Qhov Tseem Ceeb Qhov txawv - RT PCR vs QPCR
Polymerase Chain Reaction yog cov txheej txheem siv los nthuav qhia thaj chaw tshwj xeeb ntawm DNA hauv vitro. Vim yog kev tsim cov txheej txheem no los ntawm Kary Mullis hauv xyoo 1983, cov kws tshawb fawb muaj peev xwm ua tau ntau txhiab mus rau lab ntawm cov ntawv luam ntawm cov DNA tshwj xeeb rau kev tshawb fawb. Tam sim no nws tau dhau los ua cov txheej txheem ua haujlwm niaj hnub thiab niaj hnub ua hauv chaw kuaj mob thiab tshawb fawb rau ntau yam kev siv. Muaj kev hloov pauv ntawm cov txheej txheem PCR ib txwm muaj xws li RT PCR, nested PCR, multiplex PCR, Q PCR, RT – QPCR, thiab lwm yam. RT PCR thiab Q PCR yog ob qho kev hloov pauv tseem ceeb ntawm PCR. Qhov sib txawv tseem ceeb ntawm RT PCR thiab Q PCR yog tias RT PCR yog siv los txheeb xyuas cov noob qhia los ntawm kev tsim cov ntawv sau DNA (cDNA) los ntawm RNA thaum Q PCR siv los ntsuas qhov ntau ntawm PCR cov khoom siv lub sijhawm siv fluorescent dyes.
RT PCR yog dab tsi?
Rov qab hloov pauv cov tshuaj polymerase saw tshuaj tiv thaiv (RT PCR) yog qhov txawv ntawm PCR uas yog siv los txheeb xyuas RNA qhia. Nws yog ib txoj hauv kev tseem ceeb heev rau kev kuaj pom mRNA hauv cov ntaub so ntswg. RT PCR yog ua haujlwm thaum cov khoom pib ntawm cov qauv yog RNA. Hauv RT PCR, tus qauv mRNA yog thawj zaug hloov mus rau hauv DNA ntxiv. Cov kauj ruam no yog catalyzed los ntawm enzyme thim rov qab transcriptase thiab cov txheej txheem hu ua thim rov qab. Qhov thib ob, ib txwm siv PCR ua haujlwm rau cDNA tshiab rau kev nthuav dav.
RT PCR yog cov txheej txheem rhiab heev uas yuav tsum muaj tus lej me me ntawm RNA qauv. RT PCR feem ntau yog siv rau hauv kev kuaj mob thiab ntsuas qhov ntau ntawm RNA hom, tshwj xeeb tshaj yog cov kab mob RNA xws li tib neeg kev tiv thaiv kab mob thiab kab mob siab C.
Daim duab 01: RT PCR Technique
QPCR yog dab tsi?
Quantitative PCR (QPCR) yog qhov sib txawv ntawm PCR uas yog siv los ntsuas cov khoom PCR ntau. Nws tseem raug xa mus rau lub sijhawm tiag tiag-lub sijhawm polymerase saw cov tshuaj tiv thaiv vim nws ntsuas qhov kev nthuav dav ntawm lub sijhawm tiag tiag siv lub sijhawm PCR tshuab. Nws yog ib txoj hauv kev tsim nyog rau kev txiav txim siab tus nqi ntawm lub hom phiaj ib ntus lossis cov noob tam sim no hauv cov qauv. Qhov nthuav feature ntawm QPCR yog tias nws muab ob qho tib si amplification thiab muaj tseeb kom muaj nuj nqis rau hauv ib kauj ruam. Yog li ntawd, qhov xav tau ntawm gel electrophoresis hauv kev kuaj pom tuaj yeem raug tshem tawm los ntawm QPCR txheej txheem. QPCR siv fluorescent dyes los sau cov khoom PCR thaum lub sij hawm PCR cov tshuaj tiv thaiv uas nws thiaj li ua rau kom muaj nuj nqis ncaj qha. Thaum cov khoom PCR sib sau ua ke, cov teeb liab fluorescent kuj tau sau thiab nws yuav raug ntsuas los ntawm lub sijhawm tiag tiag tshuab. QPCR tuaj yeem ua ke nrog RT PCR. Nws yog lub npe hu ua RT - QPCR lossis QRT - PCR thiab suav tias yog ib txoj hauv kev muaj zog tshaj plaws, rhiab thiab muaj nuj nqis rau kev kuaj pom ntawm RNA qib hauv cov hlwb lossis cov ntaub so ntswg.
SYBR Green thiab Taqman yog ob txoj hauv kev ua haujlwm los ntes lossis saib cov txheej txheem nthuav dav ntawm PCR lub sijhawm. SYBR Green txoj kev yog ua los ntawm kev siv cov fluorescent dye hu ua SYBR ntsuab thiab kuaj pom qhov ua kom muaj zog los ntawm kev khi cov dye kom tsim tau ob txoj hlua DNA. Taqman yog ua los ntawm kev siv dual-labeled probes thiab kuaj pom lub amplification los ntawm degradation ntawm qhov kev sojntsuam los ntawm Taq polymerase thiab tso tawm cov fluorophore raws li qhia nyob rau hauv daim duab 02. Ob txoj kev soj ntsuam cov kev nce qib ntawm cov amplification txheej txheem thiab qhia kom muaj nuj nqis ntawm cov khoom ntawm lub sij hawm..
Lub sijhawm tiag tiag PCR muaj ntau yam kev siv xws li gene qhia quantification, microRNA thiab noncoding RNA tsom xam, SNP genotyping, nrhiav kom tau cov ntawv luam tus lej variants, nrhiav kom tsis tshua muaj kev hloov pauv, tshawb nrhiav cov kab mob hloov pauv, kuaj xyuas cov kab mob sib kis., etc.
Daim duab 02: Cov txheej txheem PCR ntau
Qhov txawv ntawm RT PCR thiab QPCR yog dab tsi?
RT PCR vs QPCR |
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| RT PCR yog cov txheej txheem siv los kuaj cov noob qhia los ntawm kev nthuav dav. | QPCR yog cov txheej txheem uas nthuav dav DNA thiab ntsuas cov khoom PCR raws sijhawm. |
| Kev koom tes ntawm Reverse Transcriptase Enzyme | |
| Enzyme thim rov qab transcriptase yog siv rau RT PCR. | Enzyme thim rov qab transcriptase tsis siv rau QPCR. |
| Siv cov fluorescently Labeled Molecules | |
| Fluorescently labeled dyes lossis probes tsis siv rau RT PCR. | Fluorescently labeled dyes lossis probes yog siv rau QPCR. |
| Kev Ntsuas ntawm PCR khoom | |
| Tshwj tsis yog ua ke nrog QPCR, RT PCR tsis suav cov khoom PCR. | QPCR ntau npaum li cas ntsuas cov khoom PCR. |
| khoom pib | |
| khoom pib yog mRNA. | Cov khoom pib yog DNA. |
| Synthesis of cDNA | |
| Complementary DNA yog tsim thaum lub sij hawm RT PCR. | Complementary DNA tsis tsim thaum lub sij hawm QPCR. |
Summary – RT PCR vs QPCR
RT PCR thiab QPCR yog ob hom ntawm PCR ib txwm muaj. RT PCR txheej txheem yog ua rau mRNA cov qauv thiab nws yog tsav los ntawm kev rov qab transcription thiab zus tau tej cov cDNA. QPCR yog siv los txheeb xyuas cov khoom PCR thaum lub sijhawm tiag tiag PCR thermal cycles siv fluorescent dyes los yog sau npe sojntsuam. Hauv QPCR, tus nqi ntawm PCR cov khoom yog sawv cev los ntawm emission fluorescent signals los ntawm cov qauv. RT PCR tau nrov siv los ua cov txheej txheem nthuav dav thaum QPCR feem ntau siv los ua cov txheej txheem kom muaj nuj nqis. Qhov no yog qhov txawv ntawm RT PCR thiab QPCR.
