Patent ID: 8906622

Claim:
A method of amplifying a nucleic acid region of interest, said method comprising: (i) contacting a nucleic acid sample with: (a) a first forward primer directed to said region of interest and a first reverse primer directed to said region of interest; and (b) a second forward primer directed to said region of interest wherein said second forward primer is directed to a nucleic acid sequence located 3′ to the sequence to which said first forward primer is directed and which second forward primer has a T m lower than the T m of said first forward primer; and a second reverse primer directed to said region of interest wherein said second reverse primer is directed to a nucleic acid sequence located 5′ to the sequence to which said first reverse primer is directed and which second reverse primer has a T m lower than the T m of said first reverse primer; and (c) at least one antisense oligonucleotide directed to said first forward primer, said first reverse primer, or each of said first forward primer and said first reverse primer, wherein each antisense oligonucleotide has a T m lower than said first primers, and wherein each antisense oligonucleotide comprises a 5′ nucleic acid tag sequence, the complementary nucleotide sequence of which tag is mismatched relative to the nucleotide sequence of the nucleic acid region adjacent to the 5′ end of the hybridization site of said first primer to which the antisense oligonucleotide is directed, and wherein each antisense oligonucleotide hybridizes to the 3′ end of the first forward primer or the 3′ end of the first reverse primer; and (ii) amplifying the nucleic acid sample of step (i) at an annealing temperature which enables hybridization of said first primers but which does not enable hybridization of said second primers and said antisense oligonucleotides; and (iii) amplifying the nucleic acid sample of step (ii) at an annealing temperature which enables hybridization of said second primers and said antisense oligonucleotides; wherein the molecules of parts (b) and (c) can be added to the reaction before, during or after the amplification of step (ii) but before the amplification of step (iii).