• I-PCR iyindlela esetshenziselwa ukukhulisa i-DNA kusuka enanini elincane lesifanekiso se-DNA.I-RT-PCR isebenzisa ukuloba okuhlanekezelwe ukukhiqiza isifanekiso se-DNA esivela kumthombo we-RNA esingase sithuthukiswe.
• I-PCR ne-RT-PCR ngokuvamile kuwukusabela kwe-endpoint, kuyilapho i-qPCR ne-RT-qPCR zisebenzisa i-kinetics yezinga lokuhlanganiswa komkhiqizo phakathi nokusabela kwe-PCR ukuze kulinganise inani lesifanekiso esikhona.
• Izindlela ezintsha, njenge-PCR yedijithali, zinikeza inani eliphelele lesifanekiso sokuqala se-DNA, kuyilapho izindlela ezifana ne-PCR ye-isothermal zinciphisa isidingo semishini ebizayo ukunikeza imiphumela ethembekile.

I-Polymerase chain reaction (PCR) iyindlela elula futhi esetshenziswa kabanzi ye-molecular biology ukukhulisa nokuthola ukulandelana kwe-DNA ne-RNA.Uma kuqhathaniswa nezindlela zendabuko ze-DNA cloning nokukhulisa, okungavamise ukuthatha izinsuku, i-PCR idinga amahora ambalwa kuphela.I-PCR izwela kakhulu futhi idinga isifanekiso esincane sokutholwa nokukhulisa ukulandelana okuthile.Izindlela eziyisisekelo ze-PCR sezithuthuke kakhulu ekutholeni i-DNA elula ne-RNA.Ngezansi, sinikeze umbono wezindlela ezihlukene ze-PCR kanye nama-reagents esiwanikeza kwa-Enzo Life Sciences ngezidingo zakho zocwaningo.Sihlose ukusiza ososayensi ukuthi bafinyelele ngokushesha ama-PCR ukuze bawasebenzise kuphrojekthi yabo elandelayo yocwaningo!

I-PCR

Ku-PCR evamile, okudingayo nje i-DNA polymerase, i-magnesium, ama-nucleotides, ama-primers, isifanekiso se-DNA esizokhuliswa, kanye ne-thermocycler.Indlela ye-PCR ilula njengenhloso yayo: 1) i-DNA enemicu ephindwe kabili (dsDNA) ishintshiwe ngokushisa, 2) ama-primer aqondana ne-DNA strands eyodwa, futhi 3) ama-primer anwetshwa nge-DNA polymerase, okuholela kumakhophi amabili i-DNA strand yasekuqaleni.Inqubo ye-denaturation, annealing, kanye ne-elongation phezu kochungechunge lwamazinga okushisa nezikhathi yaziwa njengomjikelezo owodwa wokukhulisa i-amplification (Fig. 1).

Isinyathelo ngasinye somjikelezo kufanele silungiselelwe isifanekiso kanye nesethi yokuqala esetshenzisiwe.Lo mjikelezo uphindwe izikhathi ezingaba ngu-20-40, futhi umkhiqizo okhulisiwe ungahlaziywa, ngokuvamile ngejeli ye-agarose (Fig. 2).

Njengoba i-PCR iyindlela ebucayi kakhulu futhi amavolumu amancane kakhulu ayadingeka ekuphenduleni okukodwa, ukulungiswa kwengxube eyinhloko yokusabela okuningana kuyanconywa.Ingxube eyinhloko kufanele ixutshwe kahle bese ihlukaniswa ngenani lokusabela, kuqinisekiswe ukuthi ukusabela ngakunye kuzoqukatha inani elifanayo le-enzyme, ama-dNTP, nama-primers.Abahlinzeki abaningi, njenge-Enzo Life Sciences, baphinde banikeze amamiksi e-PCR asevele equkethe yonke into ngaphandle kweziqalo kanye nesifanekiso se-DNA.

Izifunda ze-Guanine/Cytosine-rich (GC-rich) zimele inselele kumasu ajwayelekile e-PCR.Ukulandelana okucebile nge-GC kuzinze kakhulu kunokulandelana ngokuqukethwe kwe-GC ephansi.Ngaphezu kwalokho, ukulandelana okucebile nge-GC kuvame ukwakha izakhiwo zesibili, njengezihibe ze-hairpin.Ngenxa yalokhu, imicu ephindwe kabili ecebile nge-GC kunzima ukuyihlukanisa ngokuphelele phakathi nesigaba sokukhishwa kwe-denaturation.Ngenxa yalokho, i-DNA polymerase ayikwazi ukuhlanganisa umucu omusha ngaphandle kwesithiyo.Izinga lokushisa eliphakeme lokushintshashintsha kwe-denaturation lingakuthuthukisa lokhu, futhi ukulungiswa okuya emazingeni okushisa aphezulu kanye nesikhathi esifushane sokukhipha isisu kungavimbela ukubopha okungaqondile kwama-primer anothe nge-GC.Ama-reagents engeziwe angathuthukisa ukukhuliswa kokulandelana kwe-GC-rich.I-DMSO, i-glycerol, ne-betaine isiza ukuphazamisa izakhiwo zesibili ezibangelwa ukusebenzisana kwe-GC futhi ngaleyo ndlela kube lula ukuhlukaniswa kwemicu ephindwe kabili.

I-Hot Start PCR

Ukukhulisa okungacacisiwe kuyinkinga engenzeka ngesikhathi se-PCR.Iningi lama-polymerase e-DNA asetshenziselwa i-PCR asebenza kangcono emazingeni okushisa acishe abe ngu-68°C kuya ku-72°C.I-enzyme, nokho, ingasebenza futhi emazingeni okushisa aphansi, nakuba ngezinga eliphansi.Emazingeni okushisa angaphansi kakhulu kwezinga lokushisa le-anneal, ama-primers angabopha ngokungaqondile futhi aholele ekukhuliseni okungaqondile, ngisho noma ukusabela kusethwe eqhweni.Lokhu kungavinjelwa ngokusebenzisa i-polymerase inhibitor ehlukana ne-DNA polymerase uma nje izinga lokushisa elithile selifinyelelwe, yingakho igama elithi hot start PCR.I-inhibitor ingaba i-antibody ebopha i-polymerase nama-denatures ekushiseni kokuqala kokushintshashintsha kwe-denaturation (95°C ngokuvamile).

I-High Fidelity Polymerase

Ngenkathi ama-polymerase e-DNA ekhulisa ngokunembile ukulandelana kwesifanekiso sokuqala, amaphutha ekufanisweni kwe-nucleotide angenzeka.Ukungafani ezinhlelweni zokusebenza ezifana nokwenza i-cloning kungaholela ekulobeni okuncishisiwe, kanye namaprotheni angahumusheki kahle noma angasebenzi ezansi nomfula.Ukuze ugweme lokhu kungafani, ama-polymerase anomsebenzi "wokuhlola amaphutha" akhonjwe futhi afakwa ekuhambeni komsebenzi.I-polymerase yokuqala yokuhlola ukuhlolwa, i-Pfu, yatholakala ngo-1991 ku-Pyrococcus furiosus.Le enzyme ye-Pfu inomsebenzi we-3' kuya ku-5' we-exonuclease.Njengoba i-DNA ikhuliswa, i-exonuclease isusa ama-nucleotide angafani ekugcineni kwe-3' yomucu.Khona-ke i-nucleotide elungile iyashintshwa, futhi ukuhlanganiswa kwe-DNA kuyaqhubeka.Ukuhlonzwa kokulandelana kwe-nucleotide okungalungile kusekelwe ebudlelwaneni obubophezelayo be-nucleoside triphosphate elungile ne-enzyme, lapho ukubopha okungasebenzi kunciphisa ukuhlanganiswa futhi kuvumela ukushintshwa okulungile.Umsebenzi wokuhlola wokuhlola we-Pfu polymerase ubangela amaphutha ambalwa ngokulandelana kokugcina uma kuqhathaniswa ne-Taq DNA polymerase.Eminyakeni yamuva nje, kuye kwatholakala amanye ama-enzyme okuhlola ukuhlolwa, futhi kwenziwa ukuguqulwa kwe-enzyme yokuqala ye-Pfu ukuze kuqhutshekwe nokunciphisa izinga lamaphutha ngesikhathi sokukhulisa i-DNA.

I-RT-PCR

I-PCR yokuhlehlisa, noma i-RT-PCR, ivumela ukusetshenziswa kwe-RNA njengesifanekiso.Isinyathelo esengeziwe sivumela ukutholwa nokukhulisa i-RNA.I-RNA ihlehla ibhalwe ku-DNA ehambisanayo (cDNA), kusetshenziswa i-reverse transcriptase.Izinga nokuhlanzeka kwesifanekiso se-RNA kubalulekile empumelelweni ye-RT-PCR.Isinyathelo sokuqala se-RT-PCR ukuhlanganiswa kwe-DNA/RNA hybrid.I-Reverse transcriptase nayo inomsebenzi we-RNase H, owehlisa isithunzi ingxenye ye-RNA yengxubevange.I-molecule ye-DNA enomucu owodwa ibe isiqedwa umsebenzi oncike ku-DNA polymerase we-reverse transcriptase ibe yi-cDNA.Ukusebenza kahle kokusabela kwe-first-strand kungathinta inqubo yokukhulisa.Kusukela lapha kuya phambili, inqubo ye-PCR ejwayelekile isetshenziselwa ukukhulisa i-cDNA.Amathuba okubuyisela i-RNA ku-cDNA nge-RT-PCR kunezinzuzo eziningi, futhi isetshenziselwa ikakhulukazi ukuhlaziya isisho sofuzo.I-RNA inomucu owodwa futhi ayizinzile, okwenza kube inselele ukusebenza nayo.Ngokuvamile isebenza njengesinyathelo sokuqala ku-qPCR, esilinganisa imibhalo ye-RNA kusampula yebhayoloji.

qPCR kanye ne-RT-qPCR

I-Quantitative PCR (qPCR) isetshenziselwa ukuthola, ukubonisa kanye nokulinganisa ama-nucleic acid ezinhlelo zokusebenza eziningi.Ku-RT-qPCR, imibhalo ye-RNA ivamise ukulinganiswa ngokuyiguqulela ku-cDNA kuqala, njengoba kuchazwe ngenhla, bese i-qPCR isenziwa kamuva.Njengaku-PCR evamile, i-DNA ikhuliswa ngezinyathelo ezintathu eziphindaphindayo: i-denaturation, annealing, kanye ne-elongation.Nokho, ku-qPCR, ukulebula kwe-fluorescent kuvumela ukuqoqwa kwedatha njengoba i-PCR iqhubeka.Le nqubo inezinzuzo eziningi ngenxa yohlu lwezindlela namakhemistri atholakalayo.

Ku-qPCR esekelwe kudayi (imvamisa eluhlaza okotshani), ukulebula kwe-fluorescent kuvumela ukulinganisa kwama-molecule e-DNA akhulisiwe ngokusebenzisa ukusetshenziswa kwedayi ebophayo ye-dsDNA.Phakathi nomjikelezo ngamunye, i-fluorescence iyalinganiswa.Isiginali ye-fluorescence ikhuphuka ngokulingana nenani le-DNA ephindaphindwayo.Ngakho-ke, i-DNA ibalwa "ngesikhathi sangempela" (Fig. 3).Okubi ku-qPCR esekwe kudayi ukuthi okuhlosiwe okukodwa kuphela okungahlolwa ngesikhathi nokuthi udayi uzobophezela kunoma iyiphi i-ds-DNA ekhona kusampula.

Ku-qPCR esekelwe kuphenyo, okuhlosiwe okuningi kungatholwa ngesikhathi esisodwa kusampula ngayinye, kodwa lokhu kudinga ukuthuthukiswa nokuklanywa kophenyo oluqondiswe ngqo okusetshenziswa ngaphezu kwama-primers.Izinhlobo eziningana zemiklamo ye-probe ziyatholakala, kodwa uhlobo oluvame kakhulu i-hydrolysis probe, ehlanganisa i-fluorophore kanye ne-quencher.I-Fluorescence resonance energy transfer (FRET) ivimbela ukukhishwa kwe-fluorophore nge-quencher ngenkathi uphenyo lusamile.Kodwa-ke, ngesikhathi sokusabela kwe-PCR, i-probe i-hydrolyzed ngesikhathi sokunwetshwa kwe-primer kanye nokukhulisa ukulandelana okuqondile okubophezelekile kukho.I-cleavage ye-probe ihlukanisa i-fluorophore kusuka ku-quencher futhi iphumela ekukhuleni okuncike ekukhuliseni kwe-fluorescence (Fig. 4).Ngakho, isignali ye-fluorescence evela ku-probe-based qPCR reaction ilingana nenani lokulandelana okuqondiwe kophenyo elikhona kusampula.Ngenxa yokuthi i-qPCR esekwe kuphenyi icace kakhulu kune-qPCR esekwe kudayi, imvamisa iwubuchwepheshe obusetshenziswa ezivivinyweni zokuxilonga ezisuselwa ku-qPCR.

I-Isothermal Amplification

I-PCR eshiwo ngenhla idinga imishini ebizayo ye-thermocycling ukuze inyuse ngokunembile amazinga okushisa egunjini ukuze kuncishiswe izinga lokushisa, i-annealing, nezinyathelo zokunwetshwa.Kuye kwasungulwa amasu amaningana angadingi amathuluzi anjalo futhi angenziwa emanzini okugeza amanzi noma ngisho nangaphakathi kwamangqamuzana okuthakazelisa.Lawa masu ngokuhlangene abizwa ngokuthi i-isothermal amplification kanye nomsebenzi ngokusekelwe ku-exponential, linear, noma i-cascade amplification.

Uhlobo olwaziwa kakhulu lwe-isothermal amplification i-loop-mediated isothermal amplification, noma i-LAMP.I-LAMP isebenzisa i-exponential amplification ku-65⁰C ukuze ikhulise isifanekiso se-DNA noma i-RNA.Lapho kwenziwa i-LAMP, ama-primers amane kuya kwayisithupha ahambisana nezifunda ze-DNA eqondiwe asetshenziswa ne-DNA polymerase ukuze kuhlanganiswe i-DNA entsha.Okubili kwalezi ziqalo kunokulandelana okuvumelanayo okuqaphela ukulandelana kwezinye iziqalo futhi kuzibophe, okuvumela ukuthi ukwakheka “kweluphu” kwakheke ku-DNA esanda kuhlanganiswa ebese isiza ukuhlanganisa i-primer emizuliswaneni elandelayo yokukhuliswa.I-LAMP ingabonwa ngezindlela eziningi, okuhlanganisa i-fluorescence, i-agarose gel electrophoresis, noma i-colorimetry.Ubulula bokubona ngeso lengqondo nokubona ubukhona noma ukungabikho komkhiqizo nge-colorimetry kanye nokuntuleka kwemishini ebizayo edingekayo kwenza i-LAMP yaba inketho efanelekile yokuhlolwa kwe-SARS-CoV-2 ezindaweni lapho ukuhlolwa kwelebhu yasemtholampilo bekungatholakali kalula, noma ukugcinwa nokuthutha amasampula. bekungenzeki, noma kumalebhu angazange abe nayo imishini ye-PCR thermocycling ngaphambilini.