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Clinical Chemistry 32: 1696-1701, 1986;
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Clinical Chemistry, Vol 32, 1696-1701, Copyright © 1986 by American Association for Clinical Chemistry

Nonisotopic detection methods for strand displacement assays of nucleic acids

CP Vary, FJ McMahon, FP Barbone and SE Diamond

Using the enzymes terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) and polynucleotide phosphorylase (EC 2.7.7.8), we constructed polyriboadenylic acid tracts, approximately 8000 AMP residues long, attached to the 3'-terminus of a synthetic deoxynucleotide. The polyadenylated DNA, termed the "signal strand", was used in a displacement-type nucleic acid probe assay (see pp 1631-6, this issue). A probe-signal strand complex was made by hybridizing the signal strand to a deoxycytidylate-terminal probe DNA. The probe-signal strand complex was immobilized on an oligo (dG)-cellulose support and subsequently displaced from the immobilized hybrid complex with various amounts of analyte DNA. After the displacement procedure, the polyadenylate tracts were converted to ATP by the combined action of polynucleotide phosphorylase and pyruvate kinase. ATP was quantified by a bioluminescence assay with luciferase from Photinus pyralis. Displacement events were also quantified with biotinylated signal strand bound to avidin-conjugated horseradish peroxidase. Such enzyme- amplified assays offer considerable versatility: they may be coupled to a variety of detection systems including colorimetry, fluorimetry, and luminometry.


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 Nucleic Acids ResHome page
Q. Li, G. Luan, Q. Guo, and J. Liang
A new class of homogeneous nucleic acid probes based on specific displacement hybridization
Nucleic Acids Res., January 15, 2002; 30(2): e5 - e5.
[Abstract] [Full Text] [PDF]

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 ScienceHome page
U Landegren, R Kaiser, C. Caskey, and L Hood
DNA diagnostics--molecular techniques and automation
Science, October 14, 1988; 242(4876): 229 - 237.
[Abstract] [PDF]


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Copyright © 1986 by the American Association for Clinical Chemistry.