Methods and kits for reducing non-specific nucleic acid amplification

• US 8,507,662 B2
• Filed: 07/15/2011
• Issued: 08/13/2013
• Est. Priority Date: 01/19/2001
• Status: Active Grant

• Current Assignee: Global Life Sciences Solutions Operations UK Ltd. (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Nelson, John Richard, Duthie, Robert Scott, Shah, Sonali Jagdish, Smith, Clifford Leslie
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/183,480
• Publication Number:
  US 20110287510A1
• Time in Patent Office: 760 Days
• Field of Search: None
• US Class Current: 536/24.3
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important

Abstract

Methods and kits for efficient amplification of nucleic acids are provided. The methods comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having terminal mismatch primer-dimer structure. The methods also comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having nucleotide analogues. The methods enhance efficiency of nucleic acid amplification reaction by reducing non-specific amplification reactions.

Claims

1. A kit for nucleic acid amplification, comprising:

a nucleic acid polymerase; and

a primer solution,

wherein the primer solution consists essentially of a partially constrained primer having a terminal mismatch primer-dimer structure, and wherein the partially constrained primer consists of a nucleotide sequence (W)_x(N)_y(S)_z, wherein x, y and z are integer values independent of each other, and wherein the value of x is 2 or 3, the value of y is 2, 3 or 4, and the value of z is 1 or 2.

2. The kit of claim 1, wherein the nucleic acid polymerase comprises a strand displacing nucleic acid polymerase.

3. The kit of claim 1, wherein the nucleic acid polymerase is a Phi29 DNA polymerase.

4. The kit of claim 1, wherein the partially constrained primer is a hexamer primer, a pentamer primer or combination thereof.

5. The kit of claim 1, wherein the partially constrained primer is a pentamer primer, the nucleotide sequence of which consists of WWNNS.

6. The kit of claim 1, wherein the partially constrained primer is a hexamer primer, the nucleotide sequence of which consists of WWNNNS.

7. The kit of claim 1, wherein the partially constrained primer comprises a nucleotide analogue.

8. The kit of claim 1, wherein the partially constrained primer is a DNA-LNA chimera primer.

9. A kit for isothermal nucleic acid amplification, comprising:

a nucleic acid polymerase; and

a primer solution,

wherein the primer solution consists essentially of a partially constrained primer mixture, wherein the partially constrained primer mixture comprises a nucleotide analogue, and wherein the partially constrained primer mixture consists of a nucleotide sequence (W)_X(N)_y(S)_Z, wherein x, y and z are integer values independent of each other, and wherein the value of x is 2 or 3, the value of y is 2, 3 or 4, and the value of z is 1 or 2.

10. The kit of claim 9, wherein the nucleotide analogue is a LNA nucleotide.

11. The kit of claim 9, wherein the partially constrained primer comprises a 3′

terminal nucleotide and a 5′

terminal nucleotide, which are non-complementary to each other.

12. The kit of claim 11, wherein the 3′

terminal nucleotide of the partially constrained primer is further non-complementary to a nucleotide adjacent to the 5′

terminal nucleotide.

13. The kit of claim 9, wherein the partially constrained primer is a pentamer primer, the nucleotide sequence of which consists of +W+WNNS, or W+W+NNS.

14. The kit of claim 9, wherein the partially constrained primer is a hexamer primer, the nucleotide sequence of which consists of +W+WNNNS, or W+W+NNNS.

15. The kit of claim 9, wherein the partially constrained primer comprises a phosphorothioate linkage between a 3′

terminal nucleotide and a nucleotide that is adjacent to the 3′

terminal nucleotide.

16. The kit of claim 15, wherein the partially constrained primer is a pentamer primer, the nucleotide sequence of which consists of W+W+NN*S, or +W+WNN*S.

17. The kit of claim 15, wherein the partially constrained primer is a hexamer primer, the nucleotide sequence of which consists of W+W+NNN*S, +W+WNNN*S.

18. The kit of claim 15, wherein the partially constrained primer is a pentamer primer, the nucleotide sequence of which consists of W+W+N*N*S, or +W+WN*N*S.

19. The kit of claim 15, wherein the partially constrained primer is a hexamer primer, the nucleotide sequence of which consists of W+W+NN*N*S, or +W+WNN*N*S.

20. A kit for isothermal nucleic acid amplification, comprising:

a nucleic acid polymerase; and

a primer solution,

wherein the primer solution consists essentially of a nuclease-resistant, partially constrained primer mixture, wherein the nuclease-resistant partially constrained primer mixture comprises a modified nucleotide, wherein the nuclease-resistant, partially constrained primer has a terminal primer-dimer structure, and wherein the partially constrained primer mixture consists of a nucleotide sequence (W)_x(N)_y(S)_z, wherein x, y and z are integer values independent of each other, and wherein the value of x is 2 or 3, the value of y is 2, 3 or 4, and the value of z is 1 or 2.