Nucleic acid sequence detection employing amplification probes

• US 6,590,091 B2
• Filed: 01/09/2001
• Issued: 07/08/2003
• Est. Priority Date: 12/27/1994
• Status: Expired due to Fees

• Current Assignee: Douglas Y. Thien
• Original Assignee: Naxcor
• Inventors: Albagli, David, Wood, Michael, VanAtta, Reuel
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/757,563
• Publication Number:
  US 20030039961A1
• Time in Patent Office: 910 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/22.3, 536/24.3, 536/33
• US Class Current: 536/24.3
• CPC Class Codes: B01L 2300/1883 using thermal insulation
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/119 Triple helix formation
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Methods and compositions are provided for detecting nucleic acid sequences. In particular, pairs of probes are employed, where the pair defines a substantially contiguous sequence on a target nucleic acid. Each of the pairs has a side chain which forms a stem of the two side chains which non-covalently binds and is capable of forming a cross-link upon activation, when the probes and sample nucleic acid are base paired. Cross-linking of the stems when unbound to complementary DNA is inhibited. Each of the nucleic acids is initially present as single stranded nucleic acid to allow for base pairing, so that the probes bind to homologous target nucleic acid. The assay mixture is activated to provide cross-linking, the double stranded nucleic acid melted, and the process of base pairing, activation and melting repeated, a sufficient number of cycles, to provide a detectable amount of cross-linked probes. To inhibit background cross-linking, the side chains may provide for duplex formation, where a portion of the side chain binds to a different portion of the side chain or the portion of the probe homologous to the target. Also provided are kits comprising reagents, as well as automatic devices, for carrying out the subject method.

Claims

1. A probe set for detecting a target nucleic acid in a sample, said probe set comprising:

a) a first outer probe comprising

i) a sequence complementary to a first portion of a target nucleic acid sequence; and

ii) a first side chain;

b) a central probe comprising;

i) a sequence complementary to a second portion of said target nucleic acid sequence, said second portion adjacent to said first portion;

ii) a second side chain complementary to said first side chain; and

iii) a third side chain; and

c) a second outer probe comprising;

i) a sequence complementary to a third portion of said target nucleic acid sequence, said third portion adjacent to said second portion;

ii) a fourth side chain complementary to said third side chain;

wherein said first and second side chains and said third and fourth side chains non-covalently bind to form first and second stems, respectively, upon base pairing of said probes to said target nucleic acid sequence, and wherein at least one of said first and second side chains and at least one of said third and fourth side chains has a photoactivatible group, which upon activation during stem formation forms a covalent cross-link with the other side chain comprising said stem.

2. A probe set according to claim 1, wherein said second portion of said target nucleic acid defines a conserved region in said sample.

3. A probe set according to claim 1, wherein said first portion of said target nucleic acid defines a polymorphic region in said sample.

4. A probe set according to claim 1, wherein said photoactivatable group reacts with a nucleotide or analog thereof to form said first and second stems.

5. A probe set according to claim 1, wherein said photoactivatable group is a coumarin, furocoumarin or psoralen.

6. A kit comprising a probe set for detecting a target nucleic acid in a sample, said probe set comprising:

a) a first outer probe comprising

i) a sequence complementary to a first portion of a target nucleic acid sequence; and

ii) a first side chain;

b) a central probe comprising;

i) a sequence complementary to a second portion of said target nucleic acid sequence, said second portion adjacent to said first portion;

ii) a second side chain complementary to said first side chain; and

iii) a third side chain; and

c) a second outer probe comprising;

i) a sequence complementary to a third portion of said target nucleic acid sequence, said third portion adjacent to said second portion;

ii) a fourth side chain complementary to said third side chain;

wherein said first and second side chains and said third and fourth side chains non-covalently bind to form first and second stems, respectively, upon base pairing of said probes to said target nucleic acid sequence, and wherein at least one of said first and second side chains and at least one of said third and fourth side chains has a photoactivatible group, which upon activation during stem formation forms a covalent cross-link with the other side chain comprising said stem.

7. A kit according to claim 6, wherein said second portion of said target nucleic acid defines a conserved region in said sample.

8. A probe set according to claim 6, wherein said first portion of said target nucleic acid defines a polymorphic region in said sample.

9. A probe set according to claim 6, wherein said photoactivatable group reacts with a nucleotide or analog thereof to form said first and second stems.

10. A probe set according to claim 6, wherein said photoactivatable group is a coumarin, furocoumarin or psoralen.