Amplification primers with non-standard bases for increased reaction specificity
View Patent ↗Described herein are methods for increasing the annealing specificity of an amplification reaction using Iso-base Amplification Primers (“IAPs”). IAPs containing an iso-region are capable of regulating sequence-specific annealing thereby enhancing primer-template hybridization for sequence-specific amplification of nucleotides.
1. A method for amplifying a template nucleic acid in a two-stage reaction comprising:
(a) performing a first-stage amplification of a template sequence at a first annealing temperature to form a first amplification product, the first-stage amplification comprising at least two cycles of denaturing the template sequence, annealing at least one primer, and extending the at least one primer, wherein the at least one primer comprises:
(i) a 3′-segment complementary to a first region of the template sequence;
(ii) a 5′-segment complementary to a second region of the template sequence; and
(iii) an iso-region between the 3′-segment and the 5′-segment, wherein the iso-region comprises at least two contiguous or non-contiguous non-standard bases independently selected from the group consisting of: K, X, H, J, M, N, iso-C and iso-G; and
(b) performing a second-stage amplification of the first amplification product at a second annealing temperature that is higher than the first annealing temperature comprising at least one cycle of denaturing the amplification product generated from step (a), annealing the at least one primer, and extending the at least one primer.
2. The method according to claim 1 , wherein the template nucleic acid is a cDNA formed by (a) hybridizing an oligonucleotide dT primer or an anchored oligonucleotide dT primer to a poly-A tail region of a target mRNA, or hybridizing random hexamer, heptamer, and/or octomer oligonucleotides to a target mRNA; and (b) reverse transcribing the target mRNA to produce the cDNA.
3. The method according to claim 1 , wherein the two-stage amplification procedure is applied to a method selected from the group consisting of: PCR; multiplex DNA amplification; identification of differentially expressed genes; 3′-Rapid Amplification of cDNA Ends; primer extension reactions; 5′-Rapid Amplification of cDNA Ends; amplifying full-length cDNA; amplifying 5′-enriched cDNA; DNA fingerprinting; RNA fingerprinting; identification of conserved homology segments in multigene families; identification of nucleotide sequence variations; pre-miRNA amplification; rRNA amplification; high resolution melt analysis following PCR; and mutagenesis.
4. The method of claim 1 , wherein the non-standard bases are contiguous.
5. The method of claim 1 , wherein the non-standard bases are non-contiguous.
6. The method of claim 1 , wherein the iso-region consists of 2, 3, or 4 bases.
7. The method of claim 6 , wherein all of the bases in the iso-region are non-standard bases.
8. The method of claim 1 , wherein the 3′-segment is 4 to 7 bases in length.
9. The method of claim 1 , wherein the 5′-segment is 6 to 60 bases in length.
10. The method of claim 1 , wherein the 3′-segment is 4 to 7 bases in length, the 5′-segment is 6 to 60 bases in length, and the iso-region is 2 to 4 bases in length.
11. The method of claim 1 , wherein the 5′-segment further comprises a label.
12. The method of claim 1 , wherein the non-standard bases are independently selected from the group consisting of iso-C and iso-G.