• PCR yog ib txoj hauv kev siv los nthuav DNA los ntawm ib qho me me ntawm DNA qauv.RT-PCR siv cov ntawv thim rov qab los tsim cov qauv DNA los ntawm RNA qhov chaw uas tuaj yeem nthuav dav.
• PCR thiab RT-PCR feem ntau yog qhov kawg qhov tshwm sim, thaum qPCR thiab RT-qPCR siv cov kinetics ntawm tus nqi ntawm cov khoom synthesis thaum PCR cov tshuaj tiv thaiv kom muaj nuj nqis ntawm cov qauv tam sim no.
• Cov txheej txheem tshiab, xws li digital PCR, muab qhov tseeb ntawm cov qauv DNA thawj zaug, thaum cov txheej txheem xws li isothermal PCR txo qhov xav tau cov cuab yeej kim los muab cov txiaj ntsig zoo.

Polymerase saw cov tshuaj tiv thaiv (PCR) yog ib qho yooj yim thiab dav siv cov txheej txheem molecular biology kom nthuav dav thiab kuaj pom DNA thiab RNA ib ntus.Piv nrog rau cov txheej txheem ntawm DNA cloning thiab amplification, uas feem ntau tuaj yeem siv sijhawm ntau hnub, PCR tsuas yog ob peb teev xwb.PCR yog qhov rhiab heev thiab xav tau cov qauv me me rau kev kuaj pom thiab nthuav dav ntawm cov kab ke tshwj xeeb.Cov txheej txheem PCR yooj yim tau nce ntxiv los ntawm kev tshawb pom DNA thiab RNA yooj yim.Hauv qab no, peb tau muab cov ntsiab lus ntawm cov txheej txheem PCR sib txawv thiab cov tshuaj reagents peb muab ntawm Enzo Life Sciences rau koj cov kev xav tau tshawb fawb.Peb lub hom phiaj los pab cov kws tshawb fawb sai sai nkag mus rau PCR reagents los siv hauv lawv txoj haujlwm tshawb fawb tom ntej!

PCR

Rau tus qauv PCR, txhua yam koj xav tau yog DNA polymerase, magnesium, nucleotides, primers, DNA template yuav tsum tau amplified, thiab ib tug thermocycler.PCR mechanism yog yooj yim raws li nws lub hom phiaj: 1) ob-stranded DNA (dsDNA) yog tshav kub denatured, 2) primers ua raws li ib tug DNA strands, thiab 3) primers yog ncua los ntawm DNA polymerase, uas ua rau ob daim ntawv luam ntawm lub original DNA strand.Lub denaturation, annealing, thiab elongation txheej txheem dhau ib tug series ntawm kub thiab lub sij hawm yog hu ua ib lub voj voog ntawm amplification (Fig. 1).

Txhua kauj ruam ntawm lub voj voog yuav tsum tau ua kom zoo rau cov qauv thiab primer teeb siv.Lub voj voog no rov ua dua li ntawm 20-40 zaug, thiab cov khoom lag luam amplified tuaj yeem txheeb xyuas, feem ntau yog los ntawm agarose gel (Fig. 2).

Raws li PCR yog ib txoj kev rhiab heev thiab qhov ntim me me yuav tsum tau ua rau ib qho kev tawm tsam, kev npaj ntawm tus tswv sib xyaw rau ntau qhov kev xav tau pom zoo.Tus tswv sib xyaw yuav tsum sib xyaw kom zoo thiab tom qab ntawd muab faib los ntawm tus lej ntawm cov tshuaj tiv thaiv, kom ntseeg tau tias txhua qhov tshuaj tiv thaiv yuav muaj tib tus nqi ntawm enzyme, dNTPs, thiab primers.Ntau tus neeg muag khoom, xws li Enzo Life Sciences, kuj muaj PCR sib xyaw uas twb muaj txhua yam tshwj tsis yog primers thiab DNA qauv.

Guanine / Cytosine-nplua nuj (GC-nplua nuj) cheeb tsam sawv cev rau kev sib tw hauv cov txheej txheem PCR.GC-nplua nuj sequences ruaj khov dua li cov kab ke nrog qis GC cov ntsiab lus.Tsis tas li ntawd, GC-nplua nuj sequences zoo li tsim cov txheej txheem thib ob, xws li hairpin loops.Yog li ntawd, GC-nplua nuj ob strands yog qhov nyuaj rau kev sib cais tag nrho thaum lub sij hawm denaturation.Yog li ntawd, DNA polymerase tsis tuaj yeem tsim cov strand tshiab yam tsis muaj kev cuam tshuam.Qhov kub thiab txias dua tuaj yeem txhim kho qhov no, thiab kev hloov kho ntawm qhov kub thiab txias dua thiab lub sij hawm ua kom luv luv tuaj yeem tiv thaiv qhov tsis sib xws ntawm GC-nplua nuj primers.Ntxiv reagents tuaj yeem txhim kho qhov kev nthuav dav ntawm GC-nplua nuj sequences.DMSO, glycerol, thiab betaine pab cuam tshuam cov txheej txheem thib ob uas tshwm sim los ntawm GC kev cuam tshuam thiab yog li pab txhawb kev sib cais ntawm ob txoj hlua.

Kub Pib PCR

Kev nthuav dav tsis meej yog ib qho teeb meem uas tuaj yeem tshwm sim thaum PCR.Feem ntau DNA polymerases uas siv rau PCR ua haujlwm zoo tshaj plaws ntawm qhov kub ntawm 68 ° C txog 72 ° C.Txawm li cas los xij, cov enzyme tuaj yeem ua haujlwm ntawm qhov kub thiab txias, txawm tias qis dua.Ntawm qhov kub thiab txias qis dua qhov kub ntawm qhov annealing, primers tuaj yeem khi tsis yog tshwj xeeb thiab ua rau tsis muaj kev nthuav dav, txawm tias cov tshuaj tiv thaiv tau teeb tsa rau dej khov.Qhov no tuaj yeem tiv thaiv tau los ntawm kev siv cov tshuaj polymerase inhibitors uas cuam tshuam los ntawm DNA polymerase nkaus xwb thaum qee qhov kub tau mus txog, yog li lub sijhawm kub pib PCR.Tus inhibitor tuaj yeem yog cov tshuaj tiv thaiv kab mob uas khi cov polymerase thiab denatures ntawm qhov pib denaturation kub (95 ° C feem ntau).

High Fidelity Polymerase

Thaum DNA polymerases nthuav dav kom raug raws li tus qauv qauv, kev ua yuam kev hauv nucleotide txuam tuaj yeem tshwm sim.Kev tsis sib haum xeeb hauv cov ntawv thov xws li cloning tuaj yeem ua rau cov ntawv sau tsis raug, thiab cov ntaub ntawv tsis raug lossis tsis ua haujlwm hauv qab.Txhawm rau zam qhov tsis sib xws, polymerases nrog "kev ua pov thawj" tau txheeb xyuas thiab muab tso rau hauv kev ua haujlwm.Thawj proofreading polymerase, Pfu, tau pom nyob rau hauv 1991 hauv Pyrococcus furiosus.Qhov no Pfu enzyme muaj 3' mus rau 5' exonuclease kev ua.Raws li DNA tau nthuav dav, lub exonuclease tshem tawm cov nucleotides tsis sib haum ntawm 3 'kawg ntawm txoj hlua.Qhov tseeb nucleotide yog tom qab ntawd hloov, thiab DNA synthesis txuas ntxiv mus.Kev txheeb xyuas qhov tsis raug nucleotide sequences yog ua raws li kev sib koom ua ke rau qhov tseeb nucleoside triphosphate nrog cov enzyme, qhov uas tsis muaj txiaj ntsig khi ua kom qeeb cov synthesis thiab tso cai rau kev hloov pauv.Kev ua pov thawj ntawm Pfu polymerase ua rau muaj kev ua yuam kev tsawg dua hauv qhov kawg ua ntu zus piv rau Taq DNA polymerase.Nyob rau hauv xyoo tas los no, lwm cov enzymes proofreading tau raug txheeb xyuas, thiab kev hloov kho ntawm tus thawj Pfu enzyme tau tsim los txo qhov kev ua yuam kev thaum lub sij hawm DNA amplification.

RT-PCR

Reverse transcription PCR, lossis RT-PCR, tso cai siv RNA ua tus qauv.Ib kauj ruam ntxiv tso cai rau kev tshawb pom thiab nthuav dav ntawm RNA.RNA yog thim rov qab mus rau hauv cov DNA sib ntxiv (cDNA), siv cov ntawv thim rov qab.Qhov zoo thiab purity ntawm RNA qauv yog qhov tseem ceeb rau kev ua tiav ntawm RT-PCR.Thawj kauj ruam ntawm RT-PCR yog kev sib txuas ntawm DNA / RNA hybrid.Rov qab transcriptase kuj muaj RNase H muaj nuj nqi, uas degrades RNA feem ntawm hybrid.Qhov ib leeg-stranded DNA molecule yog tom qab ntawd ua tiav los ntawm DNA-dependent DNA polymerase kev ua ntawm kev thim rov qab rau hauv cDNA.Qhov ua tau zoo ntawm thawj-strand cov tshuaj tiv thaiv tuaj yeem cuam tshuam cov txheej txheem amplification.Txij ntawm no mus, tus txheej txheem PCR yog siv los ua kom nrov rau cDNA.Qhov muaj peev xwm thim rov qab RNA rau hauv cDNA los ntawm RT-PCR muaj ntau qhov zoo, thiab nws feem ntau yog siv rau kev tshuaj ntsuam gene.RNA yog ib leeg-stranded thiab tsis ruaj khov heev, uas ua rau nws nyuaj rau kev ua haujlwm nrog.Nws feem ntau ua haujlwm ua thawj kauj ruam hauv qPCR, uas suav nrog RNA cov ntawv teev lus hauv cov qauv lom neeg.

qPCR thiab RT-qPCR

Quantitative PCR (qPCR) yog siv los kuaj xyuas, txheeb xyuas thiab txheeb xyuas cov nucleic acids rau ntau daim ntawv thov.Hauv RT-qPCR, RNA cov ntawv teev lus feem ntau raug ntsuas los ntawm kev thim rov qab rau hauv cDNA ua ntej, raws li tau piav qhia saum toj no, thiab tom qab ntawd qPCR tau ua tiav.Raws li hauv PCR tus qauv, DNA tau nthuav dav los ntawm peb cov kauj ruam rov ua dua: denaturation, annealing, thiab elongation.Txawm li cas los xij, hauv qPCR, fluorescent labeling ua rau kev sau cov ntaub ntawv raws li PCR nce.Cov txheej txheem no muaj ntau yam txiaj ntsig vim muaj ntau txoj hauv kev thiab chemistry muaj.

Hauv dye-raws li qPCR (feem ntau ntsuab), fluorescent labeling tso cai rau kom muaj nuj nqis ntawm amplified DNA molecules los ntawm kev siv dsDNA khi dye.Thaum txhua lub voj voog, cov fluorescence raug ntsuas.Lub teeb liab fluorescence nce proportionally rau tus nqi ntawm DNA replicated.Yog li ntawd, DNA thiaj li muaj nyob rau hauv "lub sijhawm tiag tiag" (Fig. 3).Qhov tsis zoo rau dye-raws li qPCR yog tias tsuas yog ib lub hom phiaj tuaj yeem kuaj xyuas ib lub sijhawm thiab cov xim yuav khi rau ib qho ds-DNA tam sim no hauv cov qauv.

Hauv kev sojntsuam raws qPCR, ntau lub hom phiaj tuaj yeem tshawb pom ib txhij hauv txhua tus qauv, tab sis qhov no yuav tsum tau ua kom zoo dua qub thiab tsim cov phiaj xwm tshwj xeeb uas siv los ntxiv rau cov primers.Muaj ntau hom kev sojntsuam tsim muaj, tab sis hom ntau tshaj plaws yog hydrolysis sojntsuam, uas suav nrog fluorophore thiab quencher.Fluorescence resonance zog hloov (FRET) tiv thaiv emission ntawm fluorophore ntawm lub quencher thaum lub sojntsuam tsis zoo.Txawm li cas los xij, thaum lub sij hawm PCR cov tshuaj tiv thaiv, qhov kev sojntsuam yog hydrolyzed thaum lub sij hawm primer extension thiab amplification ntawm ib theem zuj zus nws yog khi rau.Lub cleavage ntawm qhov kev sojntsuam cais cov fluorophore los ntawm lub quencher thiab ua rau kom muaj amplification-dependant nce nyob rau hauv fluorescence (Fig. 4).Yog li, lub teeb liab fluorescence los ntawm kev sojntsuam raws qPCR cov tshuaj tiv thaiv yog proportional rau tus nqi ntawm qhov kev sojntsuam lub hom phiaj ib ntus tam sim no hauv cov qauv.Vim hais tias kev sojntsuam raws qPCR yog qhov tshwj xeeb tshaj li dye-based qPCR, nws feem ntau yog cov thev naus laus zis siv hauv qPCR-raws li kev tshuaj ntsuam xyuas.

Isothermal Amplification

PCR tau hais los saum toj no cov tswv yim yuav tsum muaj cov khoom siv thermocycling kim kom raug nce thiab nqis chamber kub rau qhov denaturation, annealing, thiab txuas ntxiv cov kauj ruam.Ntau cov tswv yim tau tsim los uas tsis xav tau cov cuab yeej zoo li no thiab tuaj yeem ua tau hauv chav da dej yooj yim lossis txawm nyob hauv cov hlwb ntawm kev txaus siab.Cov txheej txheem no yog hu ua isothermal amplification thiab ua hauj lwm raws li exponential, linear, los yog cascade amplification.

Qhov zoo tshaj plaws-paub hom isothermal amplification yog voj-mediated isothermal amplification, los yog LAMP.LAMP siv exponential amplification ntawm 65⁰C los nthuav cov qauv DNA lossis RNA.Thaum ua haujlwm LAMP, plaub mus rau rau tus primers ntxiv rau thaj tsam ntawm lub hom phiaj DNA yog siv nrog DNA polymerase los ua ke DNA tshiab.Ob qho ntawm cov primers no muaj kev sib txuas lus pub dawb uas paub txog cov kab ke hauv lwm cov primers thiab khi lawv, tso cai rau "voj" qauv tsim nyob rau hauv cov tshiab synthesized DNA uas tom qab ntawd pab primer annealing nyob rau hauv lub tom ntej rounds ntawm amplification.LAMP tuaj yeem pom los ntawm ntau txoj hauv kev, suav nrog fluorescence, agarose gel electrophoresis, lossis colorimetry.Qhov yooj yim ntawm kev pom thiab pom qhov muaj lossis tsis muaj cov khoom lag luam los ntawm colorimetry thiab tsis muaj cov cuab yeej kim uas yuav tsum tau ua LAMP qhov kev xaiv tsim nyog rau kev kuaj SARS-CoV-2 nyob rau hauv cov cheeb tsam uas qhov chaw kuaj sim kuaj tsis tau yooj yim, lossis khaws cia thiab thauj cov qauv. tsis muaj peev xwm ua tau, lossis hauv cov chaw sim uas yav dhau los tsis muaj PCR cov khoom siv thermocycling.