Agarose gel analysis of ERCA reaction products. Assay conditions were as described in Methods for specific Factor V Leiden Mutation ligation/ERCA genotyping assay. Following ERCA, reaction products were separated on a 1.5% agarose gel in 1X TBE at 200 Volts for 2 hours. FAM and Cy3 fluorescence were visualized using a Molecular Dynamics FluorImager. FAM signal was produced during ERCA in response to the normal allele, and Cy3 signal in response to the mutant. Sample in lanes 1 and 2 contained genomic DNA target homozygous for the Factor V Leiden mutation (M), lanes 3 and 4 were heterozygous for the mutation (H), and lane 5 was homozygous normal (N). Three different exposures of the same gel, lanes 1–5, are shown. On the left, the Cy3 fluorescent product is shown in red. In the center, the FAM product is shown in green. On the right, both exposures are shown overlapped. Overlapping FAM and Cy3 produces a yellow signal in this exposure. Migration of 100, 500, and 1000 base pair DNA size markers (not shown) is indicated by arrows at right.
Alsmadi et al. BMC Genomics 2003 4:21 doi:10.1186/1471-2164-4-21