Methods and kits for reducing non-specific nucleic acid amplification
View Patent ↗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.
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.