Optimization of gene expression analysis using immobilized capture probes

Abstract

Disclosed are methods of multiplexed analysis of oligonucleotides in a sample, including: methods of probe and target “engineering”, as well as methods of assay signal analysis relating to the modulation of the probe-target affinity constant, K by a variety of factors including the elastic properties of target strands and layers of immobilized (“grafted”) probes; and assay methodologies relating to: the tuning of assay signal intensities including dynamic range compression and on-chip signal amplification; the combination of hybridization-mediated and elongation-mediated detection for the quantitative determination of abundance of messages displaying a high degree of sequence similarity, including, for example, the simultaneous determination of the relative expression levels, and identification of the specific class of, untranslated AU-rich subsequences located near the 3′ terminus of mRNA; and a new method of subtractive differential gene expression analysis which requires only a single color label.

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