Figure 5

SNP identification on human genomic DNA. Alu I digested human genomic DNA (100 ng) was used as template for annealing and ligation of the OCP specific for the SNP G1822A. The ligation products were then amplified by ERCA. Three DNA samples representing the three genotypes were each analyzed in two reactions, each reaction containing an OCP/ P1 Amplifluor/ P2 combination corresponding to one of the two possible alleles. (a) Real-time profiles of the ERCA reactions indicate that an amplification signal is obtained only from reactions where the OCP/ P1 Amplifluor/ P2 combination matched the SNP nucleotide on the genomic DNA samples. When the combination OCPinC/ P1inFAM/ P2inC was used with DNA sample homozygous for the G allele, a fluorescent FAM signal was observed (top left panel). No signal was observed with the mismatched OCPocT and the corresponding primer pair P1ocTET/ P2ocT (top right panel). Similarly, when the combination OCPocT/ P1ocTET/ P2ocT was used with DNA sample homozygous for the A allele, only a fluorescent TET signal was observed (middle right panel). No signal was observed with the mismatched OCPinC and the corresponding primer pair P1inFAM/ P2inC (middle left panel). In the case of heterozygous DNA both the OCP/ primer combinations gave a fluorescent signal (bottom panels) (b) The endpoint products of these reactions were resolved on a 2% agarose gel. The characteristic double-stranded ERCA ladder was observed only from those reactions that gave a fluorescent signal in the real-time assay. (c) Scatter plot of the ERCA reaction endpoint fluorescence values obtained by screening a set of 96 different genomic DNA samples using the above assay.