Real-time gene quantification with internal standards
First Claim
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1. A method of determining mass fractions of first and second target nucleic acids present in a test sample comprising:
- obtaining an approximated standard melting signal, fi, for each of the first and second target nucleic acids, the approximated standard melting signal for the first target nucleic acid corresponding to a change in fluorescence of the first target nucleic acid over temperature and the approximated standard melting signal for the second target nucleic acid corresponding to a change in fluorescence of the second target nucleic acid over temperature,contacting the target nucleic acids in the test sample 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 acids, the indicator further configured to discriminate the target nucleic acids based on melting temperature,illuminating the test sample including the fluorescent nucleic acid indicator,cycling the illuminated test sample through an annealing temperature and a denaturing temperature that is higher than the annealing temperature,monitoring fluorescent change of the illuminated test sample while changing temperature of the illuminated test sample to obtain a fluorescence melting signal, fmix, of the test sample, the fluorescence melting signal, fmix, corresponding to a change in fluorescence of the test sample over temperature, andapproximating the fluorescence melting signal, fmix, for the test sample over temperature, T, as a combination of the approximated standard melting signals, fi, of the first and second target nucleic acids, and using fi and fmix to determine the mass fractions, mi, of the first and second target nucleic acids in the test sample according to the formula
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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.
27 Citations
16 Claims
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1. A method of determining mass fractions of first and second target nucleic acids present in a test sample comprising:
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obtaining an approximated standard melting signal, fi, for each of the first and second target nucleic acids, the approximated standard melting signal for the first target nucleic acid corresponding to a change in fluorescence of the first target nucleic acid over temperature and the approximated standard melting signal for the second target nucleic acid corresponding to a change in fluorescence of the second target nucleic acid over temperature, contacting the target nucleic acids in the test sample 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 acids, the indicator further configured to discriminate the target nucleic acids based on melting temperature, illuminating the test sample including the fluorescent nucleic acid indicator, cycling the illuminated test sample through an annealing temperature and a denaturing temperature that is higher than the annealing temperature, monitoring fluorescent change of the illuminated test sample while changing temperature of the illuminated test sample to obtain a fluorescence melting signal, fmix, of the test sample, the fluorescence melting signal, fmix, corresponding to a change in fluorescence of the test sample over temperature, and approximating the fluorescence melting signal, fmix, for the test sample over temperature, T, as a combination of the approximated standard melting signals, fi, of the first and second target nucleic acids, and using fi and fmix to determine the mass fractions, mi, of the first and second target nucleic acids in the test sample according to the formula - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
wherein σ
is a smoothing parameter, fσ
mix represents each of the number of fluorescence melting signals that do not have approximated standard melting signals and that are greater than σ
, mi is the mass fraction of each of the first and second target nucleic acids, fσ
i represents the approximated standard melting signals, and fr (T) represents the approximated fluorescence melting signals.
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12. The method of claim 1 wherein the mass fractions of the first and second target nucleic acids provides information concerning a deletion or duplication in a gene.
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13. The method of claim 1 wherein the fluorescent nucleic acid indicator comprises a fluorescently-labeled sequence specific oligonucleotide probe.
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14. The method of claim 13 wherein the sequence specific oligonucleotide probe is selected from the group consisting of a fluorescence resonance energy transfer pair probe system and a single-labeled oligonucleotide.
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15. The method of claim 1 wherein the second target nucleic acid is a competitor of the first target nucleic acid for the fluorescent nucleic acid indicator.
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16. The method of claim 1 wherein the test sample further comprises a thermostable polymerase and a pair of oligonucleotide primers configured to amplify the first target nucleic acid.
Specification