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BMC Chemical Biology 2003
Chemical cleavage reactions of DNA on solid support: application in mutation detectionKeywords: solid phase, chemical cleavage reaction, mutation detection, chemical modification of DNA Abstract: DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps.The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.The solution phase Chemical Cleavage of Mismatch (CCM) is one of few methods capable of detecting nearly all single base mismatches [1]. This method was developed in 1988 by Cotton et al. [2] and has been widely used in research and diagnosis of many inherited diseases. The technology is based on selective reactions of mismatched thymine and cytosine with KMnO4 and hydroxylamine respectively [2-5]. The modified mismatched bases are subjected to piperidine cleavage reactions and the resulting fragments are separated and identified by gel-electrophoresis. The process is time-consuming, as the method requires purification of DNA by using the standard precipitation technique after each reaction step [2,4,5]. In our earlier work [6], attempts have been successfully made to attach biotinylated DNA samples onto streptavidin-coated magnetic beads for solid-phase chemical modification and cleavage reactions. The desire to simplify this approach further by reducing the use of the cumbersome biotin dependent assay led us to the development of a new solid-phase chemical cleavage of mismatched DNA (Figure 5). The method involves attachment of DNA on to the commercially available silica solid supports. The solid-bound DNA remained intact throughout the chemical reactions and the final product was removed from solid support by treatment with the piperidine cleavage solution at 90°C as described in our experimental section. This new
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