Chimeric primers with hairpin conformations and methods of using same

• US 9,587,270 B2
• Filed: 06/29/2010
• Issued: 03/07/2017
• Est. Priority Date: 06/29/2009
• Status: Active Grant

• Current Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Original Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Inventors: Whitman, Douglas F., Zhang, Hongwei
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US12/826,189
• Publication Number:
  US 20100330574A1
• Time in Patent Office: 2,443 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

Methods and compositions for nucleic acid amplification, detection, and genotyping techniques are disclosed. In one embodiment, a nucleic acid molecule having a target-specific primer sequence; an anti-tag sequence 5′ of the target-specific primer sequence; a tag sequence 5′ of the anti-tag sequence; and a blocker between the anti-tag sequence and the tag sequence is disclosed. Compositions containing such a nucleic acid molecule and methods of using such a nucleic acid molecule are also disclosed.

Claims

1. A nucleic acid molecule comprising:

(a) a target-specific primer sequence;

(b) an anti-tag sequence 5′

of the target-specific primer sequence;

(c) a tag sequence 5′

of the anti-tag sequence; and

(d) a blocker between the anti-tag sequence and the tag sequence.

2. The nucleic acid molecule of claim 1, wherein the anti-tag sequence and the tag sequence are each from 8 to 30 nucleotides in length.

3. The nucleic acid molecule of claim 2, wherein the anti-tag sequence is 12 nucleotides in length.

4. The nucleic acid molecule of claim 2, wherein the anti-tag sequence and the tag sequence are each 24 nucleotides in length.

5. The nucleic acid molecule of claim 1, wherein the blocker is an iSp18 moiety or an iMe-isodC moiety.

6. A composition comprising:

(a) a first nucleic acid molecule, wherein the first nucleic acid molecule is a first member of a primer pair, comprising;

(i) a first target-specific primer sequence;

(ii) an anti-tag sequence 5′

of the target-specific primer sequence;

(iii) a tag sequence 5′

of the anti-tag sequence; and

(iv) a blocker between the anti-tag sequence and the tag sequence;

(b) a second nucleic acid molecule, wherein the second nucleic acid molecule is a second member of a primer pair, comprising;

(i) a second target-specific primer sequence;

(ii) a universal anti-tag sequence 5′

of the target-specific primer sequence;

(iii) a universal tag sequence 5′

of the anti-tag sequence; and

(iv) a blocker between the anti-tag sequence and the tag sequence; and

(c) a third nucleic acid molecule comprising;

(i) a universal anti-tag sequence complementary to the universal tag sequence; and

(ii) a label.

7. The composition of claim 6, wherein the label is a fluorescent label.

8. The composition of claim 6, comprising a plurality of different primer pairs for the amplification of a plurality of different target sequences, and a plurality of different anti-tag nucleic acid molecules covalently attached to a plurality of encoded microspheres, wherein the plurality of different anti-tag molecules comprise anti-tag sequences that are complementary to the different tag sequences in the plurality of different primer pairs, and wherein the identity of each of the different anti-tag nucleic acid molecules can be determined from the encoding of the encoded microsphere to which it is covalently attached.

9. The composition of claim 6, further comprising an anti-tag sequence covalently attached to a microsphere.

10. A composition comprising:

(a) a first primer pair comprising;

(i) a first primer comprising;

a first target-specific primer sequence;

a universal anti-tag sequence 5′

of the target-specific primer sequence;

a universal tag sequence 5′

of the universal anti-tag sequence;

a blocker between the universal anti-tag sequence and the universal tag sequence; and

a chromophore attached to the universal tag sequence; and

(ii) a second primer comprising;

a second target-specific primer sequence;

a universal anti-tag sequence 5′

of the target-specific primer sequence;

a universal tag sequence 5′

of the universal anti-tag sequence;

a blocker between the universal anti-tag sequence and the universal tag sequence; and

a chromophore attached to the universal tag sequence; and

(b) a labeled nucleic acid molecule comprising;

(i) a universal anti-tag sequence complementary to the universal tag sequences of the first primer pair; and

(ii) a chromophore capable of Forster Resonance Energy Transfer with the chromophores of the first primer pair.

11. The composition of claim 10, wherein the tag and anti-tag sequences are each from 8 to 30 nucleotides in length.

12. The composition of claim 10, wherein the blocker is an iSp18 moiety or an iMe-isodC moiety.