Nucleic Acids Research, 1987, Vol. 15, No. 17 6883-6897
© 1987
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A homogeneous nucleic acid hybridization assay based on stand displacement
Allied-Signal Inc. PO Box 1021R, Morristown, NJ 07960, USA
Received May 11, 1987. Revised August 12, 1987. Accepted August 12, 1987.
A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is describe. The assay is based on the concept of Strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe stand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of poly; nucleotide phosphorylase (EC 2.7.7.8 [EC] ) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40 [EC] ). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement.
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