Abstract
An improved method for mapping RNA transcript boundaries by the nuclease protection technique is presented. This method exploits the large (>20°C) difference in the thermal stability of RNA:DNA and DNA:DNA duplexes in concentrated chaotropic salt solutions. At 45°C in 3.0 m sodium trichloroacetate RNA:DNA hybridization is very efficient but DNA:DNA duplexes remain completely denatured. For many applications, this solvent system can eliminate the need to prepare probes that are free of competing or irrelevant DNA molecules. Fifty- to 100-fold more RNA:DNA hybridization is observed when reassociation is performed in 3.0 m sodium trichloroacetate than in solutions containing high concentrations of formamide. A comparison of the use of S1 nuclease or mung bean nuclease suggests that mung bean nuclease can produce more precise and less ambiguous nuclease protection patterns.
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