PCR, ntau PCR, Hauv PCR, Rov qab PCR, RT-PCR, qPCR (1)-PCR

Peb yuav txheeb xyuas cov ntsiab lus, cov kauj ruam thiab cov ntsiab lus ntawm ntau yam PCR

Ⅰ. PCR

Polymerase Chain Reaction, hu ua PCR, yog lub tshuab hluav taws xob molecular uas siv los nthuav cov DNA tshwj xeeb.Nws tuaj yeem suav tias yog qhov tshwj xeeb DNA replication hauv vitro.DNA polymerase (DNA Polymerase I) tau tshawb pom thaum ntxov li xyoo 1955, thiab Klenow Fragment ntawm E. Coli, uas muaj cov txiaj ntsig kev sim thiab kev ua tau zoo, tau tshawb pom los ntawm Dr. H. Klenow thaum ntxov xyoo 1970, tab sis vim tias qhov enzyme no tsis kam rau qhov kub thiab txias, qhov kub thiab txias tuaj yeem degenerate nws, yog li nws tsis ua raws li cov tshuaj tiv thaiv polymer degeneration.Cov enzymes uas siv niaj hnub no (hu ua Taq polymerase), raug cais tawm ntawm Thermus aquaticus, cov kab mob kub kub hauv xyoo 1976. Nws cov yam ntxwv yog tias nws tuaj yeem tiv taus qhov kub thiab txias thiab yog ib qho zoo tagnrho enzyme, tab sis nws yog dav siv tom qab 1980s.Lub tswv yim tseem ceeb ntawm cov qauv qub qub ntawm PCR zoo ib yam li kev kho cov noob caj noob ces thiab kev luam tawm, uas tau hais los ntawm Dr. KJell Kleppe hauv xyoo 1971. Nws tau luam tawm thawj qhov yooj yim thiab luv-term gene copy (zoo ib yam li thawj ob lub voj voog ntawm PCR).PCR tsim niaj hnub no yog tsim los ntawm Dr. Kary B. Mullis hauv xyoo 1983. Dr. Mullis tau ua haujlwm rau PE cov tuam txhab xyoo ntawd, yog li PE muaj xwm txheej tshwj xeeb hauv PCR kev lag luam.Dr. Mullis tau tshaj tawm thawj daim ntawv cuam tshuam nrog Saiki thiab lwm tus hauv xyoo 1985. Txij thaum ntawd los, kev siv PCR yog ntau txhiab mais hauv ib hnub, thiab cov ntaub ntawv zoo sib xws tuaj yeem hais tau ua rau ntau lwm txoj kev tshawb fawb tsis txaus ntseeg.Tom qab ntawd, PCR thev naus laus zis tau siv dav hauv kev tshawb fawb txog kev tshawb fawb lom neeg thiab kev siv tshuaj kho mob, dhau los ua cov thev naus laus zis tseem ceeb tshaj plaws ntawm kev tshawb fawb molecular biology.Mullis kuj yeej 1993 Nobel nqi zog hauv Chemistry.

PCRTxoj Cai

Lub hauv paus ntsiab lus ntawm PCR thev naus laus zis zoo ib yam li cov txheej txheem rov ua dua tshiab ntawm DNA, thiab nws qhov tshwj xeeb yog nyob ntawm oligonucleotide primer uas yog ntxiv rau ob qho kawg ntawm lub hom phiaj sib lawv liag.PCR yog tsim los ntawm degeneration-annealing-extending peb theem pib tshwm sim: ①Degeneration ntawm template DNA: Tom qab lub template DNA yog rhuab mus txog 93 ° C rau ib lub sij hawm ntawm lub sij hawm, lub dual DNA tov rau lub dual-chain DNA tsim los ntawm PCR amplification ntawm lub template DNA tawm, ua rau nws ib tug tib saw hlau rau lub sij hawm ntawm lub sij hawm.② Lub annealing (compound) ntawm template DNA thiab primer: Tom qab lub template DNA yog rhuab thiab degenerated rau hauv ib lub saw, qhov kub poob mus txog 55 ° C.Qhov sib ntxiv ntawm cov primer thiab cov qauv DNA ib leeg-chain.③Qhov kev txuas ntxiv ntawm cov primer: DNA template-lub primer binding yog raws li qhov kev txiav txim ntawm TaqDNA polymerase, nrog dNTP raws li cov tshuaj tiv thaiv raw khoom.Khaws lub hauv paus ntsiab lus ntawm kev rov ua dua tshiab, ua ke cov saw tshiab ib nrab khaws cia uas ua tiav cov qauv DNA cov saw, thiab rov ua lub voj voog degeneration-annealing-extension peb txheej txheem tuaj yeem tau txais ntau "semi-reserved copy chain", thiab cov saw tshiab no muaj dua los ua tus qauv rau lub voj voog tom ntej.Nws yuav siv sij hawm 2-4 feeb los ua kom tiav lub voj, lub hom phiaj gene tuaj yeem nthuav dav ntau lab zaug hauv 2-3 teev.

TxujPCRReaction System

Tsib yam ntawm PCR cov tshuaj tiv thaiv

Feem ntau muaj tsib yam tshuaj koom nrog hauv PCR cov tshuaj tiv thaiv, uas yog primer, enzyme, dNTP, template thiab tsis (Mg2+ yuav tsum tau).[Cov txheej txheem PCR]

Tus txheej txheem PCR tau muab faib ua peb kauj ruam

1. DNA degeneration (90°C-96°C): Dual-chain DNA templates nyob rau hauv thermal ua, hydrogen bonds tawg, tsim ib leeg-chain DNA.

2. Annealing (25 ℃ -65 ℃): Lub kaw lus kub yog txo, cov primer yog ua ke nrog DNA template los tsim ib lub zos dual-chain.

3. Extension (70 ℃ -75 ℃): Raws li kev ua ntawm Taq enzyme (kwv yees li 72 ° C, kev ua haujlwm zoo tshaj plaws), dNTP yog siv los ua cov khoom siv raw, txuas ntxiv los ntawm 5′ kawg ntawm primer → 3′ kawg, synthesis thiab template complements ib leeg DNA saw.

Txhua lub voj voog yog denatured, annealed thiab txuas ntxiv, ob npaug ntawm cov ntsiab lus DNA.Tam sim no, vim yog qhov luv luv amplification cheeb tsam, qee qhov PCR tuaj yeem rov ua dua nyob rau lub sijhawm luv luv txawm tias Taq enzyme kev ua haujlwm tsis zoo, yog li nws tuaj yeem hloov mus rau ob kauj ruam, uas yog, qhov annealing thiab txuas ntxiv tuaj yeem ua tiav ntawm 60 ° C-65 ° C tib lub sijhawm.Txhawm rau txo cov txheej txheem nqa thiab txias thiab txhim kho cov lus teb ceev.

PCR Reaction nta

● High-Specificity

Cov kev txiav txim siab tshwj xeeb ntawm PCR cov lus teb yog: ①Kev sib xyaw tshwj xeeb ntawm cov primer thiab cov qauv DNA.② Lub hauv paus ntsiab lus ntawm kev sib koom ua ke.③ Kev ntseeg siab ntawm TaqDNA polymerase synthesis cov tshuaj tiv thaiv.④ Qhov tshwj xeeb thiab kev txuag ntawm lub hom phiaj gene.

Kev sib xyaw kom raug ntawm primers thiab templates yog qhov tseem ceeb.Kev khi ntawm cov primer thiab cov qauv thiab kev txuas ntxiv ntawm cov saw hlau primer yog raws li lub hauv paus ntsiab lus ntawm alkaline puag txuam.Lub siab ncaj ncees ntawm polymerase synthesis cov tshuaj tiv thaiv thiab qhov kub thiab txias tsis kam ntawm Taq DNA polymerase ua rau kev khi (compound) ntawm cov qauv thiab primer hauv cov tshuaj tiv thaiv tuaj yeem ua tau ntawm qhov kub siab dua.Qhov tshwj xeeb ntawm kev sib xyaw ua ke yog ntau ntxiv.Cov clip tuaj yeem tswj tau qib siab ntawm qhov raug.Los ntawm kev xaiv lub hom phiaj genetic cheeb tsam nrog siab conservativeness thiab siab conservativeness, nws qhov tshwj xeeb yog siab dua.

● Siab rhiab heev

Cov khoom ntim ntawm PCR tau nce los ntawm qhov ntsuas, uas tuaj yeem nthuav dav cov qauv pib ntawm Picker (PG = 10-12) kom nce qib ntawm microcontroller mus rau qib ntawm micrograms (μg = -6).Lub hom phiaj hlwb tuaj yeem kuaj pom los ntawm 1 lab hlwb;nyob rau hauv kev tshawb nrhiav cov kab mob, qhov rhiab heev ntawm PCR tuaj yeem ncav cuag 3 RFUs (qhov chaw tsis muaj chaw tsim);qhov tsawg kawg nkaus nrhiav tus nqi hauv kab mob science yog 3 kab mob.

● Yooj yim thiab nrawm

Qhov kev xav ntawm PCR siv qhov kub-kub Taq DNA polymerase, uas ntxiv cov tshuaj tiv thaiv ib zaug, uas yog, cov tshuaj tiv thaiv degeneration-anneal-extension ntawm DNA amplification tov thiab dej da dej.Feem ntau, cov tshuaj tiv thaiv amplification tiav hauv 2 mus rau 4 teev.Cov khoom lag luam augmented feem ntau raug tshuaj xyuas los ntawm rab ntaj hluav taws xob, thiab tsis tas yuav siv cov isotopes, tsis muaj hluav taws xob muaj kuab paug, thiab kev txhawb nqa yooj yim.

● Lub purity ntawm cov qauv yog tsawg

Tsis tas yuav cais cov kab mob lossis kab mob thiab kab lis kev cai.DNA crude khoom thiab RNA tuaj yeem siv ua amplifiers.Kev kuaj DNA amplification tuaj yeem siv ncaj qha siv cov qauv kuaj mob xws li cov ntshav, lub cev ua kua, hnoos ntxhua khaub ncaws, plaub hau, hlwb, thiab cov ntaub so ntswg nyob.

PCRtej teeb meem

● Tsis muaj qhov tsis zoo, tsis muaj lub suab nrov nrov

Cov theem tseem ceeb ntawm PCR cov tshuaj tiv thaiv muaj xws li: ① kev npaj ntawm template nucleic acids, ② zoo thiab tshwj xeeb ntawm primers, ③ qhov zoo ntawm enzymes ④ PCR lub voj voog.Nrhiav qhov laj thawj yuav tsum tau soj ntsuam thiab kawm rau cov kev txuas saum toj no.

Templates: ① Cov qauv muaj cov protein ntau, ② Cov qauv muaj Taq enzyme inhibitor, ③ Cov protein nyob hauv cov qauv tsis raug tshem tawm, tshwj xeeb tshaj yog pab pawg protein hauv chromosome.⑤ Deminer nucleic acid degeneration tsis meej.Thaum qhov zoo ntawm cov enzymes thiab primers zoo, tsis muaj amplification band, uas yog feem ntau yuav digestive kev kho mob ntawm cov qauv.Muaj ib yam dab tsi tsis ncaj ncees lawm nrog tus qauv nucleic acid extraction txheej txheem, yog li txhawm rau npaj cov kev zom zaub mov zoo thiab ruaj khov, nws cov txheej txheem yuav tsum tau kho thiab tsis hloov pauv.

Enzyme inactivation: ib qho enzyme tshiab lossis ob qho tib si qub thiab cov enzymes tshiab yuav tsum tau siv ua ke los tshuaj xyuas seb cov enzyme puas poob lossis tsis txaus, ua rau muaj qhov tsis zoo.Nws yuav tsum raug sau tseg tias Taq enzyme lossis ethidium bromide qee zaum tsis nco qab.

Primer: qhov zoo ntawm primer, qhov concentration ntawm primer, thiab seb qhov concentration ntawm ob primers yog symmetrical.Nws yog ib qho laj thawj vim li cas rau PCR tsis ua haujlwm lossis qhov nce band tsis zoo tagnrho thiab nws yooj yim rau diffuse.Muaj teeb meem nrog qhov zoo ntawm cov primers ntawm qee tus lej batch.Ob lub primers muaj siab concentration thiab tsis tshua muaj concentration, ua rau tsawg-efficiency asymmetric amplification.Cov tshuaj tiv thaiv yog: ① Xaiv cov primer zoo los ua ke cov chav nyob.② Lub concentration ntawm cov primer tsis tsuas yog nyob ntawm tus nqi OD, tab sis kuj them nyiaj rau cov primer cov kua qub los ua agar qab zib gel electrophoresis.Yuav tsum muaj ib cheeb tsam primer, thiab qhov ci ntsa iab ntawm ob lub primers yuav tsum yog feem ntau sib xws.Txoj siv sia, PCR tuaj yeem ua tsis tiav nyob rau lub sijhawm no, thiab nws yuav tsum tau daws nrog primer synthesis unit.Yog tias primer siab, qhov ci ntsa iab qis, thiab nws cov concentration yuav tsum sib npaug thaum diluted.③ Cov primer yuav tsum tau them thiab khaws cia ntawm qhov siab siab kom tiv thaiv ntau yam khov los yog lub sijhawm ntev ntawm lub tub yees, uas yuav ua rau cov primer deteriorate thiab degrade.④ Cov qauv tsim ntawm primer yog qhov tsis tsim nyog, xws li qhov ntev ntawm cov primer tsis txaus, thiab di cluster yog tsim los ntawm cov primers.

Mg2 + concentration: Mg2 + ion concentration muaj qhov cuam tshuam zoo rau PCR amplification efficiency.Cov concentration ntau dhau tuaj yeem txo qhov kev sib deev ntawm PCR amplification.Yog tias qhov concentration tsawg dhau, PCR amplification tso zis yuav ua rau PCR amplification tsis ua haujlwm yam tsis muaj qhov nthuav dav.

Hloov cov tshuaj tiv thaiv ntim: Lub ntim siv hauv PCR amplification yog 20ul, 30ul, thiab 50ul lossis 100uL, qhov loj ntim ntawm daim ntawv thov rau PCR amplification yog teem raws li cov hom phiaj sib txawv ntawm kev tshawb fawb thiab kev kuaj mob.Tom qab ua cov ntim me me xws li 20ul, nws yog ib qho tsim nyog yuav tsum tau ua cov kab mob thaum ua qhov loj, txwv tsis pub nws yuav poob.

Yog vim li cas lub cev: Kev hloov pauv tseem ceeb heev rau PCR amplification.Yog hais tias lub degeneration kub yog tsawg, lub sij hawm degeneration yog luv, nws yuav tshwm sim nyob rau hauv tsis muaj tseeb negatives;Tsis tshua muaj annealing kub tuaj yeem ua rau tsis yog-specific amplification thiab txo cov kev ua haujlwm amplification.Muaj kev cuam tshuam rau kev sib xyaw ua ke ntawm primers thiab templates kom txo tau PCR amplification efficiency.Qee lub sij hawm nws yog ib qho tsim nyog yuav tsum siv cov ntsuas ntsuas ntsuas ntsuas ntsuas ntsuas ntsuas ntsuas qhov ntsuas qhov sib txawv, ntsuas thiab ntsuas qhov ntsuas kub hauv qhov txuas ntxiv lossis dej-soluble cooker, uas yog ib qho laj thawj rau qhov tsis ua haujlwm ntawm PCR.

Target sequence variants: Yog hais tias lub hom phiaj ib ntus tshwm sim, kev hloov pauv lossis tshem tawm, kev sib txuas ntawm cov qauv thiab cov qauv sib xyaw ua ke, lossis vim tsis muaj lub hom phiaj ib ntus, cov primer thiab tus qauv yuav poob qhov sib txuas ntxiv, thiab nws cov PCR amplification yuav tsis ua tiav.

● Qhov zoo tsis raug

PCR amplification band zoo nkaus li zoo ib yam nrog lub hom phiaj ua ntu zus band, thiab qee zaum nws cov band zoo dua thiab siab dua.

Primer tsim tsis tsim nyog: qhov kev xaiv amplification sequence thiab non-purpose amplification sequence muaj homologous, yog li thaum PCR amplification, cov khoom PCR amplified yog non-purposeful sequences.Lub hom phiaj ib ntus luv dhau los yog cov primer luv dhau, thiab nws ua rau qhov tsis zoo.Yuav tsum tau kho dua tshiab.

Hla kev ua qias tuaj ntawm lub hom phiaj ib ntus lossis cov khoom lag luam amplification: Muaj ob qho laj thawj rau qhov kev ua qias tuaj no: Ua ntej, hla kev ua paug ntawm tag nrho cov genome lossis cov ntu loj, ua rau muaj qhov tsis zoo.Qhov zoo ntawm qhov tsis zoo no tuaj yeem daws tau los ntawm cov txheej txheem hauv qab no: Ceev faj thiab maj mam ua haujlwm thaum lub sijhawm ua haujlwm los tiv thaiv lub hom phiaj ntawm kev nqus mus rau hauv cov qauv phom lossis txaws tawm ntawm lub raj centrifugal.Tsuas yog cov enzymes thiab cov khoom uas tsis tuaj yeem tiv taus qhov kub thiab txias, tag nrho cov reagents lossis cov khoom siv yuav tsum tau muab tshuaj tua kab mob siab.Cov kav dej centrifugal thiab cov qauv yuav tsum tau siv ib zaug.Thaum tsim nyog, ua ntej ntxiv cov qauv, cov tshuaj tiv thaiv raj thiab reagent raug rau ultraviolet rays los rhuav tshem cov nucleic acid uas twb muaj lawm.Qhov thib ob, cov khoom me me hauv cov pa phem.Cov tawg me me no luv dua li lub hom phiaj ib ntus, tab sis lawv muaj qee yam homology.Nws tuaj yeem txuas nrog ib leeg.Tom qab ua tiav cov primers, cov khoom PCR tuaj yeem nthuav dav, uas yuav ua rau cov khoom tsis zoo.Nws tuaj yeem siv los txo lossis tshem tawm cov zes PCR txoj kev.

● Tshwm sim nonspecific amplification band

Cov bands uas tshwm sim tom qab PCR amplification tsis sib haum nrog qhov xav tau qhov loj me, lossis loj lossis me, lossis tib lub sijhawm, lossis tib lub sijhawm, tshwj xeeb amplification bands thiab non-specific amplification bands.Qhov tshwm sim ntawm cov tsis tshwj xeeb bands yog: Ua ntej, cov primers tsis tiav ua tiav rau lub hom phiaj sib lawv liag, lossis cov polymerization ntawm primer los ua ib pawg di.Qhov thib ob yog tias qhov concentration ntawm MG2 + ions yog siab dhau lawm, qhov kub thiab txias annealing tsawg dhau, thiab tus naj npawb ntawm PCR mus txog.Qhov thib ob, qhov zoo thiab tus nqi ntawm cov enzymes.Feem ntau, cov enzymes ntawm qee qhov chaw ua rau cov kab tsis tshwj xeeb thiab cov enzymes ntawm lwm qhov chaw tsis tshwm sim.Qee lub sij hawm tsis muaj kev nthuav dav ntawm cov enzymes kuj tshwm sim.Cov kev tiv thaiv yog: rov tsim kev nyiam yog tias tsim nyog.Txo cov enzyme los yog hloov cov enzyme ntawm lwm qhov chaw.Txo tus nqi ntawm thawj, nce tus nqi ntawm cov qauv tsim nyog, thiab txo cov voj voog.Ua kom qhov kub thiab txias los yog siv ob txoj kev ntsuas kub (93 ° C degeneration, annealing thiab txuas ntxiv ntawm 65 ° C).

● Pom cov hlua khi los yog smear daim kab xev

PCR amplification qee zaum zoo li siv los yog plhaub lossis ntaub pua plag-zoo li siv.Yog vim li cas, vim yog cov enzymes ntau dhau los yog qhov tsis zoo ntawm cov enzyme, dNTP concentration siab dhau lawm, Mg2 + concentration siab dhau lawm, qhov kub thiab txias annealing tsawg dhau, thiab cov voj voog ntau dhau.Cov tshuaj tiv thaiv yog: ① Txo cov enzymes, lossis hloov cov enzyme ntawm lwm qhov.② Txo qhov concentration ntawm dNTP ③ kom txo qis Mg2+ concentration.④ Ua kom muaj ntau ntawm cov qauv thiab txo cov voj voog.

Yam khoom

PCR Hero ᵀᴹ (Nrog Dye)

◮ Kev ntseeg siab dua: 6 npaug ntawm Taq enzyme zoo tib yam;

◮ Amplification ceev dua

◮ Ntau template adaptability

◮ Ua kom muaj zog ntau dua

◮ Kev kam rau ib puag ncig muaj zog: muab tso rau ntawm 37 ° C rau ib lub lim tiam, tswj ntau dua 90% kev ua haujlwm;

◮ Nws muaj 5' → 3' DNA polymerase kev ua haujlwm thiab 5' → 3' exonuclease kev ua, tsis muaj 3' → 5' exonuclease kev ua.

PCR Ib qho yooj yim rau kev teeb tsa (nrog Dye)

Cov tshuaj tiv thaiv tshwj xeeb thiab kev ua haujlwm siab Taq DNA Polymerase ua rau PCR cov tshuaj tiv thaiv muaj kev ua kom zoo dua qub, tshwj xeeb thiab rhiab heev.

RT-qPCR Easyᵀᴹ (Ib kauj ruam)-SYBR Green I

◮ Cov khoom siv ib kauj ruam ua rau rov qab sau dua thiab qPCR ob qhov kev cuam tshuam hauv tib lub raj, tsuas yog xav tau ntxiv cov qauv RNA, tshwj xeeb PCR primers thiab RNase-Free ddH₂O.

◮ Cov khoom siv tau sai thiab muaj txiaj ntsig zoo txheeb xyuas tus kab mob RNA lossis taug RNA.

◮ Cov khoom siv siv qhov tshwj xeeb Foregene thim rov qab reagent thiab Foregene HotStar Taq DNA Polymerase ua ke nrog cov tshuaj tiv thaiv tshwj xeeb txhawm rau txhim kho kev ua kom zoo dua qub thiab tshwj xeeb ntawm cov tshuaj tiv thaiv.

◮ Cov tshuaj tiv thaiv zoo tshaj plaws ua rau cov tshuaj tiv thaiv muaj kev tshawb pom ntau dua, muaj zog thermal stability, thiab kam rau siab dua.

◮ RT-qPCR Ib qho yooj yim^TM(Ib kauj ruam)-SYBR Green I cov khoom siv los nrog ROX sab hauv siv dye, uas tuaj yeem siv los tshem tawm cov teeb liab keeb kwm yav dhau thiab cov teeb liab tsis raug ntawm qhov dej, uas yooj yim rau cov neeg siv khoom siv hauv cov qauv sib txawv ntawm cov khoom siv PCR ntau.

RT Ib qho yooj yim^TMII (Master Premix rau thawj-strand cDNA synthesis rauLub Sijhawm PCR)

-Kev muaj peev xwm tshem tawm gDNA, uas tuaj yeem tshem gDNA hauv cov qauv hauv 2 feeb.

-Txoj kev rov qab ua haujlwm tau zoo, nws tsuas yog siv sijhawm 15 feeb los ua kom tiav cov synthesis ntawm thawj strand cDNA.

-Cov qauv tsis yooj yim: cov qauv nrog cov ntsiab lus GC siab thiab cov qauv theem nrab kuj tuaj yeem thim rov qab nrog kev ua haujlwm siab.

-High-rhiab heev rov qab transcription system, pg-theem templates kuj tau txais high-zoo cDNA.

-Qhov kev hloov pauv rov qab muaj qhov thermal stability siab, qhov pom kev zoo ntawm qhov ntsuas kub yog 42 ℃, thiab nws tseem muaj qhov zoo rov qab ua haujlwm ntawm 50 ℃.