Qhov txawv ntawm qhov tseem ceeb – SYBR Green vs Taqman
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 yog ib txoj kev raws li intercalating nucleic acid staining dye thaum Taqman yog ib txoj kev raws li hydrolysis sojntsuam. Ob lub thev naus laus zis yog tsim los tsim cov fluorescence thaum lub sijhawm PCR, uas tso cai rau lub sijhawm PCR lub sijhawm los saib xyuas cov tshuaj tiv thaiv hauv "lub sijhawm tiag tiag". 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 los ua ob txoj hlua DNA. Taqman yog nqa tawm siv ob-labeled probes thiab kuaj pom lub amplification los ntawm degradation ntawm lub sojntsuam los ntawm Taq polymerase thiab tso tawm ntawm fluorophore. Nov yog qhov sib txawv tseem ceeb ntawm SYBR Green thiab Taqman.
SYBR Green yog dab tsi?
SYBR Ntsuab yog cov xim fluorescent siv los stain nucleic acids, tshwj xeeb tshaj yog muab ob npaug rau DNA hauv Molecular Biology. SYBR Green txoj kev siv los ntsuas cov khoom PCR thaum lub sijhawm PCR tiag tiag. Thaum nws khi nrog DNA, qhov ua rau DNA-dye complex absorbs xiav lub teeb thiab emits ntsuab lub teeb. Nws tshwm sim vim qhov kev hloov pauv uas tshwm sim hauv cov xim molecule thaum khi nrog ob-stranded DNA. Thaum PCR tsim ntau thiab ntau DNA, ntau dye molecules khi nrog DNA, tsim ntau fluorescence. Yog li, cov fluorescence nce nrog PCR cov khoom tsub zuj zuj. Li no, tus nqi ntawm PCR cov khoom tuaj yeem ntsuas qhov ntau los ntawm SYBR Green fluorescence detection.
SYBR ntsuab dye kuj tuaj yeem siv rau DNA labeling hauv cytometry thiab fluorescent microscopy. Ethidium bromide tau ua tiav los ntawm SYBR Green txij li Ethidium bromide yog cov xim carcinogenic nrog cov teeb meem pov tseg thaum pom DNA hauv cov gel electrophoresis.
Nws muaj qhov zoo thiab qhov tsis zoo ntawm SYBR ntsuab txoj kev. Txoj kev no yog rhiab heev, pheej yig thiab siv tau yooj yim. Txawm li cas los xij, vim nws lub peev xwm los khi rau ib qho kev sib tw ob leeg DNA, kev khi tsis muaj tseeb tuaj yeem ua rau ntau dua ntawm cov khoom PCR.
Daim duab 01: SYBR Green Technique
Taqman yog dab tsi?
Taqman yog lwm txoj hauv kev rau SYBR Green los saib xyuas cov txheej txheem PCR lub sijhawm. Txoj kev no nyob ntawm 5' - 3' exonuclease kev ua ntawm Taq polymerase enzyme kom degrade cov kev sojntsuam thaum lub sijhawm txuas ntxiv ntawm cov hlua tshiab thiab tso tawm fluorophore. Dual-labeled probes yog siv nyob rau hauv txoj kev no thiab nws yog raws li hydrolysis ntawm probes. Cov tshuaj fluorescently sau npe DNA oligonucleotides muaj cov fluorescent reporter molecule (fluorophore) ntawm 5' kawg thiab ib qho quencher molecule ntawm 3' kawg. Lawv yog tsim los khi rau ib leeg stranded template ntawm opposite sab ntawm primer anneals. Taq polymerase ntxiv nucleotides rau cov primer thiab txuas cov strand tshiab rau ntawm ob-labeled probes. Thaum Taq polymerase ua tau raws li kev sojntsuam, exonuclease kev txiav txim ntawm Taq polymerase activates thiab degrades qhov kev sojntsuam. Thaum nws ua tiav cov synthesis ntawm cov strand tshiab, qhov kev sojntsuam yuav raug ua kom tiav degradation thiab tso cov fluorophore. Kev tso tawm ntawm fluorophore tsim fluorescence. Fluorescent Quencher molecule zoo quenches lub teeb emitted thiab tsim cov zis rau kom muaj nuj nqis ntawm cov khoom PCR. Kev tso tawm ntawm fluorophores thiab qhov ntau ntawm cov khoom PCR yog proportional. Li no, qhov ntau npaum tuaj yeem ua tau yooj yim los ntawm txoj kev Taqman.
Daim duab 02: Taqman Method
Taqman txoj kev yog siv nyob rau hauv lub sijhawm PCR, kom muaj nuj nqis ntawm cov noob qhia, nrhiav kom tau cov noob caj noob ces, kom muaj nuj nqis ntawm chromosomal DNA deletions, kab mob identification, pov thawj ntawm microarray tsom xam, SNP genotyping thiab lwm yam.
Qhov txawv ntawm SYBR ntsuab thiab Taqman yog dab tsi?
SYBR Green vs Taqman |
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| SYBR Ntsuab yog raws li DNA khi dye. | Taqman nyob ntawm hybridization probes thiab 5' rau 3' exonuclease kev ua ntawm Taq polymerase. |
| Fluorescently Labeled Probes | |
| Tsis tas yuav muaj cov tshuaj tiv thaiv fluorescently. | Dual-labeled probes yuav tsum tau. |
| Multiplex Gene Analysis | |
| Nws siv tsis tau rau multiplex noob hom phiaj. | Nws tuaj yeem siv tau rau ntau lub hom phiaj gene. |
| Ntawm no tsis tshua kim. | Nyob no kim dua. |
| Specificity | |
| Qhov no tsis tshua meej thiab khi nrog ob txoj hlua DNA | Cov no yog qhov tshwj xeeb heev txij li kev sojntsuam kuaj pom cov khoom lag luam tshwj xeeb. |
| effectiveness | |
| Qhov no tsis zoo. | Qhov no ua tau zoo heev. |
| Daim ntawv thov | |
| Qhov no yog siv rau hauv lub sijhawm tiag tiag PCR, agarose gel visualization, DNA labeling thiab lwm yam. | Qhov no yog siv rau hauv lub sijhawm PCR, kev txheeb xyuas cov noob qhia, kuaj pom cov noob caj noob ces, thiab lwm yam. |
Summary – SYBR Green and Taqman
Taqman thiab SYBR ntsuab yog ob txoj hauv kev ua haujlwm hauv lub sijhawm PCR (quantitative PCR). Ob txoj hauv kev pab kom muaj nuj nqis ntawm PCR cov khoom lag luam zoo thiab tso siab rau cov emission ntawm fluorescence. Taqman txoj kev siv dual-labeled sojntsuam rau kev kuaj pom cov DNA sib sau thaum SYBR Green txoj kev siv cov xim fluorescent. Ob txoj kev no kuj muaj kev siv sib txawv hauv molecular biology.
