Allied Corporation

Genetics Institute, Inc.

Calvin P Vary

Steven E Diamond

Neil M Wolfman

Astrid P Koudelka

A probe polynucleotide binds to a target nucleotide sequence in the nucleic acid of a biological sample, and then is enzymatically extended in the 3'-direction with a mixture of nucleoside triphosphates including at least one nucleoside triphosphate that has been detectably labeled. After separating extended hybrid from unreacted nucleoside triphosphates, detectably-modified nucleotides which have been incorporated are determined. In some forms, the 3'-terminal nucleotide of the probe polynucleotide is selected to form a matched pair with some sample strands, but a mismatched pair with other sample strands. In such cases, if the primer dependent enzyme used for extension is one lacking 3'-exonuclease activity, then only those hybrids forming such a matched pair will be extended and subsequently determined.

Method and kit for polynucleotide assay including primer-dependant DNA

An RNA signal strand is displaced from a probe strand by a target nucleotide sequence. Without separation, a digestion enzyme selective for displaced RNA, e.g., a polynucleotide phosphorylase, digests the displaced RNA to nucleoside phosphates including ADP. The digestion products are detected, e.g. by phosphorylating the ADP to ATP and detecting the ATP by bioluminescence.

Homogeneous polynucleotide displacement assay method kit and reagent

A reagent complex is disclosed containing (1) a probe polynucleotide having a target binding region complementary to a target nucleotide sequence and (2) a signal strand polynucleotide bound by base pairing to a portion of the target binding region. The signal strand is displaced from the reagent complex by the target nucleotide sequence and separated from intact reagent complexes. A digestable ribonucleotide segment of the displaced signal strand, such as a 3' , terminal segment, is digested to ribonucleotide phosphates, which are phosphorylated to ATP. The ribonucleotide phosphates or ATP are detected.

Diagnostic reagent, kit and method employing polynucleotide

RNA such as messenger RNA is digested to nucleotide phosphates including AMP or ADP. The ATP or a byproduct of the phosphorylation, e.g., pyruvate, is detected. Exemplary enzymes used (with appropriate co-reactants and co-factors) are: (1) polynucleotide phosphorylase, pyruvate kinase and luciferase, or (2) phosphodiesterase (or RNase), myokinase, pyruvate kinase and luciferase. The phosphorylation to ATP (e.g., with pyruvate kinase) is preferably coupled with the previous (reversible) enzymatic step.

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Califon, NJ, United States of America

Calvin P Vary