Nuclease reaction conditions

RPA are most reliable for relatively short probes (200-400 bases) the longer the probes, the stronger the need for intact mRNA. In contrast to SI analysis, gel puriHcation of the probe is not required, although recommended, after runoff synthesis. Another important advantage of RPA is that probes are internally labeled so that the specific activity of RNA probes is determined by specific activity of precursors and not by the activity of kinase (as in common SI probes) although internally labeled DNA probes have also been used in SI analysis (Ley et al., 1982). Internally labeled probes have a much higher specific activity than end-labeled probes and improve the detectability considerably. Finally, RNases are not as sensitive to reaction conditions as SI nuclease and thus yield more reproducible results. 

When the ability of nucleases SI and MB to trim the single-stranded portion of the cohesive termini of X DNA was compared (25), nuclease SI (at pH 4.5) made a complete cleavage of the 12-nt ends at 10°C as well as at 30°C without any digestion of the dsDNA. Nuclease MB (at pH 5.0) cleaved off the single-stranded ends completely at 30°C but, at 5°C, 4 nt remained undigested even at high enzyme concentrations. Under both reaction conditions, nuclease MB introduced some nicks into dsDNA. 

The optimal pH for nuclease MB lies between pH 4.7 and 5.3, and is dependent on the NaAc concentration (1). Although the enzyme is less active at higher pH values, reaction conditions at neutral pH (7.0-7.5) may be more useful and even necessary in some applications. At neutral pH, the single-strand specificity of the enzyme, in terms of the relative cleavage rate of supercoiled versus relaxed phage PM2 DNA, increases substantially over that at low pH conditions. 

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

Li, H.-H., He, Y.-H., Yuan, Y., and Guan, Z., Nuclease pi A new biocatalyst for direct asymmetric aldol reaction under solvent-free conditions. Green Chem. 2011, 13 (1), 185-189. 

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