Families of non-cross-hybridizing polynucleotides for use as tags and tag complements, manufacture and use thereof
First Claim
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1. A set of minimally cross-hybridizing oligonucleotide tags or tag complements, the set of tags or tag complements being characterized in that:
- (a) each tag or tag complement either is free of cytosine residues and comprises guanosine residues or is free of guanosine residues and comprises cytosine residues;
(b) in each tag or tag complement that comprises cytosine residues, the cytosine residues are separated by between one and six non-cytosine residues, and in each tag or tag complement that comprises guanosine residues, the guanosine residues are separated by between one and six non-guanosine residues;
(c) the number of cytosine and guanosine residues present in each tag or tag complement does not exceed L/4 where L is the number of bases in the oligonucleotide;
(d) the length of each oligonucleotide differs by no more than five bases from the average length of all oligonucleotides in the set;
(e) each tag or tag complement contains no more than 4 contiguous identical nucleotides;
(f) the number of guanosine and cytosine residues present in each tag or tag complement does not vary from the average number of guanosine and cytosine residues present in all other tags or tag complements of the set by more than one; and
(g) when each tag or tag complement of the set is exposed to hybridization conditions comprising 0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0 at 37°
C., the degree of cross-hybridization between the tag or tag complement and an oligonucleotide of the set that is not fully complementary to the tag or tag complement does not exceed 30% of the degree of hybridization between the tag or tag complement and a fully complementary oligonucleotide to said tag or tag complement,wherein each tag or tag complement is between 10 and 50 nucleotides in length.
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Abstract
A family of minimally cross-hybridizing nucleotide sequences, methods of use, etc. A specific family of 1168 24mers is described.
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28 Claims
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1. A set of minimally cross-hybridizing oligonucleotide tags or tag complements, the set of tags or tag complements being characterized in that:
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(a) each tag or tag complement either is free of cytosine residues and comprises guanosine residues or is free of guanosine residues and comprises cytosine residues; (b) in each tag or tag complement that comprises cytosine residues, the cytosine residues are separated by between one and six non-cytosine residues, and in each tag or tag complement that comprises guanosine residues, the guanosine residues are separated by between one and six non-guanosine residues; (c) the number of cytosine and guanosine residues present in each tag or tag complement does not exceed L/4 where L is the number of bases in the oligonucleotide; (d) the length of each oligonucleotide differs by no more than five bases from the average length of all oligonucleotides in the set; (e) each tag or tag complement contains no more than 4 contiguous identical nucleotides; (f) the number of guanosine and cytosine residues present in each tag or tag complement does not vary from the average number of guanosine and cytosine residues present in all other tags or tag complements of the set by more than one; and (g) when each tag or tag complement of the set is exposed to hybridization conditions comprising 0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0 at 37°
C., the degree of cross-hybridization between the tag or tag complement and an oligonucleotide of the set that is not fully complementary to the tag or tag complement does not exceed 30% of the degree of hybridization between the tag or tag complement and a fully complementary oligonucleotide to said tag or tag complement,wherein each tag or tag complement is between 10 and 50 nucleotides in length. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A composition comprising minimally cross-hybridizing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide comprising a sequence of ten to fifty nucleotide bases in length, wherein each sequence comprises guanosine residues and, for each sequence, the number of guanosine residues does not exceed L/4 where L is the number of bases in said sequence, and for which,
under a single set of hybridization conditions, the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 15% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide, and wherein the single set of hybridization conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° - C., and the oligonucleotides are covalently linked to microparticles.
- View Dependent Claims (14, 15, 16, 17, 18, 19, 20)
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21. A composition comprising minimally cross-hybridizing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide comprising a sequence of ten to fifty nucleotide bases in length, wherein each sequence comprises cytosine residues and, for each sequence, the number of cytosine residues does not exceed L/4 where L is the number of bases in said sequence, and for which,
under a single set of hybridization conditions, the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 15% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide, and wherein the single set of hybridization conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° - C., and the oligonucleotides are covalently linked to microparticles.
- View Dependent Claims (22, 23, 24, 25, 26, 27, 28)
Specification