Method for suppressing nonspecific hybridization in primer extension method

• US 6,033,851 A
• Filed: 05/30/1997
• Issued: 03/07/2000
• Est. Priority Date: 12/09/1994
• Status: Expired due to Fees

• Current Assignee: Wakunaga Seiyaku Kabushiki Kaisha
• Original Assignee: Wakunaga Seiyaku Kabushiki Kaisha
• Inventors: Yamane, Akio
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/849,075
• Time in Patent Office: 1,012 Days
• Field of Search: 435/91.2, 435/5, 435/6, 935/77, 935/78, 536/23.1, 536/24.3
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety

Abstract

An object of the present invention is to suppress nonspecific extension reaction of the primer in the primer extension method.

The primer extension reaction to form a nucleic acid strand complementary to a nucleic acid template strand with the use of a primer according to the present invention is characterized in that the reaction between the primer and the template strand is carried out in the presence of a nucleic acid or a derivative thereof which is complementary to said primer and has an affinity for said primer is equivalent to or less than that of said primer for the nucleic acid template strand.

Claims

1. A method for extending a primer comprising the steps of:

(a) providing a primer, a template, and a third nucleic acid molecule, wherein the primer is capable of specifically hybridizing to the third nucleic acid molecule, wherein the affinity of the primer for the third nucleic acid molecule is less than or equal to the affinity of the primer for the template, wherein the third nucleic acid molecule contains an inosine residue; and

(b) incubating the primer, the template, and the third nucleic acid molecule together under conditions sufficient to extend the primer, thereby extending the primer.

2. A method for extending a primer comprising the steps of:

(a) providing a primer, a template, and a third nucleic acid molecule, wherein the primer is capable of specifically hybridizing to the third nucleic acid molecule, wherein the affinity of the primer for the third nucleic acid molecule is less than or equal to the affinity of the primer for the template, wherein the third nucleic acid molecule is covalently bonded to the primer; and

(b) incubating the primer, the template, and the third nucleic acid molecule together under conditions sufficient to extend the primer, thereby extending the primer.

3. The method of claim 2, wherein the primer, the template, and the third nucleic acid molecule are each DNA or RNA.

4. The method of claim 2, wherein the third nucleic acid molecule is shorter than the primer.

5. The method of claim 2, wherein the third nucleic acid molecule contains a nucleotide which is not complementary to the primer.

6. The method of claim 2, wherein the third nucleic acid molecule contains a nucleotide which is capable of forming a weak hydrogen bond with the primer.

7. The method of claim 2, wherein the third nucleic acid molecule contains an inosine residue.

8. The method of claim 2, wherein the third nucleic acid molecule and the primer are bound to each other by a linker.

9. The method of claim 2, wherein the linker is a saturated hydrocarbon group, an unsaturated hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, or a heterocyclic group.

10. The method of claim 9, wherein the linker is a saturated hydrocarbon group.

11. The method of claim 10, wherein the linker is an alkyl group containing from 5 to 30 carbon atoms.

12. The method of claim 2, wherein the nucleotide residue at the 3'"'"' end of the third nucleic acid molecule does not contain a hydroxyl group directly bonded to the carbon atom at position 3 of the ribose moiety, wherein a pair of hydrogen atoms, a functional group, or a protecting group is bonded to the carbon atom at position 3 of the ribose moiety.

13. The method of claim 2, wherein the third nucleic acid molecule is peptide nucleic acid (PNA).

14. The method of claim 2, wherein the primer is extended by the polymerase chain reaction method, the 3SR method, the NASBA method, the RCR method, the SDA method, the P/LCA method, or the Sanger method.

15. A method for extending a primer comprising the steps of:

(a) providing a primer, a template, and a third nucleic acid molecule, wherein the primer is capable of specifically hybridizing to the third nucleic acid molecule, wherein the T_m of the primer binding to the third nucleic acid molecule is lower than the T_m of the primer binding to the template; and

(b) incubating the primer, the template, and the third nucleic acid molecule together under conditions sufficient to extend the primer, thereby extending the primer.

16. The method of claim 15, wherein the third nucleic acid molecule contains an inosine residue.

17. The method of claim 15, wherein the third nucleic acid molecule is covalently bonded to the primer.

18. The method of claim 15, wherein the primer, the template, and the third nucleic acid molecule are each DNA or RNA.

19. The method of claim 15, wherein the third nucleic acid molecule is shorter than the primer.

20. The method of claim 15, wherein the third nucleic acid molecule contains a nucleotide which is not complementary to the primer.

21. The method of claim 15, wherein the third nucleic acid molecule contains a nucleotide which is capable of forming a weak hydrogen bond with the primer.

22. The method of claim 15, wherein the third nucleic acid molecule and the primer are bound to each other by a linker.

23. The method of claim 15, wherein the linker is a saturated hydrocarbon group, an unsaturated hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, or a heterocyclic group.

24. The method of claim 15, wherein the linker is a saturated hydrocarbon group.

25. The method of claim 15, wherein the linker is an alkyl group containing from 5 to 30 carbon atoms.

26. The method of claim 15, wherein the nucleotide residue at the 3'"'"' end of the third nucleic acid molecule does not contain a hydroxyl group directly bonded to the carbon atom at position 3 of the ribose moiety, wherein a pair of hydrogen atoms, a functional group, or a protecting group is bonded to the carbon atom at position 3 of the ribose moiety.

27. The method of claim 15, wherein the third nucleic acid molecule is peptide nucleic acid (PNA).

28. The method of claim 15, wherein the primer is extended by the polymerase chain reaction method, the 3SR method, the NASBA method, the PCR method, the SDA method, the P/LCA method, or the Sanger method.