Quantitative microarray hybridizaton assays
First Claim
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1. A hybridization assay comprising the steps of:
- contacting an array of probe molecules stably associated with the surface of a solid support with an end labeled target nucleic acid sample under hybridization conditions sufficient to produce a hybridization pattern, wherein each of said end labeled target nucleic acids is capable of generating a signal of substantially the same specific activity; and
detecting said hybridization pattern.
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Abstract
Methods are provided for quantitative gene expression analysis. In the subject methods, end-labeled target nucleic acid is contacted with an array of probe molecules stably associated with the surface of a solid support under hybridization conditions sufficient to produce a hybridization pattern. The resultant hybridization pattern can be used to obtain a quantitative information about the genetic profile of the end-labeled target nucleic acid sample, as well as the physiological source from which it is derived. As such, the subject methods find use in a variety of applications.
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Citations
17 Claims
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1. A hybridization assay comprising the steps of:
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contacting an array of probe molecules stably associated with the surface of a solid support with an end labeled target nucleic acid sample under hybridization conditions sufficient to produce a hybridization pattern, wherein each of said end labeled target nucleic acids is capable of generating a signal of substantially the same specific activity; and detecting said hybridization pattern. - View Dependent Claims (2, 3, 4, 5, 6)
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7. An assay to determine the genetic profile of a physiological source, said assay comprising the steps of:
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(a) preparing an end labeled target nucleic acid sample by contacting mRNA from a physiological source with an end-labeled oligo(dT) primer, a reverse transcriptase and nucleotides under conditions sufficient for reverse transcription of said mRNA into said end-labeled target nucleic acid sample, wherein said end-labeled oligo (dT) primer is comprises a known number of labeled nucleotides; (b) preparing end labeled standard DNA by contacting standard RNA with said end-labeled oligo(dT) primer, a reverse transcriptase and nucleotides under conditions sufficient for reverse transcription of said standard RNA into said end labeled standard DNA; (c) combining said end labeled standard DNA with said end-labeled target nucleic acid sample; (d) contacting said end labeled target nucleic acid sample with an array of probe molecules stably associated with the surface of a solid support under hybridization conditions to produce a hybridization pattern; (e) separating unhybridized target from said array; (f) contacting said array with a nuclease; and (g) detecting said hybridization pattern. - View Dependent Claims (8, 9, 10, 11)
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12. A kit for use in the preparation of end-labeled target nucleic acids to be used in an array based assay, said kit comprising:
end labeled oligo(dT) primers capable of generating a signal of known value; and
a polymerase.- View Dependent Claims (13, 14, 15, 16, 17)
Specification