Real-time gene quantification with internal standards
First Claim
1. A method of determining mass fractions of first and second target nucleic acids present in a test sample, said method comprising the steps of (a) contacting the target nucleic acids with a fluorescent nucleic acid indicator, the indicator being configured to provide a signal related to the quantity of indicator hybridized to the target nucleic acid, the indicator further configured to discriminate the target nucleic acids based on melting temperature, (b) illuminating the test sample, (c) monitoring fluorescent change to generate a melting curve, and (d) using a thermodynamically based signal processing algorithm to determine the mass fraction of the target nucleic acids.
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Abstract
The present invention is directed to a nucleic acid quantification kit and method for determining the initial concentration or mass fraction of a target nucleic acid present in a sample. Illustrative embodiments include real-time competitive quantitative polymerase chain reaction (PCR) to determine the copy number or mass fraction of a target nucleic acid sequence in a sample and use of a thermodynamically based signal processing algorithm, with or without PCR, to provide mass fraction information.
57 Citations
19 Claims
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1. A method of determining mass fractions of first and second target nucleic acids present in a test sample, said method comprising the steps of
(a) contacting the target nucleic acids with a fluorescent nucleic acid indicator, the indicator being configured to provide a signal related to the quantity of indicator hybridized to the target nucleic acid, the indicator further configured to discriminate the target nucleic acids based on melting temperature, (b) illuminating the test sample, (c) monitoring fluorescent change to generate a melting curve, and (d) using a thermodynamically based signal processing algorithm to determine the mass fraction of the target nucleic acids.
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9. A method of quantifying a target nucleic acid present in a biological sample, said method comprising the steps of
(a) combining in a single reaction vessel at least a portion of said sample, a thermostable polymerase, a known concentration of a competitor nucleic acid, a pair of oligonucleotide PCR primers, and an oligonucleotide probe; -
wherein said pair of oligonucleotide PCR primers is configured for amplifying a selected segment of the target nucleic acid and the competitor nucleic acid;
wherein said competitor nucleic acid has a unique section having a different sequence from a corresponding region of the target nucleic acid; and
wherein the competitor nucleic acid and the target nucleic acid are amplified with essentially equal efficiency;
said oligonucleotide probe labeled with a first fluorophore and configured to hybridize to the unique section of the competitor nucleic acid and the corresponding region of the target nucleic acid;
wherein hybridization of the oligonucleotide probe to at least one of its respective complementary target nucleic acid and competitor nucleic acids results in a change in the magnitude of fluorescence from the fluorophore;
(b) amplifying the selected segment of the target and competitor nucleic acids; and
(c) illuminating the biological sample and monitoring fluorescent change from the first fluorophore. - View Dependent Claims (10, 11, 12, 13, 14)
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15. A kit for determining the initial copy number of a preselected target nucleic acid, said kit comprising
a competitor nucleic acid sequence, a pair of oligonucleotide PCR primers, an anchor probe, a target probe, and a competitor probe; -
wherein said pair of oligonucleotide PCR primers is configured for amplifying a selected segment of the target nucleic acid and competitor nucleic acid; and
wherein said competitor nucleic acid has a first section having an identical sequence as a corresponding first region of the target nucleic acid, and a unique section having a different sequence from a corresponding second region of the target nucleic acid; and
wherein the competitor nucleic acid and the target nucleic acid are amplified with essentially equal efficiency;
said anchor probe labeled with a first fluorophore and configured to hybridize to said first section of the competitor nucleic acid and to said first region of the target nucleic acid, adjacent to the unique section of the competitor nucleic acid and adjacent to the second region of the target nucleic acid;
said competitor probe labeled with a second fluorophore and configured to hybridize to said unique section of the competitor nucleic acid;
said target probe labeled with a third fluorophore and configured to hybridize to said second region of the target nucleic acid;
wherein hybridization of the anchor, target, and competitor probes to their respective complementary target nucleic acids and competitor nucleic acids places the first fluorophore and the second fluorophore as well as the first fluorophore and the third fluorophore in a resonance energy transfer relationship. - View Dependent Claims (16, 17)
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18. A kit for determining a mass fraction of a target nucleic acid present in a biological sample, said kit comprising
a competitor nucleic acid, and a fluorescently labeled sequence specific oligonucleotide probe, the sequence specific probe configured to be homologous with one of said target and competitor nucleic acids and having a first melting temperature, the sequence specific probe also being configured to have general homology with the other of said target and competitor nucleic acids but having at least one mismatch thereto to provide a second melting temperature different from the first melting temperature.
Specification