Optimization of gene expression analysis using immobilized capture probes
View Patent ↗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.
1. A method of obtaining similar signal intensities from hybridization of different sets of the oligonucleotide probes to a set of cognate targets, in a hybridization assay that comprises sets of probes of different lengths on different solid phase carriers and sets of targets of different lengths, wherein annealing of a set of probes with cognate targets generates a signal intensity, wherein a cumulative signal intensity from said annealing is determined, the method comprising;
(i) selecting probe lengths and densities for said sets of probes, wherein probes include different subsequences such that at least one subsequence is complementary to a subsequence in a cognate target; wherein probes for longer cognate targets are longer in length than probes for shorter cognate targets and wherein the density for longer probes is lower than the density for shorter probes,
(ii) producing said probes and affixing said probes to different solid phase carriers at a selected density,
(iii) annealing said target to said probes, wherein signal intensities of probes and targets of different lengths are about the same.
2. The method of claim 1 further including attaching a bifunctional polymeric moiety to the solid phase carriers and then attaching said capture probes to said bifunctional polymeric moiety.
3. The method of claim 2 wherein the surface area of the bifunctional polymeric moiety, when attached to the solid phase carriers, is known.
4. The method of claim 2 wherein said bifunctional polymeric moiety is a polyethylene glycol whose approximate molecular weight is known.
5. The method of claim 2 wherein said bifunctional polymeric moiety is a protein.
6. The method of claim 1 wherein the capture probe density threshold is determined based on the condition that adjacent probe-target complexes attached to the surface do not overlap each other.
7. The method of claim 1 wherein the capture probe and the targets are both either RNA or DNA.
8. The method of claim 5 wherein the protein is neutravidin.