Enzymatic synthesis of oligonucleotide tags
First Claim
1. A method of synthesizing a repertoire of oligonucleotide tags of a predetermined length, the method comprising the steps of:
- (a) providing a repertoire of oligonucleotide tag precursors in an amplicon, the oligonucleotide tag precursors each comprising one or more words, and each of the one or more words being selected from the same minimally cross-hybridizing set;
(b) cleaving the amplicon at a word in each of the oligonucleotide tag precursors to form one or more ligatable ends on each oligonucleotide tag precursor;
(c) ligating one or more words to the one or more ligatable ends to elongate each of the oligonucleotide tag precursors;
(d) amplifying the elongated oligonucleotide tag precursors in the amplicon; and
(e) repeating steps (b) through (d) until a repertoire of oligonucleotide tags having the predetermined length is formed.
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Abstract
The invention provides oligonucleotide tag compositions and methods for synthesizing repertoires of error-free oligonucleotide tags that may be used for labeling and sorting polynucleotides, such as cDNAs, restriction fragments, and the like. In accordance with the method of the invention, oligonucleotide tag precursors are provided in an amplicon, wherein the tag precursors each consists of one or more oligonucleotide “words” selected from the same minimally cross-hybridizing set of words. The oligonucleotide tag precursors are elongated by repeated cycles of cleavage, ligation of one or more words, and amplification. Cycles continue until the oligonucleotide tags of the repertoire have a desired length or complexity.
160 Citations
16 Claims
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1. A method of synthesizing a repertoire of oligonucleotide tags of a predetermined length, the method comprising the steps of:
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(a) providing a repertoire of oligonucleotide tag precursors in an amplicon, the oligonucleotide tag precursors each comprising one or more words, and each of the one or more words being selected from the same minimally cross-hybridizing set;
(b) cleaving the amplicon at a word in each of the oligonucleotide tag precursors to form one or more ligatable ends on each oligonucleotide tag precursor;
(c) ligating one or more words to the one or more ligatable ends to elongate each of the oligonucleotide tag precursors;
(d) amplifying the elongated oligonucleotide tag precursors in the amplicon; and
(e) repeating steps (b) through (d) until a repertoire of oligonucleotide tags having the predetermined length is formed. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A repertoire of oligonucleotide tags, wherein the oligonucleotide tags of the repertoire are of the form:
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w1(N)x1w2(N)x2 . . . (N)xn−
1wnwherein each of w1 through wn is a word consisting of an oligonucleotide having a length from three to fourteen nucleotides or basepairs and being selected from the same minimally cross-hybridizing set wherein a word of the set and a complement of any other word of the set has at least two mismatches;
N is a nucleotide or basepair;
each of x1 through xn−
1 is an integer selected from the group consisting of 0, 1, 2, 3, and 4, provided that at least one of x1 through xn−
1 is 1, 2, 3, or 4; and
n is an integer in the range of from 4 to 10.- View Dependent Claims (9, 10, 11, 12, 13, 14)
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15. A repertoire of cloning vectors for attaching oligonucleotide tags to polynucleotides, wherein each of the vectors comprises a double stranded element corresponding to an oligonucleotide tag of the form:
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w1(N)x1w2(N)x2 . . . (N)xn−
1wnwherein each of w1 through wn is a word consisting of an oligonucleotide having a length from three to fourteen nucleotides and being selected from the same minimally cross-hybridizing set wherein a word of the set and a complement of any other word of the set has at least two mismatches;
N is a nucleotide;
each of x1 through xn−
1 is an integer selected from the group consisting of 0, 1, 2, 3, and 4, provided that at least one of x1 through xn−
1 is 1, 2, 3, or 4; and
n is an integer in the range of from 4 to 10.- View Dependent Claims (16)
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Specification