Patent ID: 8192940
Filing Date: 2012-06-05
Classification: C12Q

Abstract:
1. A method for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, which comprises the step of (a) performing a primary amplification of the unknown nucleotide sequence using a DNA walking annealing control primer (DW-ACP) and a first target specific primer (TSP) hybridizable with a site on the known nucleotide sequence, and the step of (b) performing a secondary amplification; in which the step (a) comprises: (a-1) performing a first-stage amplification of the unknown nucleotide sequence at a first annealing temperature, said first-stage amplification comprising at least one cycle of primer annealing, primer extending, and denaturing, using a first degenerate DW-ACP containing (i) a degenerate random nucleotide sequence to hybridize with the unknown nucleotide sequence and (ii) a hybridizing nucleotide sequence substantially complementary to a site on the unknown nucleotide sequence, wherein the first annealing temperature enables the first degenerate DW-ACP to function as a primer, whereby a first degenerate DW-ACP extension product is generated; and (a-2) performing a second-stage amplification of the amplification product generated from step (a-1) at a second annealing temperature which is higher than the first annealing temperature, comprising at least one cycle of primer annealing, primer extending, and denaturing using the first degenerate DW-ACP as used in the step (a-1) and the first TSP, under conditions in which each primer anneals to its target nucleotide sequence, whereby a primary amplification product is generated; wherein the first degenerate DW-ACP has the general formula I: wherein, X in which the step (b) comprises performing a secondary amplification at a third annealing temperature, said secondary amplification comprising at least one cycle of primer annealing, primer extending, and denaturing, using (i) a second DW-ACP and (ii) a second TSP designed to amplify an internal region of the primary amplification product; wherein the second DW-ACP has the general formula II: wherein, X′