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

✍ Scribed by Neil J. Gibson; Clive R. Newton; Stephen Little

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 158 KB
• Volume: 254
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1997.2324

✦ Synopsis

using 30-mer amplimers and accumulates to 50 nM, We describe a rapid colorimetric method for the dethen the orthophosphate concentration will be 14 mM.

tection of PCR products. Color generation is complete

Sensitive assays for phosphate require it to complex within 5 min of mixing reagents with a PCR. The with molybdate ions to form phosphomolybdate. The method is simple and does not require affinity capture addition of a dye such as crystal violet or malachite steps or special labeling to be carried out. The color green gives rise to a brightly colored complex which development can be monitored by eye or a simple speccan then be detected and quantified (4, 5). A malachite trophotometer can be used to read sample absorbance. green assay has been used to detect the generation of We demonstrate the method by its use in the amplifiphosphate in biological systems such as protein phoscation refractory mutation system (ARMS) analysis phatase assays (6), immunoassays ( 7), and assay of pro-[C. R. Newton, A. Graham, L. E. Heptinstall, S. J. Powell, tein-bound phosphate (8). The detection of pyrophos-C. Summers, N. Kalsheker, et al. (1989) Nucleic Acids phate released by DNA polymerase has also been Res. 17, 2503-2515] of cystic fibrosis [J. R. Riordan, applied in a minisequencing format for mutation detec-