We here describe a rapid and convenient sample preparation in a single tube for detecting single nucleotide variation based on oligonucleotide ligation assay (OLA) using a biotinylated primer and a set of four different fluorophore-labeled primers. Only one of the fluorophore-labeled primers that was fully complementary to a DNA template was ligated with the biotin-primer by DNA ligase. We isolated the ligated fragments that carried a biotin at the 3'-end by capture with streptavidin-coated magnetic beads. The distinct fluorescence signature of each ligation product coded the single nucleotide differences.