Optimization of Gene Expression Analysis using Immobilized Capture Probes

• US 20090263820A1
• Filed: 06/08/2009
• Published: 10/22/2009
• Est. Priority Date: 10/28/2003
• Status: Active Grant

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: Seul, Michael, Vener, Tatiana, Banerjee, Sukanta, Yang, Jiacheng
• Granted Patent: US 8,795,960 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

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.

Claims

1-62. -62. (canceled)

63-1. The method of claim 63 wherein the analyte polynucleotide is a cDNA produced from a corresponding mRNA by reverse transcription and target length is adjusted by adjusting the placement of a reverse transcription primer on the mRNA.

64. A method of expanding the dynamic range of detection of a quantitative assay for an analyte polynucleotide using one or more probe reagents for quantifying the amount of the analyte polynucleotide, wherein the polynucleotide is quantitated directly or following an in-vitro nucleic acid amplification reaction, and wherein the probe reagents consist of encoded solid phase carriers functionalized with oligonucleotides, comprising:

adjusting the amount of analyte polynucleotide captured by the probe reagents by using lower quantities of probe reagents when the analyte polynucleotide is anticipated to be present in low concentration so as to increase the amount of captured analyte polynucleotide associated with individual solid phase carriers and using greater quantities of probe reagents when the analyte polynucleotide is anticipated to be present in high concentration so as to decrease the amount of captured analyte polynucleotide associated with individual solid phase carriers, and wherein the amount of captured analyte polynucleotide captured on individual solid phase carriers is inversely proportional to the amount of probe reagents.