Optimization of gene expression analysis using immobilized capture probes

• US 8,795,960 B2
• Filed: 06/08/2009
• Issued: 08/05/2014
• 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, Banerjee, Sukanta, Yang, Jiacheng, Vener, Tatiana
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US12/480,215
• Publication Number:
  US 20090263820A1
• Time in Patent Office: 1,884 Days
• Field of Search: 435/6.1, 435/6.12, 435/91.1, 435/91.2, 435/287.2, 536/24.3, 536/24.33
• US Class Current: 435/6.1
• 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. 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;

or

ii. 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=al^x ;

wherein a is a constant and x<

0; and

e) Quantifying the target using one or more probe reagents.

2. The method of claim 1 wherein the target 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.

3. The method of claim of 2, wherein said primer is placed towards the 5′

terminus of the mRNA.

4. The method of claim 2, wherein said primer is placed adjacent to the 5 ′

terminus of the mRNA.

5. The method of claim 1, wherein quantification occurs directly or following an in-vitro nucleic acid amplification reaction.

6. The method of claim 1, wherein said short target length is less than about 150 nucleotides.

7. The method of claim 1, wherein said long target length is more than about 150 nucleotides.

8. The method of claim 1, wherein said probe reagents comprise microparticles.

9. The method of claim 8, wherein said microparticles are encoded.