPCR yochokera ku utoto ndikuti chandamale chimodzi chokha chikhoza kuyesedwa panthawi imodzi ndikuti utotowo umamangiriza ku ds-DNA iliyonse yomwe ilipo pachitsanzocho.

Mu probe-based qPCR, mipherezero yambiri imatha kuzindikirika nthawi imodzi pachitsanzo chilichonse, koma izi zimafunikira kukhathamiritsa ndi kapangidwe ka kafukufuku wa chandamale (ma) omwe amagwiritsidwa ntchito kuwonjezera pa zoyambira.Mitundu ingapo ya mapangidwe a probe ilipo, koma mtundu wodziwika kwambiri ndi kafukufuku wa hydrolysis, womwe umaphatikizapo fluorophore ndi quencher.Fluorescence resonance energy transfer (FRET) imalepheretsa kutuluka kwa fluorophore kudzera pa quencher pamene kafukufukuyo sali bwino.Komabe, panthawi ya PCR, kafukufukuyu amapangidwa ndi hydrolyzed panthawi yowonjezera yoyambira ndikukulitsa mndandanda wazomwe akuyenera kuchita.The cleavage wa kafukufuku umalekanitsa fluorophore ndi quencher ndi kuchititsa ndi amplification amadalira kuwonjezeka fluorescence (mkuyu. 4).Chifukwa chake, siginecha ya fluorescence yochokera ku probe-based qPCR reaction ndi yolingana ndi kuchuluka kwa zomwe zafufuzidwa zomwe zili pachitsanzocho.Chifukwa qPCR yochokera ku probe ndiyokhazikika kwambiri kuposa qPCR yopangidwa ndi utoto, nthawi zambiri imakhala ukadaulo womwe umagwiritsidwa ntchito poyesa mayeso a qPCR.

Isothermal Amplification

Njira za PCR zomwe tazitchula pamwambapa zimafuna zida zamtengo wapatali zopangira thermocycling kuti zikwere bwino ndi kutsika kutentha kwa chipindacho potengera masitepe a denaturation, annealing, and extensions.Njira zingapo zapangidwa zomwe sizifunikira zida zenizeni zotere ndipo zitha kuchitidwa mumadzi osamba osavuta kapena ngakhale mkati mwa maselo okonda.Njira izi pamodzi zimatchedwa isothermal amplification ndi ntchito kutengera exponential, linear, kapena cascade amplification.

Mtundu wodziwika bwino wa isothermal amplification ndi loop-mediated isothermal amplification, kapena LAMP.LAMP imagwiritsa ntchito kukulitsa kwachidziwitso pa 65⁰C kukulitsa template ya DNA kapena RNA.Mukamapanga LAMP, zoyambira zinayi mpaka zisanu ndi chimodzi zophatikizana ndi zigawo zomwe mukufuna DNA zimagwiritsidwa ntchito ndi DNA polymerase kupanga DNA yatsopano.Awiri mwa oyambirawa ali ndi katsagana kakang'ono komwe kamazindikira kutsatizana muzoyambira zina ndikuzimanga, zomwe zimapangitsa kuti "lupu" ipangike mu DNA yomwe yangopangidwa kumene yomwe imathandizira kukulitsa kakulidwe kotsatira.LAMP imatha kuwonedwa ndi njira zingapo, kuphatikiza fluorescence, agarose gel electrophoresis, kapena colorimetry.Kusavuta kuwona ndikuzindikira kupezeka kapena kusakhalapo kwa chinthu ndi colorimetry komanso kusowa kwa zida zodula zomwe zimafunikira zidapangitsa LAMP kukhala njira yoyenera yoyezera SARS-CoV-2 m'malo omwe kuyezetsa kwa labu sikunapezeke, kapena kusunga ndi kutumiza zitsanzo. sizinali zotheka, kapena m'ma lab omwe kale analibe zida za PCR thermocycling.