Colorimetric method for detecting amplified nucleic acids.
Methods for detecting and measuring the success of nucleic acid sequence amplifications can be developed by detecting the by-products of amplification procedures. One method includes the detection of inorganic phosphate (Pi) during or on completion of the PCR. The method requires modification of assay conditions to prevent thermal- and template-independent enzymatic activity from nonspecifically hydrolyzing dNTPs. Detection of Pi by the traditional Fiske-SubbaRow method provides a sensitivity similar to ethidium bromide staining of amplified products. The method offers a simple and rapid assay for amplified nucleic acids and can be useful in assays where confirmation of the amplified DNA product is not essential.
Durward
E
E
University of Aberdeen, Institute of Medical Sciences, Foresterhill, Scotland, UK.
Harris
W J
WJ
D000818:Animals; D004247:DNA / Q000032:analysis*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003843:Deoxycytidine Monophosphate / Q000494:pharmacology; D004996:Ethidium; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D008982:Molybdenum / Q000378:metabolism; D010710:Phosphates / Q000032:analysis* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011755:Pyrophosphatases / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013698:Templates, Genetic
D003838:Deoxyadenine Nucleotides; D007140:Immunoglobulin Fab Fragments; D010710:Phosphates; D003843:Deoxycytidine Monophosphate; C012873:2'-deoxy-5'-adenosine monophosphate; D008982:Molybdenum; D004247:DNA; D011755:Pyrophosphatases; D004996:Ethidium; C022175:ammonium molybdate