DOL assay with FRET detection. All of the reagents, including DNA polymerase, DNA ligase, PCR primers, and ligation probes, are added to the test DNA sample when the reaction is first set up. In a two-stage thermal cycling sequence, PCR is allowed to proceed without the ligation probe annealing to the target followed by the ligation reaction when enough PCR product has accumulated. At the end of the assay, there are four possible outcomes for each test sample: (A) homozygous for the first allele leading to the formation of a ligation product containing the G allele in this example in the absence of the ligation product corresponding to the second allele (A allele in this example); (B) heterozygous with ligation product of both alleles formed; (C) homozygous for the second allele with the ligation product containing the A allele formed in this example in the absence of theG allele ligation product; (D) PCR or ligation failure leading to no ligation product being formed. (Blue) FAM; (green) TAMRA; (red) ROX.
