PRIMER FOR NUCLEIC ACID DETECTION

• US 20090118490A1
• Filed: 11/10/2008
• Published: 05/07/2009
• Est. Priority Date: 01/03/2005
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: Government of the United States of America, US Government Health and Human Services For Disease Control and Prevention Centre
• Inventors: Narayanan, Jothikumar, Hill, Vincent
• Granted Patent: US 7,709,626 B2
• Time in Patent Office: Days
• US Class Current: 536/24.330
• 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 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation

Abstract

This application provides universal labeled primers for detection and amplification of nucleic acid molecules. These universal primers can be attached to the 5′-end of a target sequence-specific primer. In particular examples, the universal primer includes a labeled nucleotide flanked on both sides a nucleotide whose complement nucleotides changes a detectable signal from the label when the universal primer hybridizes with its complementary nucleic acid molecule. Also disclosed are methods of using the universal primer in nucleic acid amplification, such as real-time PCR.

Claims

1. A method of making a labeled sequence-specific primer, comprising:

adding a universal nucleic acid sequence to a sequence-specific primer, thereby generating a labeled sequence-specific primer,

wherein the universal nucleic acid sequence comprises

a 5′

end,

a 3′

end; and

a labeled nucleotide flanked on each side by at least one nucleotide, wherein nucleotides complementary to the nucleotides flanking the labeled nucleotide quench a detectable signal from the label when the labeled sequence-specific primer is hybridized with its complementary nucleic acid molecule and the complementary nucleic acid is synthesized,

wherein the sequence-specific primer can hybridize to a target nucleic acid sequence, and

wherein the universal nucleic acid sequence does not substantially hybridize to the target nucleic acid sequence recognized by the sequence-specific primer.

2. The method of claim 1, wherein the universal nucleic acid sequence comprises the sequence 5′

-X_1(n)X₂X₃X₄X_5(n)-3′

, wherein X₃ is the labeled nucleotide, wherein X₂ and X₄ are the nucleotides flanking the labeled nucleotide, and wherein n is zero or more nucleotides.

3. The method of claim 1, wherein the universal nucleic acid sequence comprises the sequence 5′

-X_1(n)X₂X₃X₄X_5(n)-3′

, wherein X₃ is the labeled nucleotide, wherein X₂ and X₄ are the nucleotides flanking the labeled nucleotide, and wherein n is one or more nucleotides.

4. The method of claim 3, wherein X_1(n) is at least one nucleotide and wherein X_5(n) is at least two nucleotides.

5. The method of claim 2, wherein X_1(n) is zero nucleotides and wherein X_5(n) is at least four nucleotides.

6. The method of claim 2, wherein the universal nucleic acid sequence comprises the sequence 5′

-X₂X₃X₄X_5(n)-3′

, wherein X₃ is the labeled nucleotide, wherein X₂ and X₄ are the nucleotides flanking the labeled nucleotide, and wherein n is one or more nucleotides.

7. The method of claim 6, wherein the universal nucleic acid sequence comprises the sequence 5′

-X₂X₃X₄X_5(n)-3′

, wherein X₂ and X₄ are “

C”

.

8. The method of claim 6, wherein the universal nucleic acid sequence comprises the sequence 5′

-X₂X₃X₄X_5(n)-3′

, wherein X₃ is “

T”

.

9. The method of claim 6, wherein the universal nucleic acid sequence comprises the sequence 5′

-X₂X₃X₄X_5(n)-3′

, wherein X₃ is “

T” and

wherein X₂ and X₄ are “

C”

.

10. The method of claim 5, wherein the universal nucleic acid sequence comprises the sequence 5′

-CTCSX_(n)-3′

(SEQ ID NO;

     20), wherein X_(n) is any number of nucleotides, wherein S is G or C, and wherein T is the labeled nucleotide and is the second nucleotide from the 5′

-end of the universal nucleic acid sequence.

11. The method of claim 10, wherein n comprises 3-50 nucleotides.

12. The method of claim 10, wherein n comprises 3-20 nucleotides.

13. The method claim 10, wherein n comprises 3-11 nucleotides.

14. The method of claim 1, wherein the universal nucleic acid sequence is at least 4 nucleotides.

15. The method of claim 14, wherein the universal nucleic acid sequence is 4 to 25 nucleotides.

16. The method of claim 14, wherein the universal nucleic acid sequence is 4 to 15 nucleotides.

17. The method of claim 10, wherein the universal nucleic acid sequence comprises the sequence 5′

-CTCSXXX-3′

(SEQ ID NO;

     21), wherein X is any nucleotide, wherein S is G or C, and wherein T is the labeled nucleotide and is the second nucleotide from the 5′

-end of the universal nucleic acid sequence.

18. The method of claim 10, wherein the universal nucleic acid sequence comprises the nucleic acid sequence shown in SEQ ID NO:

1, 6, 14, 18, 19, 21, 22, 31, or 32.

19. The method of claim 1, wherein the universal nucleic acid sequence comprises the sequence 5′

-CTCSXXX_(n)-3′

(SEQ ID NO;

     22), wherein T is the labeled nucleotide and is the second nucleotide from the 5′

-end of the universal nucleic acid sequence and n comprises 0-11 nucleotides.

20. The method of claim 1, wherein the labeled nucleotide is thymidine.

21. The method of claim 1, wherein the label is a fluorophore.

22. The method of claim 21, wherein a signal emitted by the fluorophore is decreased by guanosine in the complementary nucleic acid molecule.

23. The method of claim 21, wherein the fluorophore comprises 6-carboxyfluorescein (6-FAM);

5-carboxyfluorescein (5-FAM);

boron dipyrromethene difluoride (BODIPY);

N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine (TAMRA);

acridine;

stilbene;

6-carboxyfluorescein hexachloride (HEX);

or derivatives thereof.

24. The method of claim 1, wherein a 5′

-end of the sequence-specific primer is added to the 3′

-end of the universal nucleic acid sequence.

25. The method of claim 1, wherein sequence-specific primer comprises at least nine nucleotides.

26. The method of claim 1, wherein the sequence-specific primer comprises a forward primer.

27. The method of claim 1, wherein the sequence-specific primer comprises a reverse primer.

28. The method of claim 1, wherein the labeled sequence-specific primer comprises 5′

-CTCSSSSX_(n)RRRRGAG-3′

(SEQ ID NO;

     31), wherein X_(n) is any number of nucleotides comprising the sequence-specific primer, wherein S is G or C, wherein R is the complementary nucleotide to S, and wherein T is the labeled nucleotide and is the second nucleotide from the 5′

end of the labeled sequence-specific primer.

29. The method of claim 28, wherein the labeled sequence-specific primer comprises 5′

-CTCSSSSX_(n)RRRRGAG(Z_(n))X_(n)-3′

(SEQ ID NO;

     32), wherein X_(n) at position 8 is any number of nucleotides comprising a target-specific nucleic acid sequence, wherein Z_(n) is a carbon spacer arm and n of Z_(n) is a spacer phosphoramidite containing at least 3 carbons, and wherein X_(n) at position 17 is any number of nucleotides comprising the sequence-specific primer.

30. The method of claim 29, wherein Z_(n) is a blocking moiety that can reduce or prevent copying of the 5′

tail sequence.