Method and kit for detecting polyriboadenosine segments and messenger RNA
AssigneesAllied Corporation, Genetics Institute, Inc.
InventorsCalvin P. H. Vary, Steven E. Diamond, Neil M. Wolfman, Astrid P. Koudelka
Life of this patent
Estimating the term…
Abstract
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.
Claims25
A method for the determination of polynucleotides having 3'-terminal polyriboadenosine segments in a sample which comprises the steps:
- (a) digesting the nucleic acid of a sample with a polynucleotide phosphorylase in the presence of inorganic phosphate to convert 3'-terminal polyriboadenosine segments to adenosine diphosphate (ADP);
- (b) phosphorylating the ADP produced in the digesting step (a) to produce ATP;
- and
- (c) detecting either the ATP produced in the phosphorylating step (b) or a by-product of the phosphorylating step.
A kit for the determination of polynucleotides having 3'-terminal polyriboadenosine segments which comprises:
- (a) polynucleotide phosphorylase,
- (b) a kinase capable of converting ADP to ATP and its organophosphate co-reactant, and
- (c) reactants for the detection of ATP.
A method for the determination of polynucleotides having single-stranded ribonucleotide segments which comprises the steps:
- (a) digesting the nucleic acid of a sample with a phosphodiesterase which is a ribonuclease to convert single-stranded ribonucleotide segments to ribonucleotide monophosphates including AMP;
- (b) phosphorylating the AMP produced in the digesting step (a) to ADP;
- (c) further phosphorylating the ADP produced in the phosphorylating step (c) to ATP;
- and
- (d) detecting either the ATP produced in the further phosphorylating step (c) or a by-product of the further phosphorylating step (c).
A kit for the determination of polynucleotides having single-stranded ribonucleotide segments comprising reagents and enzymes effective to:
- (a) digest the single-stranded ribonucleotide segments to ribonucleoside monophosphates including AMP;
- (b) phosphorylate AMP to ADP;
- (c) further phosphorylate ADP to ATP;
- and
- (d) detect either ATP or a byproduct of the further phosphorylation reaction.
Dependencies are inferred from the claim text.