Patent ID: 8058032
Filing Date: 2011-11-15
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 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: wherein said first degenerate DW-ACP has a general formula I: wherein, X p represents a 5′-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 3′-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.