Digital profiling of polynucleotide populations
First Claim
1. A method of determining relative amounts of target polynucleotides in a population, the method comprising the steps of:
- providing for each target polynucleotide a plurality of probes, each probe of the same plurality being specific for the same target polynucleotide and each probe of the same or different plurality having a different oligonucleotide tag, the oligonucleotide tags of all the pluralities belonging to the same minimally cross-hybridizing set;
combining in a reaction mixture the pluralities of probes with the population so that substantially every target polynucleotide specifically hybridizes to one or more probes of its corresponding plurality and so that probes specifically hybridized to a target polynucleotide are enzymatically modified to form selectable probes;
removing a sample of selectable probes from the reaction mixture;
amplifying and labeling the oligonucleotide tags of the sample;
specifically hybridizing the labeled oligonucleotide tags to their respective tag complements on one or more solid phase supports having addressable hybridization sites; and
determining for each plurality of probes a proportion of hybridization sites on the one or more solid phase supports that contain labeled oligonucleotide probes to give a relative amount of the corresponding target polynucleotide in the population.
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Abstract
The invention provides methods and compositions for hybridization-based assays that employ oligonucleotide tags, wherein probes specific for the same target polynucleotide are labeled with a plurality of different oligonucleotide tags. When probes are used in conjunction with a microarray, or like, readout platform, containing hybridization sites of tag complements, assay of a target polynucleotide results in a signal being generated from any of a plurality hybridization sites with predetermined addresses and the number of such sites generating a signal is proportional to the relative amount of the target polynucleotide in a population, test sample, or reaction volume, as the case may be. The invention provides methods and compositions for measuring amounts of selected target polynucleotides in a sample and for providing a digital readout of such amounts. Statistical confidence in measurements made by the present invention may be increased as much as desired by increasing the size of the sample of successfully hybridized and selected probes from which signals are generated.
302 Citations
17 Claims
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1. A method of determining relative amounts of target polynucleotides in a population, the method comprising the steps of:
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providing for each target polynucleotide a plurality of probes, each probe of the same plurality being specific for the same target polynucleotide and each probe of the same or different plurality having a different oligonucleotide tag, the oligonucleotide tags of all the pluralities belonging to the same minimally cross-hybridizing set;
combining in a reaction mixture the pluralities of probes with the population so that substantially every target polynucleotide specifically hybridizes to one or more probes of its corresponding plurality and so that probes specifically hybridized to a target polynucleotide are enzymatically modified to form selectable probes;
removing a sample of selectable probes from the reaction mixture;
amplifying and labeling the oligonucleotide tags of the sample;
specifically hybridizing the labeled oligonucleotide tags to their respective tag complements on one or more solid phase supports having addressable hybridization sites; and
determining for each plurality of probes a proportion of hybridization sites on the one or more solid phase supports that contain labeled oligonucleotide probes to give a relative amount of the corresponding target polynucleotide in the population. - View Dependent Claims (2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17)
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- 8. A probe composition for detecting one or more target polynucleotides in a sample, the composition comprising a plurality of probes for each target polynucleotide, each probe of the same plurality being specific for the same target polynucleotide and each probe of the same or different plurality having a different oligonucleotide tag, wherein each probe specifically hybridizes to a region of a target polynucleotide and the oligonucleotide tags each belong to the same minimally cross-hybridizing set.
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11. A method of determining absolute concentrations of target polynucleotides in a test sample, the method comprising the steps of:
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providing one or more nucleic acid standards each having a concentration;
providing for each target polynucleotide and each nucleic acid standard a plurality of probes, each probe of the same plurality being specific for the same target polynucleotide or the same nucleic acid standard and each probe of the same or different plurality having a different oligonucleotide tag, the oligonucleotide tags of all the pluralities belonging to the same minimally cross-hybridizing set;
combining in a reaction mixture the pluralities of probes with the test sample so that substantially every target polynucleotide and nucleic acid standard specifically hybridizes to one or more probes of its corresponding plurality and so that probes specifically hybridized to a target polynucleotide or a nucleic acid standard are enzymatically modified to form selectable probes;
removing a sample of selectable probes from the reaction mixture;
amplifying and labeling the oligonucleotide tags of the sample;
specifically hybridizing the labeled oligonucleotide tags to their respective tag complements on one or more solid phase supports having addressable hybridization sites; and
determining an absolute concentration of each target polynucleotide in the test sample by comparing a number of hybridization sites generating signals from a specifically hybridized oligonucleotide tag of a target polynucleotide to a number of hybridization sites generating signals from one or more nucleic acid standards and their respective concentrations.
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Specification