A system for the quantitation of DNA using a microtiter plate-based hybridization and enzyme amplification technology.
A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P1. The pH-dependent specificity of this enzyme for 3'-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection.
Fisher
M
M
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, United Kingdom. M.Fisher@Biochem.ucl.ac.uk
Harbron
S
S
Taylorson
C J
CJ
D003124:Colorimetry; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D005949:Glucose Oxidase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008832:Microchemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity
D004247:DNA; D005949:Glucose Oxidase; D012259:Ribonuclease, Pancreatic; D015719:Single-Strand Specific DNA and RNA Endonucleases