CHIMERIC PRIMERS WITH HAIRPIN CONFORMATIONS AND METHODS OF USING SAME

• US 20100330574A1
• Filed: 06/29/2010
• Published: 12/30/2010
• 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: Zhang, Hongwei, Whitman, Douglas F.
• Granted Patent: US 9,587,270 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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. (canceled)

10. 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.

11. The composition of claim 10, wherein the label is a fluorescent label.

14. The composition of claim 10, comprising a plurality of primer pairs for the amplification of a plurality of different target sequences and a plurality of anti-tag nucleic acid molecules covalently attached to a plurality of encoded microspheres, wherein the plurality of anti-tag molecules comprise anti-tag sequences that are complementary to the tag sequences in the plurality of primer pairs, and wherein the identity of each of the anti-tag nucleic acid molecules can be determined from the encoding of the encoded microsphere to which it is covalently attached.

12-13. -13. (canceled)

15. A method for amplifying a target nucleic acid comprising:

(a) providing a first primer pair comprising;

(i) a first primer comprising;

a first 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; and

(ii) a second primer comprising;

a second target-specific primer sequence; and

(b) providing a reporter;

(c) providing a capture complex comprising an anti-tag sequence attached to a solid support; and

(d) amplifying the target nucleic by combining the first primer pair, the reporter, the capture complex, and a sample comprising the target nucleic acid under conditions suitable for amplification of the target nucleic acid.

16. The method of claim 15, further comprising hybridizing the amplified nucleic acid to the anti-tag sequence of the capture complex.

23. The method of claim 16, further comprising detecting the amplified nucleic acid.

24. The method of claim 23, wherein detecting the amplified nucleic acid comprises imaging the amplified target nucleic acid sequence bound to the capture complex.

25. The method of claim 15, wherein the sample comprises at least a second target nucleic acid and at least a second primer pair is combined with the first primer pair, the reporter, the capture complex, and the sample comprising the target nucleic acids under conditions suitable for amplification of the target nucleic acids.

17-22. -22. (canceled)

26. (canceled)

27. The method of claim 26, further comprising hybridizing the different amplified target nucleic acids to different anti-tag sequences of distinguishable capture complexes.

28. The method of claim 27, wherein the capture complexes are spatially distinguishable or optically distinguishable.

29-40. -40. (canceled)

41. A method for quantifying gene expression comprising:

(a) immobilizing cDNA reverse transcribed from mRNA;

(b) hybridizing to the immobilized cDNA a first oligonucleotide comprising a universal primer-binding sequence and a cDNA-specific sequence, and a second oligonucleotide comprising a unique primer-binding sequence and a cDNA-specific sequence;

(c) ligating the first oligonucleotide to the second oligonucleotide to form a ligated oligonucleotide;

(d) amplifying the ligated oligonucleotide using a universal primer and a unique primer, the unique primer comprising a unique primer sequence, an anti-tag sequence 5′

of the unique primer sequence, a tag sequence 5′

of the anti-tag sequence; and

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

(e) capturing the amplicon by hybridizing the tag sequence of the amplicon to an anti-tag sequence of a capture complex;

(f) labeling the amplicon; and

(g) detecting and quantifying the labeled, captured amplicon.

42. The method of claim 41, comprising capturing and reverse transcribing the mRNA on an oligo-dT coated well or bead.

43. The method of claim 41, wherein the universal primer is a T3 primer.

44. The method of claim 41, wherein the labeling of the captured amplicon is with a DNA-binding dye.

45. The method of claim 41, wherein the expression is quantified for a plurality of different genes, and for each of the different genes there is a different tag, anti-tag, and capture complex.

46. The method of claim 41, wherein the amplicon is labeled prior to capturing.

47. The method of claim 41, wherein the amplicon is labeled after capturing.

48-66. -66. (canceled)

67. A method for detecting a target nucleic acid comprising:

(a) providing an oligonucleotide probe comprising;

(i) a target-specific sequence;

(ii) an anti-tag sequence 5′

of the target-specific sequence;

(iii) a tag sequence 5′

of the anti-tag sequence;

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

(v) a label;

(b) hybridizing the oligonucleotide probe to the target nucleic acid sequence; and

(c) detecting the hybridization of the oligonucleotide probe to the target nucleic acid sequence.

68. The method of claim 67, wherein the target nucleic acid sequence is a cleavage product of a nucleic acid cleavage reaction.

69. The method of claim 68, wherein the cleavage product is made by a structure-specific flap endonuclease or a mung bean nuclease or an S1 nuclease.

70. The method of claim 67, wherein the target nucleic acid sequence is a ligation product.

71. The method of claim 67, wherein the label is a FRET donor or acceptor molecule.

72. The method of claim 67, further comprising immobilizing the oligonucleotide probe on a solid support.

73. The method of claim 72, wherein the oligonucleotide probe is immobilized on a solid support by hybridization of the tag sequence to a complementary anti-tag sequence coupled to the solid support.