Patent ID: 8440406

Claim:
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.