Method for amplifying unknown DNA sequence adjacent to known sequence
First Claim
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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;
(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;
5′
-Xp-Yq-Zr-Qs-3′
(I)wherein, Xp represents a 5′
-end portion having a pre-selected nucleotide sequence, Yq represents a regulator portion comprising contiguous nucleotides having at least two universal base or non-discriminatory base analog residues, Zr represents a degenerate random sequence portion having a degenerated random nucleotide sequence, Qs 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 Yq consists of deoxyinosine or inosine.
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Abstract
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 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; 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.
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27 Claims
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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; (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;
5′
-Xp-Yq-Zr-Qs-3′
(I)wherein, Xp represents a 5′
-end portion having a pre-selected nucleotide sequence, Yq represents a regulator portion comprising contiguous nucleotides having at least two universal base or non-discriminatory base analog residues, Zr represents a degenerate random sequence portion having a degenerated random nucleotide sequence, Qs 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 Yq consists of deoxyinosine or inosine.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
- in which said step (a) comprises;
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24. A DNA walking annealing control primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, which is represented by the following general formula I:
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5′
-Xp-Yq-Zr-Qs-3′
(I)wherein, Xp represents a 5′
-end portion having a pre-selected nucleotide sequence, Yq represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues, Zr represents a degenerate random sequence portion having a degenerated random nucleotide sequence, Qs 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.
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25. A DNA walking annealing control primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, which is represented by the following general formula II:
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5′
-X′
p-Su-Y′
v-Z′
w-3′
(II)wherein, X′
p represents a 5′
-end portion having a nucleotide sequence corresponding to the 5′
-end portion of the first degenerate DW-ACP, Su represents a supplementary annealing portion comprising a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first degenerate DW-ACP in the target-specific primer extension product of the step (a-2-1), Y′
v represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues and prevents annealing of said X′
p and Su portions to non-target sequences of the amplified product of the step (a) except to the nucleotide sequence complementary to the first degenerate DW-ACP, Z′
w represents a 3′
-end portion having a nucleotide sequence corresponding to the 3′
-end portion of the first degenerate DW-ACP, p, u, v and w represent the number of nucleotides, and X′
, S, Y′
, and Z′
are deoxyribonucleotide or ribonucleotide.
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26. A DNA walking annealing control primer for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, which is represented by the following general formula III:
-
5′
-X″
p-Y″
q-Cc-3′
(III)wherein, X″
p represents a 5′
-end portion having a nucleotide sequence corresponding to all or a part of the 5′
-end portion of said second DW-ACP, Y″
q represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues corresponding to the supplementary annealing portion, a part of the 5′
-end portion, a part of supplementary annealing portion plus a part of the 5′
-end portion, or a part of supplementary annealing portion plus a part of the regulator portion of the second DW-ACP of the formula II and prevents annealing of said X″
p portion to non-target sequences of said primary amplification product except to the nucleotide sequence complementary to said second DW-ACP, Cc represents a 3′
-end portion having a nucleotide sequence to hybridize with the opposite-sense nucleotide sequence to all or a part of the 3′
-end portion and regulator portion sequences of said second DW-ACP, p, q and c represent the number of nucleotides, and X″
, Y″
, and C are deoxyribonucleotide or ribonucleotide.
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27. A kit for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, which comprises a primer wherein the primer is selected from the group consisting of:
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(a) a DNA walking annealing control primer which is represented by the following general formula I;
5′
-Xp-Yq-Zr-Q5-3
(I)wherein Xp represents a 5′
-end portion having a re-selected nucleotide sequence, Yq represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues, Zr represents a degenerate random sequence portion having degenerated random nucleotide sequence, Qs 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,(b) a DNA walking annealing control primer which is represented by the following general formula II;
5′
-X′
p-Su-Y′
v-Z′
w-3′
(II)wherein X′
p represents a 5′
-end portion having a nucleotide sequence corresponding to the 5′
-end portion of the first degenerate DW-ACP, Su represents a supplementary annealing portion comprising a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first degenerate DW-ACP in the target-specific primer extension product of the step (a-2-1), Y′
v represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues and prevents annealing of said X′
p and Su portions to non-target sequences of the amplified product of the step (a) except to the nucleotide sequence complementary to the first degenerate DW-ACP, Z′
w represents a 3′
-end portion having a nucleotide sequence corresponding to the 3′
-end portion of the first degenerate DW-ACP, p, u, v and w represent the number of nucleotides, and X′
, S, Y′
, and Z′
are deoxyribonucleotide or ribonucleotide,(c) a DNA walking annealing control primer which is represented by the following general formula III;
5′
-X″
p-Y″
q-Cc-3′
(III)wherein X″
p represents a 5′
-end portion having a nucleotide sequence corresponding to all or a part of the 5′
-end portion of said second DW-ACP, Y″
q represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues corresponding to the supplementary annealing portion, a part of the 5′
-end portion, a part of supplementary annealing portion plus a part of the 5′
-end portion, or a part of supplementary annealing portion plus a part of the regulator portion of the second DW-ACP of the formula II and prevents annealing of said X″
p portion to non-target sequences of said primary amplification product except to the nucleotide sequence complementary to said second DW-ACP, Cc represents a 3′
-end portion having a nucleotide sequence to hybridize with the opposite-sense nucleotide sequence to all or a part of the 3′
-end portion and regulator portion sequences of said second DW-ACP, p, q and c represent the number of nucleotides, and X″
, Y″
, and C are deoxyribonucleotide or ribonucleotide and(d) combinations thereof.
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Specification
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Current AssigneeSeegene, Inc.
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Original AssigneeSeegene, Inc.
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InventorsChun, Jong-Yoon
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Primary Examiner(s)Strzelecka, Teresa E
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Assistant Examiner(s)Pande, Suchira
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Application NumberUS10/578,521Publication NumberTime in Patent Office2,927 DaysField of SearchNoneUS Class Current435/91.2CPC Class CodesC12Q 1/686 Polymerase chain reaction [...C12Q 2525/161 incorporating target specif...C12Q 2525/179 incorporating arbitrary or ...
