Patent ID: 8058032

Claim:
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 said unknown nucleotide sequence using a DNA walking annealing control primer (DW-ACP) and a first target-specific primer; in which said step (a) comprises: (a-1) performing a first-stage amplification of said unknown nucleotide sequence at a first annealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a first degenerate DW-ACP containing a degenerate random nucleotide sequence to hybridize with said unknown nucleotide sequence and a hybridizing nucleotide sequence substantially complementary to a site on said unknown nucleotide sequence, wherein said first annealing temperature enables said first degenerate DW-ACP to function as a primer, the annealing portion of the first DW-ACP is restricted to the portion consisting of said degenerate random nucleotide sequence and hybridizing nucleotide sequence at the first annealing temperature, whereby a first degenerate DW-ACP extension product is generated; and (a-2) performing a second-stage amplification at a second annealing temperature to render said first degenerate DW-ACP not to function as a primer, comprising: (a-2-1) amplifying said first degenerate DW-ACP extension product using said first target-specific primer to hybridize with a target-specific nucleotide sequence substantially complementary to a site on said known nucleotide sequence, whereby a target-specific primer extension product is generated, (a-2-2) amplifying said target-specific primer extension product using a second DW-ACP to hybridize with a nucleotide sequence complementary to said first degenerate DW-ACP sequence of said target-specific primer extension product, whereby a second DW-ACP extension product is generated, and (a-2-3) amplifying said second DW-ACP extension product using said second DW-ACP and said first target-specific primer, whereby a primary amplification product without a degenerate random nucleotide sequence is generated, wherein said first degenerate DW-ACP has a general formula I: 5â€²-X p -Y q -Z r -Q s -3â€²â€ƒâ€ƒ(I) wherein, X p represents a 5â€²-end portion having a pre-selected nucleotide sequence, Y q represents a regulator portion comprising contiguous nucleotides having at least two universal base or non-discriminatory base analog residues, Z r represents a degenerate random sequence portion having a degenerated random nucleotide sequence, Q s represents a 3â€²-end portion having a hybridizing nucleotide sequence substantially complementary to a site on said unknown nucleotide sequence to hybridize therewith, p, q, r and s represent the number of nucleotides, and X, Y, Z and Q are deoxyribonucleotide or ribonucleotide, p represents an integer of 10 to 60, q is at least 3, r represents an integer from 2 to 5, s represents an integer of 3 to 10, and Y q consists of deoxyinosine or inosine.