Patent ID: 8455197
Filing Date: 2013-06-04
Classification: C12Q

Abstract:
1. A method for the selective amplification of a target nucleotide sequence located within a nucleic acid molecule, the method comprising: (a) denaturing a nucleic acid molecule (“template” nucleic acid molecule) comprising a target nucleotide sequence to produce a denatured template nucleic acid molecule and contacting the denatured template nucleic acid molecule with at least one initiator primer, wherein the initiator primer is modified such that the presence of the modification prematurely terminates complementary strand synthesis; (b) initiating second strand synthesis from the initiator primer to produce a double-stranded nucleic acid molecule, wherein the modification of the initiator primer is introduced into the newly synthesized second strand; (c) denaturing the double-stranded molecule produced in (b) and contacting the resulting denatured double-stranded molecule with a primer that binds to the opposite end of the target nucleotide sequence to that which the initiator primer binds; (d) initiating reverse strand synthesis from the primer to produce a double-stranded nucleic acid molecule; whereby reverse strand synthesis prematurely terminates due to the modification introduced in (b) to produce an incomplete reverse strand; (e) denaturing the double-stranded molecule synthesized in (d) and contacting the resulting denatured double-stranded molecule with a facilitator oligonucleotide that binds to the incomplete reverse strand, (f) initiating strand extension from the facilitator oligonucleotide to complete the reverse strand; and (g) optionally repeating steps (a) to (f) for one or more additional cycles.