A Colorimetric Assay for Phosphate to Measure Amplicon Accumulation in Polymerase Chain Reaction

Abstract We describe a rapid colorimetric method for the detection of PCR products. Color generation is complete within 5 min of mixing reagents with a PCR. The method is simple and does not require affinity capture steps or special labeling to be carried out. The color development can be monitored by eye or a simple spectrophotometer can be used to read sample absorbance. We demonstrate the method by its use in the amplification refractory mutation system (ARMS) analysis [C. R. Newton, A. Graham, L. E. Heptinstall, S. J. Powell, C. Summers, N. Kalsheker, et al. (1989) Nucleic Acids Res. 17, 2503–2515] of cystic fibrosis [J. R. Riordan, J. M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, et al. (1989) Science 245, 1066–1073] and factor V [R. M. Bertina, B. P. C. Koeleman, T. Koster, F. R. Rosendaal, R. J. Dirven, H. de Ronde, P. A. van der Velden, and P. H. Reitsma (1994) Nature 369, 64–67] allelic variants.