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genomics For more information on these products contact your local VWR sales office, send an e-mail to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com Green I has been suggested to interfere with DNA repair mechanisms in cells and as a result it potentiates the genotoxicity of other mutagens as well as DNA damage by UV light. Although no safety data is available on other PCR and HRM dyes (e.g., SYTO9, LC Green, BRYT Green and ResoLight), these dyes are known to be cell membrane permeable, thus posing potential genotoxicity risk. With this in mind, Biotium’s scientists designed EvaGreen ® dye to be cell membrane impermeable by increasing the molecular size and charge of the dye (Figure 3). Because EvaGreen ® dye is denied the chance to interact with genomic DNA in living cells, it is much safer than other qPCR dyes. Independent laboratory testing has confirmed that EvaGreen ® is non mutagenic, non cytotoxic and safe to aquatic life. The dye meets environmental hazardous waste regulations in the state of California (CCR title 22) for easy disposal down the drain. Visit Biotium’s website to download the full EvaGreen ® dye safety report. Dye stability EvaGreen ® dye is highly stable during storage and PCR. SYBR ® Green I, on the other hand, is known to degrade following multiple freeze-thaw cycles and under PCR conditions. Moreover, decomposed SYBR ® Green I is reported to be even more inhibitory to PCR than the intact dye.7 As a result, when assessing the performance of EvaGreen ® master mix, you can eliminate the stability of the dye as a variable. Spectral compatibility EvaGreen ® dye is spectrally similar to FAM or SYBR ® Green I. No change in optical settings is required when using an EvaGreen-based master mix (Figure 4). Figure 4. Excitation and emission spectra of EvaGreen in the presence of dsDNA Other applications EvaGreen ® dye has been applied in numerous other applications, such as isothermal amplification, capillary gel electrophoresis, DNA quantitation in solution and selective staining of dead cells in cell viability tests. Biotium offers several EvaGreen ® dyebased products, including stand-alone EvaGreen dye and EvaGreen ® dye master mix products. Biotium’s EvaGreen ® dye technologies are available for licensing. References: 1. Khan, et al. Detection of aacA-aphD, qacE 1, marA, floR, and tetA genes from multidrug-resistant bacteria: comparative analysis of real-time multiplex PCR assays using EvaGreen ® and SYBR ® Green I dyes, Molecular and Cellular Probes (2011), doi: 10.1016/j.mcp.2011.01.004. 2. Cheng, et al. Detection of hemi/homozygotes through heteroduplex formation in high-resolution melting analysis, Anal. Biochem. 410, 158 (2011). 3. White, et al. Methylation-sensitive high-resolution melt-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman Syndromes. Clin. Chem. 53, 1960 (2007). 4. Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76 (2007). 5. Giglio S, et al. Demonstration of preferential binding of SYBR Green I to specific DNA fragments in realtime multiplex PCR. Nucleic Acids Res. 31(22), e136 (2003). 6. Ohta, et al. Ethidium bromide and SYBR Green I enhances the genotoxicity of UV-irradiation and chemical mutagens in E. coli, Mut. Res. 492, 91 (2001). 7. Karsai, et al. Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression. BioTechniques 32(4), 790 (2002). * Practicing HRM may require a license from Idaho Technologies, Inc.; SYBR, ResoLight, LC Green and BRYT Green are trademarks of Invitrogen, Roche, Idaho Technologies and Promega, respectively; EvaGreen technologies are covered by US patent numbers 7,601,498, 7,776,567 and other pending US and international patents. Figure 3. Comparison of cell membrane permeability between EvaGreen ® dye and SYBR ® Green I. HeLa cells were incubated with SYBR ® Green I (1,2 uM) or EvaGreen ® dye (1,2 uM) at 37 °C. Photographs were taken following incubation for 5 and 30 minutes. SYBR ® Green I entered cells rapidly while EvaGreen ® dye appeared membrane impermeable as evident from the absence of cell nuclear staining. Image taken with long photo exposure time revealed that EvaGreen ® dye only associated with cell membranes. Description Size Cat. No. EvaGreen ® dye, 20x in water 1 ml trial size BTIU31000-T 5x 1 ml BTIU31000 100 rxn trial size BTIU31020-T Fast-Plus EvaGreen ® Master mix (no ROX) 200 rxn BTIU31020 500 rxn BTIU31020-1 5000 rxn BTIU31020-2 100 rxn trial size BTIU31014-T Fast-Plus EvaGreen ® Master mix with low ROX 200 rxn BTIU31014 500 rxn BTIU31014-1 5000 rxn BTIU31014-2 100 rxn trial size BTIU31015-T Fast-Plus EvaGreen ® Master mix with high ROX 200 rxn BTIU31015 500 rxn BTIU31015-1 5000 rxn BTIU31015-2 VWR International I VWRbioMarke Issue 27 I September 2011 I 15
the market source for life science New AgilePulse in vivo system DNA vaccine development Skin electroporation: Effects on transgene expression, DNA persistence and local tissue environment Intradermal DNA injection targets the skin for efficient delivery in vaccine research. The skin is an excellent target for DNA vaccine delivery since it is easily accessible and has abundant antigenpresenting cells for a robust immunological response. This response is further increased when injection is followed by intradermal electroporation-- a series of electrical pulses that are applied through an array of small electrodes pressed onto the tissue. This paper studies the effect of intradermal electroporation on the kinetics of transgene expression and DNA persistence and reports on the response of the local tissue environment. Results 1. Intradermal electroporation speeds the onset of transgene expression to 24 hours for injection alone (Figure 2b-d). Closer examination shows especially fast expression for some of the injection sites, at less than 17 minutes (Figure 2b). 2. Electroporation enhanced DNA delivery shows no change in plasmid persistence over several months (Figure 2a). 3. There are no visible differences in the skin that might suggest inflammation of the electrovaccinated mice. 4. Gene profiling of the treated vs. untreated skin shows an up-regulation of genes that are associated with an immunological response in the treated skin (Figure 1). Both DNA injection and electroporation alone showed an upregulation of these pro-inflammatory genes but the combination of DNA injection and electroporation produced a tenfold further up-regulation. Conclusions Intradermal electroporation enhances DNA vaccine delivery with faster expression and stronger immunological response. The persistence of DNA expression is not affected by electroporation. Figure 1. Genes up-regulated at the DNA electrovaccination site. Histogram showing fold increase in gene expression, compared to non treated control skin, of the indicated genes after the specified treatments. Bars represent mean6SD. The number of independently analysed samples varied from 4 - 6; and each sample was run in duplicates or triplicates for each QPCR. The QPCR analysis was run three times. Figure 2. Time kinetics of transgene expression in skin after DNA electrovaccination. (a) Time course of in vivo luciferase expression after intradermal (i.d.) DNA delivery alone (dotted line) and after i.d. DNA delivery followed by electroporation (filled line). One representative experiment of two is shown (n = 8). (b – d) Immediate monitoring of gene expression after DNA electrovaccination. Representative bioluminescent images showing luciferase expression in skin at different time points after DNA electrovaccination. N denotes the negative control (non injected). The scale shows intensity of luminescence (photons/sec/cm2). The experiment was repeated three times. 16 I VWR International I VWRbioMarke Issue 27 I September 2011
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