A colorimetric assay for phosphate to measure amplicon accumulation in polymerase chain reaction.
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.
Gibson
N J
NJ
Zeneca Diagnostics, Gadbrook Park, Cheshire, Northwich, CW9 7RA, United Kingdom. neil.gibson@ukbla71.zeneca.com
Newton
C R
CR
Little
S
S
D000483:Alleles; D002138:Calibration; D003124:Colorimetry / Q000379:methods*; D004396:Coloring Agents; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004252:DNA Mutational Analysis; D011756:Diphosphates / Q000378:metabolism; D005165:Factor V / Q000235:genetics*; D005784:Gene Amplification; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D043564:Inorganic Pyrophosphatase; D008982:Molybdenum; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010710:Phosphates / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011755:Pyrophosphatases / Q000378:metabolism; D012394:Rosaniline Dyes; D016963:Thermus thermophilus / Q000201:enzymology
D004396:Coloring Agents; D011756:Diphosphates; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D012394:Rosaniline Dyes; C005095:malachite green; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C044659:molybdate; D008982:Molybdenum; D005165:Factor V; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase