Genomics applications for modified OLIGO nucleotides
First Claim
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1. A method for producing a stabilized double D loop at a target sequence within a double-stranded nucleic acid, the method comprising:
- contacting the double-stranded nucleic acid with a first oligonucleotide and a second oligonucleotide, said first and second oligonucleotides having at least a region of complementarity therebetween, wherein said first oligonucleotide is bound by a recombinase and has a region that is substantially complementary in sequence to a first strand of said target, and said second oligonucleotide is not substantially bound by a recombinase and has a region that is substantially complementary in sequence to a second strand of said target.
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Abstract
Methods for the production and use of stable complexes of duplex nucleic acid molecules and oligonucleotides are presented. These complexes can be used for the detection and purification of a known nucleic acid target as well as the manipulation of a defined nucleic acid target sequence.
50 Citations
41 Claims
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1. A method for producing a stabilized double D loop at a target sequence within a double-stranded nucleic acid, the method comprising:
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contacting the double-stranded nucleic acid with a first oligonucleotide and a second oligonucleotide, said first and second oligonucleotides having at least a region of complementarity therebetween, wherein said first oligonucleotide is bound by a recombinase and has a region that is substantially complementary in sequence to a first strand of said target, and said second oligonucleotide is not substantially bound by a recombinase and has a region that is substantially complementary in sequence to a second strand of said target. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method for detecting the presence of a desired target sequence within a double-stranded nucleic acid, the method comprising:
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contacting the double-stranded nucleic acid with a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase and is substantially complementary in sequence to a first strand of said desired target, said second oligonucleotide is not substantially bound by a recombinase and is substantially complementary in sequence to a second strand of said desired target, and said first oligonucleotide and said second oligonucleotide have at least a region of complementarity therebetween; and
thendetecting stabilized double D loops having said oligonucleotides. - View Dependent Claims (8, 9, 10, 11)
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12. A method for detecting the presence of a desired target sequence in a sample of double-stranded nucleic acids suspected of having sequences that differ at a target therein, the method comprising:
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contacting said sample of double-stranded nucleic acids with a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase, said second oligonucleotide is not substantially bound by a recombinase, and said first and second oligonucleotides have at least a region of complementarity therebetween, wherein both of said first and said second oligonucleotides have regions that are perfectly complementary to respective first and second strands of said desired target sequence, but at least one of said oligonucleotides is imperfectly matched in said region to each of said target sequences that differ from said desired sequence;
deproteinizing said nucleic acids; and
thendetecting stable double D loops, said stable double D loops signaling the presence of a desired target sequence. - View Dependent Claims (13, 14)
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15. A method for detecting, in a sample of double-stranded nucleic acids suspected of having sequences that differ at a target therein, the presence of at least two different target sequences, the method comprising:
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forming double D loops at said target using a mixture of first oligonucleotide species and at least one species of second oligonucleotide, wherein said mixture includes at least two species of first oligonucleotide, each of said species having a region that is perfectly complementary to a distinct one of said different target sequences, and each of said species is bound by a recombinase;
wherein each of said at least one second oligonucleotide species is not substantially bound by said recombinase; and
wherein said first oligonucleotides and said second oligonucleotides have at least a region of complementarity therebetween;
deproteinizing said nucleic acids; and
thendiscriminably detecting the species of first oligonucleotides present among stable D loops. - View Dependent Claims (16, 17, 18, 19, 20, 21, 41)
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22. A method of purifying, from a mixture of double-stranded nucleic acids having sequences that differ at a target therein, double-stranded nucleic acids having a desired target sequence, the method comprising:
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forming double D loops at said target using a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase, said second oligonucleotide is not substantially bound by said recombinase, and said first and second oligonucleotides have at least a region of complementarity therebetween, wherein said first oligonucleotide is perfectly complementary to a first strand of said desired target sequence, said second oligonucleotide is perfectly complementary to a second strand of said desired target sequence, and at least one of said oligonucleotides is imperfectly matched at each of said target sequences that differ from said desired target sequence; and
thenpurifying double-stranded nucleic acids having stable D loops. - View Dependent Claims (23, 24, 25, 26)
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27. A method of protecting a restriction site target within double-stranded nucleic acids from cleavage during a restriction digest, comprising:
forming double D loops at said target using a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase and has at least a region that is substantially complementary in sequence to a first strand of said target;
wherein said second oligonucleotide is not substantially bound by said recombinase and has at least a region that is substantially complementary in sequence to a second strand of said target; and
wherein said double D loop is resistant to restriction cleavage at said target; and
thendigesting said double-stranded nucleic acids with a restriction enzyme that recognizes said target sequence. - View Dependent Claims (28, 29, 30, 31, 32)
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33. A method of cleaving at or near a target sequence within a double-stranded nucleic acid, the method comprising:
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forming a double D loop at said target using a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide has at least a region that is substantially complementary in sequence to a first strand of said target and is bound by a recombinase;
wherein said second oligonucleotide has at least a region that is substantially complementary in sequence to a second strand of said target and is not substantially bound by said recombinase; and
thenreacting said double-stranded nucleic acid with an enzyme that cleaves the double-stranded nucleic acid. - View Dependent Claims (34)
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35. A method of cleaving at or near a target sequence within a double-stranded nucleic acid, the method comprising:
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forming a double D loop at said target using a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase and has at least a region that is substantially complementary in sequence to a first strand of said target;
wherein said second oligonucleotide is not substantially bound by said recombinase and has at least a region that is substantially complementary in sequence to a second strand of said target; and
thenreacting said double-stranded nucleic acid with an enzyme that cleaves at or near said double D loop.
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36. A kit for forming stabilized double D loops at a target sequence in double-stranded nucleic acids, comprising:
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a first composition comprising a first oligonucleotide, said first oligonucleotide being bound by a recombinase and having a region that is substantially complementary in sequence to a first strand of said target; and
a second composition comprising a second oligonucleotide, said second oligonucleotide being not substantially bound by a recombinase and having a region that is substantially complementary in sequence to a second strand of said target. - View Dependent Claims (37, 38, 39)
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40. A kit for detecting the presence of at least two different target sequences in a sample of double-stranded nucleic acids suspected of having sequences that differ at a target therein, comprising:
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a composition comprising (i) a mixture of first oligonucleotide species and (ii) at least a first species of second oligonucleotide, wherein said mixture includes at least two species of first oligonucleotides, each of said species having a region that is perfectly complementary to a distinct one of said differing target sequences, and each of said first oligonucleotide species in said mixture being bound by a recombinase;
wherein each of said at least one second oligonucleotide species is not substantially bound by said recombinase; and
wherein each of said first oligonucleotide species in said mixture and said at least one second oligonucleotide has at least a region of complementarity therebetween.
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Specification