Increasing hybridization efficiencies

• US 7,919,239 B2
• Filed: 07/01/2005
• Issued: 04/05/2011
• Est. Priority Date: 07/01/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wang, Hui
• Primary Examiner(s): Kapushoc; Stephen
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US11/173,078
• Publication Number:
  US 20070003937A1
• Time in Patent Office: 2,104 Days
• Field of Search: 435/6, 435/91.3, 435/91.51, 435/87.2, 536/23.1, 536/24.3
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

Aspects of the disclosure are generally directed to probes and probe compositions for detecting or quantifying a target. One aspect provides a method for selectively hybridizing a probe to a polynucleotide by contacting a sample containing a first and second polynucleotide with a probe. The probe includes a number of nucleotides complementary to the first or second polynucleotide in the region of mismatch between the first and second polynucleotides. Another aspect provides arrays including the disclosed probes and methods of using the arrays and the probes.

Claims

1. A method comprising:

a) contacting a sample comprising a plurality of labeled miRNA molecules with an array of probes, each of which probes is;

i. bound to a substrate

ii. complementary to said labeled miRNA molecules starting at the 3′

terminal nucleotide of said labeled miRNA molecules, to produce a plurality of duplexes between said probes and said labeled miRNA molecules,

wherein said plurality of duplexes comprises;

first duplexes in which the 5′

end of said labeled miRNA molecules is hybridized with said probe and

second duplexes in which the 5′

end of said labeled miRNA molecules is not hybridized with said probe, and

wherein the melting temperatures of said duplexes are within 15°

C. of each other; and

b) detecting binding of said miRNA molecules with each of said probes.

2. The method of claim 1, wherein binding of said labeled miRNA to each of said probes forms a duplex that is longer by 1, 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides than the complementary region formed between said labeled miRNA and its respective target.

3. The method of claim 1, wherein said melting temperatures of said duplexes are within 5°

C. of each other.

4. The method of claim 1, wherein each probe of said array comprises 10 to 32 nucleotides that are complementary to said labeled miRNA molecules.

5. The method of claim 1, wherein said each probe of said array comprises at least 10 consecutive nucleotides that are complementary to said miRNA.

6. The method of claim 1, wherein the number of said probes on said array is proportional to the expected relative amount of said labeled miRNA molecules in said sample.

7. The method of claim 1, wherein each probe of said array is complementary to a different labeled miRNA molecules in said sample.

8. The method of claim 1, wherein each of said probes comprises a nucleotide clamp coupled to consecutive nucleotides complementary to said miRNA.