Assays for short sequence variants

• US 9,260,761 B2
• Filed: 11/09/2009
• Issued: 02/16/2016
• Est. Priority Date: 10/22/1999
• Status: Expired due to Term

• Current Assignee: PHRI Properties, Inc.
• Original Assignee: PHRI Properties, Inc.
• Inventors: Tyagi, Sanjay, Kramer, Fred, Alland, David
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US12/614,917
• Publication Number:
  US 20100120048A1
• Time in Patent Office: 2,290 Days
• Field of Search: 435/6, 435/91.2, 435/6.12, 536/25.32, 536/26.6, 536/24.3
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The invention provides assays that can detect multiple genetic variants of a gene (e.g., a mycobacterium gene) in a sample using a pool (using 2, 3, 4, or more) of oligonucletide hybridization probes.

Claims

1. A kit of reagents capable of identifying which variant among multiple variants of a first region of a gene sequence is present in a sample, said multiple variants differing from one another by one or more changes selected from the group consisting of nucleotide substitutions, deletions, and additions, said kit comprising

a) reagents for amplifying said first region and

b) a set of hybridization probes for said first region, said set containing at least three probes that

(i) comprise a single-stranded loop target binding sequence and an arms hybrid,

(ii) comprise a nucleotide sequence that is distinct from the other probes, such that each probe has a different target binding sequence,

(iii) are detectably differently fluorescently labeled,

(iv) hybridize in the presence of said reagents at temperatures in the range of 40-55°

C. to more than one variant of said first region with varying degrees of complementarity to form probe-target hybrids having unique melting temperatures, and

(v) are separably detectable when hybridized without separating unbound probes

wherein said set of hybridized probes is capable through their combined hybridizations to said first region of identifying said variant among multiple variants.

2. The kit of claim 1, wherein the at least three probes are DNA, RNA or a combination of DNA and RNA.

3. The kit of claim 1 wherein at least one of said at least three probes includes modified nucleotides or modified nucleotide linkages.

4. The kit according to claim 1 wherein at least one probe in said set is a wavelength-shifting molecular beacon probe.

5. The kit of claim 1 further comprising a supplemental molecular beacon probe for a second region of said gene sequence, said supplemental probe being detectably differently labeled.

6. The kit according to claim 1, wherein the single-stranded loop target-binding sequence of the at least three probes each probes are 25-50 nucleotides.

7. The kit according to claim 1, wherein said at least three probes are immobilized at preselected locations on a solid surface.

8. The kit according to claim 1, wherein the reagents for amplifying said first region are PCR reagents.

9. The kit according to claim 8 wherein the means to detect the presence of said variant provided by the probes in combination is a pattern of fluorescence intensities of said detectable fluorescent signals at a defined temperature.