| 摘要 |
<p>The present invention relates to 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; in which the step (a) comprises: (a-1) performing a first-stage amplification of the unknown nucleotide sequence at a first annealing temperature, 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, using the first degenerate DW-ACP as used in the step (a-1) and the first TSP, under conditions in which each primer anneals the its target nucleotide sequence, whereby a primary amplification product is generated.</p> |
