Methods and apparatus for biomolecule detection, identification, quantification and/or sequencing
First Claim
1. A method comprising:
- a) obtaining at least one sample suspected of containing one or more target biomolecules;
b) using the target biomolecule to generate pyrophosphate (PPi) by an enzyme-catalyzed reaction;
c) using the PPi to produce light by a luciferase dependent process;
d) accumulating the number of photons produced over a time interval; and
e) detecting the target biomolecules in the sample.
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Accused Products
Abstract
The present invention concerns methods, compositions and apparatus for detecting. Identifying, quantifying and/or sequencing target biomolecules, such as nucleic acids or proteins. Where the target biomolecule is not a nucleic acid, the target or a ligand that binds to the target may be tagged with an oligonucleotide or nucleic acid. The presence of target molecules in samples may be detected by a variety of enzymatic processes that generate a detectable product, such as pyrophosphate (PPi) or ATP. In preferred embodiments of the invention, the product is detected by a bioluminescence regenerative cycle (BRC), utilizing luciferase mediated bioluminescence. In other preferred embodiments, thermostable enzymes may be used in either isothermal or cyclic thermal reactions, such as terminal transferase activity or nucleic acid polymerization, to generate PPi. Apparatus and compositions for biomolecule analysis are also disclosed. Methods for analysis of generated data are also disclosed herein.
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Citations
63 Claims
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1. A method comprising:
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a) obtaining at least one sample suspected of containing one or more target biomolecules;
b) using the target biomolecule to generate pyrophosphate (PPi) by an enzyme-catalyzed reaction;
c) using the PPi to produce light by a luciferase dependent process;
d) accumulating the number of photons produced over a time interval; and
e) detecting the target biomolecules in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 62, 63)
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31. A method of nucleic acid sequencing comprising:
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a) replicating multiple copies of a nucleic acid template molecule;
b) obtaining a complex signal from the nucleic acid replication;
c) deconvoluting the signal by statistical signal processing; and
d) determining the sequence of the template nucleic acid. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39)
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40. A method comprising:
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a) obtaining at least one sample suspected of containing one or more target proteins and/or peptides;
b) obtaining an assay mixture with at least one enzyme or substrate inactivated by peptide linkage;
c) exposing the sample to the assay mixture;
d) activating the inactivated enzyme or substrate;
e) generating pyrophosphate (PPi); and
f) producing light by a luciferase dependent process. - View Dependent Claims (41, 42, 43, 44, 45)
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46. A method comprising:
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a) obtaining at least one sample suspected of containing one or more target nucleic acids;
b) generating pyrophosphate (PPi) by terminal transferase activity;
c) measuring the pyrophosphate generated; and
e) detecting the target nucleic acid. - View Dependent Claims (47, 48, 49, 50)
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51. A method for biomolecule detection comprising:
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a) generating pyrophosphate in a biomolecule dependent process;
b) using thermostable ATP sulfurylase and luciferase to produce light from the pyrophosphate; and
c) measuring the light output to detect the biomolecule. - View Dependent Claims (52, 53, 54, 55)
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56. A method comprising:
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a) obtaining at least one sample suspected of containing one or more target nucleic acids;
b) adding labeled nucleotides to the one or more target nucleic acids with a thermostable terminal transferase; and
c) detecting the labeled nucleic acids. - View Dependent Claims (57, 58)
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59. A method of biomolecule detection comprising:
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a) attaching a target molecule to a substrate;
b) binding a first binding moiety to the target molecule;
c) binding a second binding moiety to the first binding moiety, wherein the second binding moiety is attached to a dextran or dendromer molecule labeled with oligonucleotides;
d) generating pyrophosphate by terminal transferase mediated addition of nucleotides to the oligonucleotides; and
e) detecting the pyrophosphate. - View Dependent Claims (60)
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61. A system comprising:
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a) one or more reaction chambers;
b) a microfluidic system;
c) one or more photodectectors d) a thermostable luciferase; and
e) a thermostable ATP sulfurylase.
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Specification