Lub Sijhawm PCR, tseem hu ua quantitative PCR lossis qPCR, yog ib txoj hauv kev rau kev saib xyuas lub sijhawm thiab kev tshuaj xyuas ntawm PCR amplification khoom.
Vim hais tias ntau PCR muaj qhov zoo ntawm kev ua haujlwm yooj yim, ceev thiab yooj yim, siab rhiab heev, rov ua tau zoo, thiab tsis tshua muaj paug tus nqi, nws yog dav siv hauv kev kuaj mob, tshuaj ntsuam xyuas kev ua tau zoo, kev tshawb fawb noob caj noob ces, kev tshawb fawb transgenic, kuaj noob, kuaj kab mob, kuaj tsiaj thiab cog., kuaj zaub mov thiab lwm yam kev lag luam.
Yog li ntawd, txawm hais tias koj tau koom nrog hauv kev tshawb fawb hauv lub neej, lossis cov neeg ua haujlwm ntawm cov tuam txhab tshuaj, cov tuam txhab tsiaj txhu, cov tuam txhab noj zaub mov, lossis txawm tias cov neeg ua haujlwm ntawm kev nkag-tawm kev tshuaj xyuas thiab cais tawm chaw ua haujlwm, chaw saib xyuas ib puag ncig, tsev kho mob thiab lwm chav, koj yuav raug ntau lossis tsawg dua lossis koj yuav tsum paub txog kev paub txog kev paub txog PCR ntau npaum li cas.

Txoj Cai ntawm Real Time PCR

Lub Sijhawm PCR yog ib txoj hauv kev uas cov tshuaj fluorescent tau ntxiv rau PCR cov tshuaj tiv thaiv, thiab cov teeb liab fluorescence siv nyob rau hauv cov txheej txheem ntawm PCR cov tshuaj tiv thaiv yog saib xyuas nyob rau lub sijhawm los ntawm PCR ntau npaum li cas, thiab thaum kawg cov ntaub ntawv sim tau tshuaj xyuas thiab ua tiav.

【Amplification nkhaus】yog qhov nkhaus piav txog cov txheej txheem dynamic ntawm PCR.Qhov kev nthuav dav ntawm PCR tsis yog tus qauv nthuav dav, tab sis yog qhov nkhaus sigmoid.

[Platform theem ntawm amplification nkhaus]Nrog rau qhov nce ntawm cov PCR mus, qhov inactivation ntawm DNA polymerase, depletion ntawm dNTPs thiab primers, thiab inhibition ntawm synthesis cov tshuaj tiv thaiv los ntawm cov tshuaj tiv thaiv los ntawm cov khoom pyrophosphate, thiab lwm yam, PCR tsis tas yuav nthuav dav exponentially., thiab thaum kawg yuav nkag mus rau lub toj siab.

[Exponential Kev Loj Hlob Thaj Av ntawm Amplification Curve]Txawm hais tias lub toj siab theem sib txawv heev, nyob rau hauv ib cheeb tsam ntawm lub exponential kev loj hlob cheeb tsam ntawm lub amplification nkhaus, lub repeatability yog zoo heev, uas yog ib qho tseem ceeb heev rau kev ntsuam xyuas ntawm PCR.

[Tus nqi pib thiab Ct tus nqi]Peb teeb tsa tus nqi txwv ntawm kev kuaj pom fluorescence ntawm qhov tsim nyog nyob rau hauv qhov chaw loj hlob ntawm qhov nthuav dav ntawm qhov nkhaus, uas yog tus nqi pib (Threshold).Qhov kev sib tshuam ntawm tus nqi pib thiab qhov kev nthuav dav yog qhov Ct tus nqi, uas yog, tus nqi Ct yog hais txog tus lej ntawm lub voj voog (Threshold Cycle) thaum tus nqi pib mus txog.

Daim duab hauv qab no qhia meej txog kev sib raug zoo ntawm txoj kab pib thiab kev nthuav dav nkhaus, qhov pib thiab Ct tus nqi.

【Yuav ntsuas li cas?】

Nws tau raug pov thawj los ntawm kev tshawb nrhiav lej uas tus nqi Ct muaj qhov sib txawv ntawm cov kab sib txuas nrog lub logarithm ntawm tus lej ntawm cov qauv pib.Lub Sijhawm PCR saib PCR cov khoom lag luam nthuav dav hauv lub sijhawm tiag tiag thiab suav lawv thaum lub sijhawm nthuav dav nthuav dav.

Rau txhua lub voj voog ntawm PCR, DNA nce exponentially los ntawm 2 zaug, thiab sai sai mus txog toj siab.

Piv txwv tias tus nqi pib DNA yog A₀ , tom qab n cycles, qhov theoretical npaum li cas ntawm cov khoom DNA tuaj yeem qhia tau tias:

A _n =A ₀ × 2ⁿ

Tom qab ntawd, qhov ntau dua qhov pib DNA ntau npaum li cas A 0, qhov sai npaum li cas ntawm cov khoom lag luam tau nce mus txog qhov ntsuas tus nqi An, thiab tus lej ntawm lub voj voog thaum ncav cuag An yog tus nqi Ct.Ntawd yog, qhov ntau dua qhov pib DNA tus nqi A 0 yog, qhov ua ntej qhov kev nthuav dav nkhaus peaks, thiab qhov sib npaug ntawm qhov xav tau ntawm cov voj voog n yog me dua.

Peb nqa tawm gradient dilution ntawm tus qauv paub qhov concentration thiab siv nws ua tus qauv rau Real Time PCR, thiab cov kev nthuav dav ntawm cov nkhaus yuav tau txais ntawm qhov sib npaug ntawm qhov kev txiav txim ntawm qhov pib DNA ntawm ntau mus rau tsawg dua.Raws li kev sib raug zoo ntawm tus nqi Ct thiab lub logarithm ntawm tus naj npawb ntawm cov qauv pib, a[tus qauv nkhaus] tuaj yeem tsim tau.

Los ntawm kev hloov tus nqi Ct ntawm tus qauv nrog qhov tsis paub concentration rau hauv tus qauv nkhaus, thawj tus qauv ntawm tus qauv uas tsis paub meej tuaj yeem tau txais, uas yog lub hauv paus ntsiab lus ntau ntawm Real Time PCR.

Kev txheeb xyuas txoj kev ntawm Real Time PCR

Lub Sijhawm PCR tshawb pom PCR cov khoom lag luam nthuav dav los ntawm kev kuaj xyuas cov fluorescence siv hauv cov tshuaj tiv thaiv.

Txoj Cai ntawm Fluorescent Dye Embedding Method】

Fluorescent dyes, xws li TB Green ® , tuaj yeem khi tsis tau tshwj xeeb rau ob txoj hlua DNA hauv PCR systems thiab fluoresce thaum khi.

Kev siv fluorescence hauv cov tshuaj tiv thaiv tau nce ntxiv nrog kev nce ntawm PCR mus.Los ntawm kev txheeb xyuas qhov kev siv fluorescence, tus nqi ntawm DNA amplification nyob rau hauv cov tshuaj tiv thaiv kab mob tuaj yeem soj ntsuam lub sijhawm, thiab tom qab ntawd tus nqi ntawm cov qauv pib hauv cov qauv tuaj yeem kwv yees rov qab.

【Txoj cai ntawm txoj kev fluorescent sojntsuam】

fluorescent sojntsuamyog nucleic acid ib ntus nrog ib pawg fluorescent ntawm 5′ kawg thiab pawg quenching ntawm 3′ kawg, uas tuaj yeem khi rau tus qauv.Thaum qhov kev sojntsuam tsis zoo, cov fluorescence tawm los ntawm fluorophore yog quenched los ntawm pawg quenching thiab tsis tuaj yeem fluoresce.Thaum qhov kev sojntsuam yog decomposed, cov tshuaj fluorescent yuav dissociate thiab emit fluorescence.

Ib qho fluorescent sojntsuam ntxiv rau cov tshuaj tiv thaiv PCR.Thaum lub sij hawm annealing txheej txheem, fluorescent sojntsuam yuav khi rau qhov chaw tshwj xeeb ntawm tus qauv.Thaum lub sijhawm txuas ntxiv, 5′ → 3′ exonuclease kev ua ntawm PCR enzyme tuaj yeem decompose cov fluorescent sojntsuam hybridized nrog cov qauv, thiab cov tshuaj fluorescent dissociated rau emit fluorescence.Los ntawm kev txheeb xyuas qhov kev siv fluorescence ntawm qhov kev sojntsuam hauv cov tshuaj tiv thaiv, lub hom phiaj ntawm kev saib xyuas cov khoom lag luam ntawm PCR tuaj yeem ua tiav.

【Kev xaiv ntawm Fluorescence Detection Method】

Yog tias nws yog siv los txheeb xyuas qhov sib txawv nrog cov homology siab thiab ua ntau yam PCR nrhiav pom xws li SNP typing tsom xam, fluorescent sojntsuam txoj kev yog irreplaceable.
Rau lwm qhov kev sim PCR tiag tiag, siv txoj kev yooj yim, yooj yim thiab tus nqi qis fluorescent chimera.

Kev sojntsuam muaj zog tshwj xeeb, muaj peev xwm ntawm multiplex PCR

Ua tsis tau

High tshwj xeeb yuav tsum tau rau amplification;

multiplex PCR tsis tuaj yeem ua tiavNyob rau hauv kev tsim tshwj xeeb probes, tus nqi siab;

Qee zaum kev sojntsuam tsim yog qhov nyuaj

Yam khoom: