Use of primers containing nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules

Claim 1

An improved method for amplifying the concentration of a target nucleic acid molecule by using a first and a second primer to form a double-stranded nucleic acid molecule, wherein said first primer hybridizes to a first strand of said double-stranded nucleic acid molecule, and said second primer hybridizes to a second strand of said double-stranded nucleic acid molecule, and wherein said primers exhibit different T m s with respect to one another, when hybridized to said-double stranded nucleic acid molecule, said improved method comprises the steps: (a) replacing at least one residue in at least one of said primers with at least one nucleotide having altered base pairing characteristics, wherein said replacement equalizes the T m s of the first and second primers with respect to one another, when hybridized to said double-stranded nucleic acid molecule; (b) performing the template-dependent extension of said first primer, said first primer being hybridized to a first strand of said double-stranded molecule, wherein said extension forms a second strand of nucleic acid complementary to said first strand of said double-stranded nucleic acid molecule; (c) performing the template-dependent extension of said second primer, by extending said second primer, said second primer being hybridized to said second strand of said double-stranded nucleic acid molecule, wherein said extension forms a copy of said first strand of said double-stranded nucleic acid molecule; (d) performing the template-dependent extension of said copy of said first stand, to thereby form a copy of said second strand of said double-stranded nucleic acid molecule; and (e) repeating steps (b), (c) and (d) to thereby achieve said amplification of said target nucleic acid molecule.