| 摘要 |
<p>A method for amplifying the unknown nucleotide sequence adjacent to the known nucleotide sequence, and a kit used in the method are provided to remove the high background problem completely and to improve confidence. A method for amplifying the unknown nucleotide sequence adjacent to the known nucleotide sequence comprises the steps of first-stage amplifying the unknown nucleotide sequence with the first degenerate DNA working annealing control primer (DW-ACP) at a first annealing temperature where the first degenerate DW-ACP acts as a primer by carrying out at least one cycle comprising primer annealing, primer extension and denaturing processes; and second-stage amplifying the amplified product at a second annealing temperature higher than the first annealing temperature by carrying out at least one cycle comprising primer annealing using the DW-ACP and the first target-specific primer (TSP), primer extension and denaturing processes, wherein the first degenerate DW-ACP comprises the degenerate random nucleotide hybrided with the known nucleotide sequence and the hybrid nucleotide sequence complementary to the one position of the known nucleotide sequence.</p> |
