Optimization of gene expression analysis using immobilized capture probes
First Claim
1. A method comprising:
- a) Providing a sample;
b) Providing probe reagents complementary to a target polynucleotide in the sample;
c) Estimating the concentration of the target in the sample;
d) Adjusting the affinity constant (K) governing the interaction of the target with the probe reagents by;
i. using short target length when the target is anticipated to be present in low concentration;
orii. using long target length when the target is anticipated to be present in high concentration;
wherein the relationship between target length (I) and K is;
K=alx ;
wherein a is a constant and x<
0; and
e) Quantifying the target using one or more probe reagents.
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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.
554 Citations
9 Claims
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1. A method comprising:
-
a) Providing a sample; b) Providing probe reagents complementary to a target polynucleotide in the sample; c) Estimating the concentration of the target in the sample; d) Adjusting the affinity constant (K) governing the interaction of the target with the probe reagents by; i. using short target length when the target is anticipated to be present in low concentration;
orii. using long target length when the target is anticipated to be present in high concentration;
wherein the relationship between target length (I) and K is;
K=alx ;
wherein a is a constant and x<
0; ande) Quantifying the target using one or more probe reagents. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification