• PCR inzira inoshandiswa kukudza DNA kubva padiki diki reDNA template.RT-PCR inoshandisa reverse transcription kugadzira template yeDNA kubva kuRNA sosi inogona kukwidziridzwa.
• PCR uye RT-PCR ndiwo maitiro ekupedzisira, nepo qPCR neRT-qPCR vachishandisa kinetics yechiyero chechigadzirwa synthesis panguva yePCR maitiro kuverengera huwandu hwetemplate iripo.
• Mitoo mitsva, yakadai sedhijitari PCR, inopa huwandu hwakakwana hwekutanga DNA template, nepo nzira dzakadai se isothermal PCR dzinoderedza kudiwa kwemidziyo inodhura kuti ipe mhedzisiro yakavimbika.

Polymerase chain reaction (PCR) inzira iri nyore uye inoshandiswa zvakanyanya mamorekuru biology kukudza uye kuona kutevedzana kweDNA neRNA.Kuenzaniswa nemaitiro echinyakare eDNA cloning uye amplification, iyo inogona kazhinji kutora mazuva, PCR inoda maawa mashoma chete.PCR inonzwa zvakanyanya uye inoda kushoma template yekuona uye kukwidziridzwa kweakatevedzana akatevedzana.Basic PCR nzira dzakaenderera mberi kubva nyore DNA uye RNA yekuona.Pazasi, takapa mucherechedzo wenzira dzakasiyana dzePCR uye mareagents atinopa kuEnzo Life Sayenzi kune zvaunoda pakutsvaga.Isu tine chinangwa chekubatsira masayendisiti kukurumidza kuwana PCR reagents kuti vashandise muchirongwa chavo chinotevera chekutsvagisa!

PCR

Kune yakajairwa PCR, zvese zvaunoda iDNA polymerase, magnesium, nucleotides, primers, template yeDNA kuti ikwidziridzwe, uye thermocycler.Muchina wePCR uri nyore sechinangwa chayo: 1) DNA ine tambo mbiri (dsDNA) inopisa kupisa, 2) maprimer anowirirana kune imwechete DNA tambo, uye 3) maprimers anowedzerwa neDNA polymerase, zvichiita kuti makopi maviri yekutanga DNA strand.The denaturation, annealing, uye elongation muitiro pamusoro nhevedzano tembiricha uye nguva inozivikanwa seimwe denderedzwa rekudzamisa (Fig. 1).

Imwe neimwe nhanho yekutenderera inofanirwa kugadziridzwa kune template uye primer seti inoshandiswa.Kutenderera uku kunodzokororwa kanenge 20-40 nguva, uye chigadzirwa chakasimudzirwa chinogona kuongororwa, kazhinji neagarose gel (Fig. 2).

Sezvo PCR iri nzira ine hanya zvikuru uye mavhoriyamu madiki kwazvo anodiwa pakuita kumwe chete, kugadzirira kwemusanganiswa mukuru wekuita kwakati wandei kunokurudzirwa.Musanganiswa mukuru unofanirwa kusanganiswa zvakanaka uye wozopatsanurwa nehuwandu hwemabatiro, kuve nechokwadi chekuti maitiro ega ega achange aine huwandu hwakafanana hweenzyme, dNTPs, uye maprimers.Vazhinji vatengesi, vakadai seEnzo Life Sayenzi, vanopawo PCR misanganiswa inotova nezvose kunze kwemaprimers uye DNA template.

Guanine/Cytosine-rich (GC-rich) matunhu anomiririra dambudziko mune akajairwa PCR matekiniki.GC-yakapfuma sequence yakagadzikana kupfuura kutevedzana ine yakaderera GC zvemukati.Uyezve, kutevedzana kweGC-rakapfuma kunowanzoita zvimiro zvechipiri, senge hairpin loops.Nekuda kweizvozvo, GC-yakapfuma tambo mbiri yakaoma kupatsanura zvachose panguva ye denaturation chikamu.Nekuda kweizvozvo, DNA polymerase haigone kugadzira tambo nyowani pasina chipingamupinyi.Iyo tembiricha yepamusoro ye denaturation inogona kuvandudza izvi, uye kugadzirisa kune yakakwira tembiricha yekudziya uye ipfupi nguva yekumisikidza inogona kudzivirira kusungirirwa kusingatarisike kweGC-rich rich primers.Mamwe mareagents anogona kusimudzira kukwidziridzwa kweGC-yakapfuma sequences.DMSO, glycerol, uye betaine inobatsira kukanganisa zvigadziro zvechipiri zvinokonzerwa nekubatana kweGC uye nokudaro kufambisa kuparadzaniswa kwetambo mbiri.

Kupisa Kutanga PCR

Unspecific amplification idambudziko rinogona kuitika panguva yePCR.Mazhinji maDNA polymerases ayo anoshandiswa kuPCR anoshanda zvakanyanya pakudziya kunosvika 68°C kusvika 72°C.Iyo enzyme inogona, zvisinei, zvakare inoshanda kune yakaderera tembiricha, kunyangwe kune yakaderera degree.Pane tembiricha iri kure pasi petembiricha yekumisa, maprimers anogona kusunga zvisiri-chaiyo uye kutungamira kune kusiri-chaiyo amplification, kunyangwe kana maitiro akaiswa paaizi.Izvi zvinogona kudzivirirwa nekushandisa mapolymerase inhibitors anopatsanura kubva kuDNA polymerase kana imwe tembiricha yasvika, nekudaro izwi rinopisa rinotanga PCR.Iyo inhibitor inogona kuva antibody inosunga polymerase uye denatures panguva yekutanga denaturation tembiricha (95 ° C kazhinji).

High Fidelity Polymerase

Nepo DNA polymerases inokwidziridza zvakaringana kune yekutanga template kutevedzana, zvikanganiso mukufananidza nucleotide zvinogona kuitika.Kusapindirana mumashandisirwo akaita se cloning kunogona kukonzera zvinyorwa zvidiki, uye kusaturikirwa kana kusashanda mapuroteni kuzasi.Kuti udzivise kusawirirana uku, mapolymerase ane "proofreading" chiitiko akaonekwa uye akaiswa mukufambiswa kwebasa.Yekutanga proofreading polymerase, Pfu, yakaonekwa muna 1991 muPyrococcus furiosus.Iyi Pfu enzyme ine 3' kusvika 5' exonuclease chiitiko.Sezvo DNA ichiwedzerwa, iyo exonuclease inobvisa nucleotides isina kufanana pa 3 'kumagumo kwetambo.Iyo chaiyo nucleotide inobva yatsiviwa, uye DNA synthesis inoenderera mberi.Kuzivikanwa kweiyo nucleotide kutevedzana kwakashata kunobva pakusunga hukama hweiyo chaiyo nucleoside triphosphate ine enzyme, uko kusungirirwa kusingashande kunononoka kuumbwa uye kunobvumira kutsiva kwayo.Iyo yekudzokorora chiitiko chePfu polymerase inokonzeresa zvikanganiso zvishoma mukutevedzana kwekupedzisira zvichienzaniswa neTaq DNA polymerase.Mumakore achangopfuura, mamwe maenzayimu ekuongorora akaonekwa, uye kugadziridzwa kweiyo Pfu enzyme yekutanga kwakaitwa kuti kuwedzere kudzikisa mwero wekukanganisa panguva yekukwidziridzwa kweDNA.

RT-PCR

Reverse transcription PCR, kana RT-PCR, inobvumira kushandiswa kweRNA setemplate.Imwe nhanho yekuwedzera inobvumira kuonekwa uye kukwidziridzwa kweRNA.Iyo RNA inodzokororwa yakanyorwa kuita inowirirana DNA (cDNA), ichishandisa reverse transcriptase.Hunhu uye kuchena kweiyo RNA template kwakakosha pakubudirira kweRT-PCR.Nhanho yekutanga yeRT-PCR ndeyekuumbwa kweDNA/RNA yakasanganiswa.Reverse transcriptase ine zvakare RNase H basa, rinodzikisira chikamu cheRNA chehybrid.Iyo imwe-stranded DNA molecule inozopedzwa neDNA-inotsamira DNA polymerase chiitiko che reverse transcriptase mu cDNA.Kushanda kweyekutanga-strand reaction kunogona kukanganisa maitiro ekusimudzira.Kubva pano zvichienda mberi, iyo yakajairwa PCR maitiro inoshandiswa kukwidziridza cDNA.Iko kugona kudzoreredza RNA mu cDNA neRT-PCR kune zvakawanda zvakanaka, uye inonyanya kushandiswa pakuongorora gene expression.RNA ine tambo imwe uye haina kugadzikana, izvo zvinoita kuti zviome kushanda nayo.Inowanzo shanda senhanho yekutanga muqPCR, iyo inoverengera zvinyorwa zveRNA mumuenzaniso webhayoloji.

qPCR uye RT-qPCR

Quantitative PCR (qPCR) inoshandiswa kuona, kuratidza uye kuyera nucleic acids kune akawanda maapplication.MuRT-qPCR, zvinyorwa zveRNA zvinowanzoyeverwa nekudzoserwa kuanyora mu cDNA kutanga, sezvatsanangurwa pamusoro, uyezve qPCR inozoitwa.Sezviri muPCR yakajairika, DNA inokwidziridzwa nematanho matatu anodzokorora: denaturation, annealing, uye elongation.Nekudaro, muqPCR, fluorescent labeling inogonesa kuunganidzwa kwedata sezvo PCR inofambira mberi.Iyi tekinoroji ine mabhenefiti akawanda nekuda kwehuwandu hwenzira uye chemistries iripo.

Mudhayi-yakavakirwa qPCR (kazhinji girini), fluorescent labeling inobvumira kuverengerwa kweakawedzera DNA mamorekuru nekushandisa kushandiswa kwe dsDNA inosunga dhayi.Munguva yega yega kutenderera, fluorescence inoyerwa.Chiratidzo chefluorescence chinowedzera zvakaenzana nehuwandu hweDNA yakadzokororwa.Nokudaro, DNA inotaridzirwa mu "nguva chaiyo" (Fig. 3).Izvo zvisingabatsiri kune dhayi-based qPCR ndezvekuti chinangwa chimwe chete chinogona kuongororwa panguva uye kuti dhayi inosunga kune chero ds-DNA iripo mumuenzaniso.

Muprobe-based qPCR, zvinangwa zvakawanda zvinogona kuonekwa panguva imwe chete mumuenzaniso wega wega, asi izvi zvinoda optimization uye dhizaini yechinangwa-chaicho probe (s) inoshandiswa kuwedzera kune yekutanga.Mhando dzinoverengeka dzemagadzirirwo eprobe dziripo, asi mhando yakajairika ndeye hydrolysis probe, iyo inosanganisira fluorophore uye quencher.Fluorescence resonance energy transfer (FRET) inodzivirira kubuda kwefluorophore kuburikidza ne quencher apo probe inenge iripo.Nekudaro, panguva yekuitwa kwePCR, iyo probe inoitwa hydrolyzed panguva yekuwedzera kweprimer uye kukwidziridzwa kweiyo chaiyo kutevedzana kwainosungirwa kwairi.Iyo cleavage yeprobe inoparadzanisa fluorophore kubva kune quencher uye inoguma nekuwedzera-kunoenderana nekuwedzera kwe fluorescence (Fig. 4).Saka, chiratidzo chefluorescence kubva kune probe-based qPCR reaction inoenzanirana nehuwandu hweiyo probe target sequence iripo mumuenzaniso.Nekuti probe-based qPCR yakanyanya kujeka kupfuura dhayi-yakavakirwa qPCR, kazhinji ndiyo tekinoroji inoshandiswa muqPCR-based diagnostic assays.

Isothermal Amplification

Iyo PCR yataurwa pamusoro apa nzira inoda inodhura thermocycling yekukwira nekudzika tembiricha yemukamuri ye denaturation, annealing, uye matanho ekuwedzera.Nzira dzinoverengeka dzakagadzirwa dzisingadi michina yakadaro uye inogona kuitwa mubhati remvura iri nyore kana kunyange mukati memasero ekufarira.Aya matekiniki akaunganidzwa anodaidzwa kunzi isothermal amplification uye basa rinobva pane exponential, linear, kana cascade amplification.

Iyo inonyanya kuzivikanwa mhando yeisothermal amplification ndeye loop-mediated isothermal amplification, kana LAMP.LAMP inoshandisa exponential amplification pa65⁰C kukudza template DNA kana RNA.Paunenge uchiita LAMP, mana kusvika matanhatu maprimers anoenderana nematunhu echinangwa cheDNA anoshandiswa neDNA polymerase kugadzira DNA nyowani.Maviri eaya maprimers ane anotevedzana anotevedzana anoona kutevedzana mune mamwe maprimers uye anoasunga, zvichibvumira kuti "loop" chimiro chiumbe muDNA ichangobva kugadzirwa iyo inozobatsira kutanga kudzoreredza mumashure anotevera ekukudza.LAMP inogona kuonekwa nenzira dzakawanda, kusanganisira fluorescence, agarose gel electrophoresis, kana colorimetry.Kureruka kwekuona uye kuona kuvepo kana kusavapo kwechigadzirwa ne colorimetry uye kushomeka kwemidziyo inodhura inodiwa kwakaita kuti LAMP ive sarudzo yakakodzera yekuongororwa kweSARS-CoV-2 munzvimbo dzainge dzisingawanikwe nyore nyore, kana kuchengetedza uye kutakura masampuli. zvaisakwanisika, kana mumalabhu akange asina kare PCR thermocycling michina.