Chemical cleavage reactions of DNA on solid support: application in mutation detection
Genomic Disorders Research Centre (the University of Melbourne) 7th Floor, Daly Wing, St. Vincent's Hospital, 35 Victoria parade, Fitzroy, Vic. 3065, Australia
BMC Chemical Biology 2003, 3:1 doi:10.1186/1472-6769-3-1Published: 13 May 2003
The conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports.
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.