Patent ID: 7579146

Claim:
A method of generating a nucleic acid molecule, the method comprising steps of: providing a first precursor nucleic acid molecule including a first double stranded portion flanked by first and second single-stranded overhangs; providing a second precursor nucleic acid molecule including a second double-stranded portion flanked by third and fourth single-stranded overhangs, the third single-stranded overhang being complementary with the second overhang of the first precursor nucleic acid molecule; providing a third precursor nucleic acid molecule including a third double-stranded portion flanked by fifth and sixth single-stranded overhangs, the fifth overhang being complementary with the fourth overhang of the second precursor nucleic acid molecule and the sixth overhang being complementary with the first overhang of the first nucleic acid molecule; and combining the first, second, and third precursor nucleic acid molecules together in a single reaction under conditions that allow for hybridization of the complementary overhangs, so that a new nucleic acid molecule is generated; wherein at least one of the precursor nucleic acid molecules is generated by extending first and second primers in a polymerase-mediated extension reaction, at least one of the first and second primers including at least one base-containing residue, which base-containing residue is not used as a template by the polymerase employed in the polymerase-mediated extension reaction, but which is capable of being used as a template by a different polymerase.