کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1925052 | 1536332 | 2015 | 7 صفحه PDF | دانلود رایگان |
• Two assays for monitoring DNA digestion by λ-exonuclease and EcoRV in real-time.
• Assays rely on the different fluorescence intensities between ss- and ds-DNA–YOYO.
• Sensitive determination of nuclease activity and quantification of reaction products.
Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA–protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA–YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.
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Journal: Archives of Biochemistry and Biophysics - Volume 570, 15 March 2015, Pages 40–46