Internal references measurements
First Claim
1. A method of detecting a binding event between a probe and a target sample comprising steps of:
- contacting unstructured nucleic acid (UNA) targets derived from at least a first and second sample with a first and second UNA probe;
wherein the UNA targets corresponding to the same gene of interest derived from the first and second samples comprise different UNA nucleotide compositions;
wherein the nucleotide composition of the first UNA probe is selected to specifically hybridize to the UNA target derived from the first sample and not to the UNA target derived from the second sample, both UNA targets corresponding to the same gene of interest; and
the nucleotide composition of the second UNA probe is selected to specifically hybridize to the UNA target derived from the second sample and not to the UNA target derived from the first sample, both UNA targets corresponding to the same gene of interest;
detecting the extent of hybridization between the UNA targets derived from the first sample and the first UNA probe, and between the UNA targets derived from the second sample and the second UNA probe.
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Abstract
The present invention provides an improved method of detecting differential expression of a gene of interest using modified nucleotides that reduce the levels of secondary structure in a nucleic acid molecule. In certain embodiments of the invention, multiple genes of interest are provided on the surface of a solid support, such as in the form of a microarray. The presence of carefully chosen unstructured nucleic acid bases (UNAs) in the samples being assayed and in the probes on the surface of the solid support provides an internal referenced measurement that is suitable for detecting the differential expression of a gene of interest in the samples. Also provided are arrays of pairs UNA probes that are capable of detecting differential expression of a particular gene of interest in two samples of nucleic acid.
11 Citations
62 Claims
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1. A method of detecting a binding event between a probe and a target sample comprising steps of:
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contacting unstructured nucleic acid (UNA) targets derived from at least a first and second sample with a first and second UNA probe;
wherein the UNA targets corresponding to the same gene of interest derived from the first and second samples comprise different UNA nucleotide compositions;
wherein the nucleotide composition of the first UNA probe is selected to specifically hybridize to the UNA target derived from the first sample and not to the UNA target derived from the second sample, both UNA targets corresponding to the same gene of interest; and
the nucleotide composition of the second UNA probe is selected to specifically hybridize to the UNA target derived from the second sample and not to the UNA target derived from the first sample, both UNA targets corresponding to the same gene of interest;
detecting the extent of hybridization between the UNA targets derived from the first sample and the first UNA probe, and between the UNA targets derived from the second sample and the second UNA probe. - 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)
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31. A method of producing an array of UNA probes stably associated with a surface of a solid support, wherein the UNA probes are designed in pairs comprising at least a first and a second probe, wherein the first and second probes have different UNA nucleotides and are capable of hybridizing to UNA targets from different samples, the method comprising the steps of:
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selecting at least one gene that is likely to be present in different samples; and
generating at least a pair of UNA probes for the gene, wherein the first probe of the pair is capable of hybridizing to the target derived from the first sample and not the target UNA derived from the second sample, and the second probe of the pair is capable of hybridizing to the target UNA derived from the second sample and not the target UNA derived from first sample; and
creating an array containing at least the pair of UNA probes. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39)
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40. An array of a plurality of UNA probes, wherein the UNA probes are designed in pairs, wherein each member of a pair has a different UNA nucleotide content and is capable of detecting a UNA target corresponding to the same gene derived from different samples, wherein the plurality of UNA probes are:
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stably associated with a surface of a solid support; and
synthesized such that the first member of the pair of UNA probes can be differentiated from the second member of the pair by a difference in UNA nucleotide content, wherein the first member of the pair of UNA probes is capable of hybridizing to a UNA target derived from the first sample and cannot hybridize to a UNA target derived from second sample, and the second member of the pair of UNA probes is capable of hybridizing to a UNA target derived from the second sample and cannot hybridize to the UNA target derived from the first sample. - View Dependent Claims (41, 42, 43, 44, 45, 46)
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47. A kit for carrying out differential gene expression analysis comprising:
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an array of a plurality of UNA probes, wherein the UNA probes are designed in pairs, wherein each member of a pair has a different UNA nucleotide content and is capable of detecting a UNA target corresponding to the same gene derived from different samples, wherein the plurality of UNA probes are;
stably associated with a surface of a solid support; and
synthesized such that the first member of the pair of UNA probes can be differentiated from the second member of the pair by a difference in UNA nucleotide content, wherein the first member of the pair of UNA probes is capable of hybridizing to a UNA target derived from the first sample and cannot hybridize to a UNA target derived from second sample, and the second member of the pair of UNA probes is capable of hybridizing to a UNA target derived from the second sample and cannot hybridize to the UNA target derived from the first sample. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55)
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56. A system for detecting a binding event on a surface comprising at least one probe attached to the surface;
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one or more UNA targets derived from two or more samples, wherein the targets corresponding to the same gene comprise different UNA nucleotide compositions, wherein the UNA targets derived from the two or more samples comprise different UNA nucleotide compositions and can compete for hybridization to the same surface probe; and
two or more labeling probes having a different UNA chemistry, wherein one UNA labeling probe is capable of hybridizing to a UNA target derived from one sample and cannot hybridize to a UNA target derived from the second sample, and the second UNA labeling probe is capable of hybridizing to a UNA target derived from the second sample and cannot hybridize to the UNA target derived from the first sample; and
wherein each labeling probe is attached to a detectable moiety. - View Dependent Claims (57, 58, 59, 60)
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61. A method for detecting a binding event on a microarray comprising the steps of:
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providing at least one array probe on an array;
contacting the array probe with targets derived from at least two samples, wherein the targets are UNAs that competitively hybridize to the same array probe;
contacting the target with labeling probes, wherein each labeling probe has a different UNA chemistry that directs sample -specific hybridization to the target, wherein each labeling probe is attached to a different detectable moity; and
detecting the labeling probes hybridized to the targets that are hybridized to the array probe. - View Dependent Claims (62)
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Specification