High specificity primers, amplification methods and kits

• US 6,365,729 B1
• Filed: 07/12/2001
• Issued: 04/02/2002
• Est. Priority Date: 05/24/1999
• Status: Expired due to Term

• Current Assignee: Rutgers University
• Original Assignee: The Public Health Research Institute of the City of New York, Inc.
• Inventors: Kramer, Fred R., Tyagi, Sanjay, Vartikian, Robert
• Primary Examiner(s): Whisenant, Ethan C.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/903,915
• Time in Patent Office: 264 Days
• Field of Search: 425/6, 425/91.1, 425/91.2, 425/183, 425/475, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 536/24.33
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

For nucleic acid amplification including extension of primers by a DNA polymerase, high specificity primers are provided. The primers include a type of hairpin structure in which a single-stranded loop separates complementary 3′ and 5′ arms and in which the loop and the 3′ arm are complementary to the target nucleic acid. Amplification methods, assays and kits including such primers are included in the invention.

Claims

1. A hairpin oligonucleotide primer for extension by a DNA polymerase comprising a 3′

arm sequence, a 5′

arm sequence and a single-stranded loop sequence separating said arm sequences, said 3′

arm sequence and said loop sequence both being perfectly complementary to a selected priming region of a target nucleic acid strand, and said arm sequences having sufficient complementarily to one another to form a double-stranded stem hybrid in the absence of the target strand, wherein said primer hybridizes to said target strand, overcoming the stem hybrid, wherein said stem hybrid is sufficiently strong that hybridization of the loop sequence to a sequence of the length of the loop and perfectly complementary to the loop sequence does not cause dissociation of the stem, and wherein said 3′

arm sequence contains insufficient nucleotides that are not complementary to said 5′

arm sequence to nucleate hybridization of the primer to the target.

2. The hairpin primer according to claim 1 additionally comprising interactive fluorescent label moieties attached to the 3′

and 5′

arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.

3. The hairpin primer according to claim 2 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.

4. The hairpin primer according to claim 1 wherein said loop sequence is 5 to 9 nucleotides in length and said complementary 3′

and 5′

arm sequences form a stem that is 5 to 12 nucleotides in length.

5. The hairpin primer according to claim 4 wherein said priming region contains a single nucleotide subject to mutation and said loop sequence is complementary to the portion of said priming region containing said nucleotide.

6. The hairpin primer according to claim 4 wherein said primer contains a terminator nucleotide between the loop sequence and the 5′

arm sequence.

7. The hairpin primer according to claim 6 wherein said 3′

arm sequence is perfectly complementary to said 5′

arm sequence.

8. The hairpin primer according to claim 4 containing modified nucleotides, modified internucleotide linkages, or both.

9. The hairpin primer according to claim 4 additionally comprising interactive fluorescent label moieties attached to the 3′

and 5′

arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.

10. The hairpin primer according to claim 6 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.

11. The hairpin primer according to claim 1 containing modified nucleotides, modified internucleotide linkages, or both.

12. The hairpin primer according to claim 1 wherein said primer contains a terminator nucleotide between the loop sequence and the 5′

arm sequence.

13. The hairpin primer according to claim 1 wherein said priming region contains a single nucleotide subject to mutation and said loop sequence is complementary to the portion of said priming region containing said nucleotide.

14. The hairpin primer according to claim 13 additionally comprising interactive fluorescent label moieties attached to the 3′

and 5′

arm sequences, whereby incorporation of the primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.

15. The hairpin primer according to claim 14 wherein said interactive fluorescent label moieties comprise a fluorophore and a non-fluorescent quencher.

16. The hairpin primer according to claim 1 wherein said 3′

arm sequence is perfectly complementary to said 5′

arm sequence.

17. A kit of reagents for performing amplification of a target nucleic acid sequence comprising amplification buffer, dNTPs, at least one primer according to claim 1, and instructions for performing said amplification.

18. The kit of reagents according to claim 17 wherein said at least one primer according to claim 1 includes a first primer and a second primer differing from said first primer by a single nucleotide of said loop sequence.

19. The kit of reagents according to claim 18, wherein in each of said first and second primers the loop sequence is 5 to 9 nucleotides in length and said complementary 3′

and 5′

arm sequences form a stem that is 5 to 12 nucleotides in length.

20. The kit of reagents according to claim 18 wherein said each of said first and second primers includes interactive fluorescent label moieties attached to the 3′

and 5′

arm sequences, whereby incorporation of each said primer into a double-stranded nucleic acid detectably alters the fluorescence emitted by said label moieties.

21. The kit of reagents according to claim 17 wherein the loop sequence is 5 to 9 nucleotides in length and said complementary 3′

and 5′

arm sequences form a stem that is 5 to 12 nucleotides in length.

22. The kit of reagents according to claim 17 wherein said at least one primer according to claim 1 includes a terminator nucleotide between the loop and the 5′

arm.

23. In a linear oligonucleotide primer for extension by a DNA polymerase when hybridized to a complementary sequence in a target strand, said primer having a 5′

terminus and a 3′

terminal region, the improvement comprising adding to said 5′

terminus a nucleotide sequence having sufficient complementarity to said 3′

terminal region to form a hairpin structure comprising a double-stranded stem and a single-stranded loop, wherein contacting said extended primer to said target causes said stem to dissociate, wherein hybridization of the loop to a model oligonucleotide having the same length as the loop and being perfectly complementary thereto does not cause said stem to dissociate, and wherein said stem lacks a 3′

single-stranded overhang sufficient to nucleate hybridization of the primer to its target.

24. The improvement according to claim 23 wherein said loop is 5 to 9 nucleotides in length and said stem is 5 to 12 nucleotides in length.

25. The improvement according to claim 23 further comprising adding a terminator nucleotide to the 5′

terminus prior to adding said nucleotide sequence that is complementary to said 3′

terminal region.