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:
- providing 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 free of a recombinase and has a region that is substantially complementary in sequence to a second strand of said target;
combining said first oligonucleotide with said double-stranded nucleic acid under conditions suitable for production of a double D-loop to form a mixture; and
adding the second oligonucleotide to the mixture, producing-said stabilized double D loop at said target sequence within said double-stranded nucleic acid.
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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.
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Citations
29 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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providing 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 free of a recombinase and has a region that is substantially complementary in sequence to a second strand of said target; combining said first oligonucleotide with said double-stranded nucleic acid under conditions suitable for production of a double D-loop to form a mixture; and adding the second oligonucleotide to the mixture, producing-said stabilized double D loop at said target sequence within said double-stranded nucleic acid. - View Dependent Claims (2, 3, 4)
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5. A method for detecting the presence of a desired target sequence within a double-stranded nucleic acid, the method comprising:
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providing a first oligonucleotide and a second oligonucleotide;
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 free of a recombinase and is substantially complementary in sequence to a second strand of said desired target, and said first oligonucleotide and second oligonucledtide have at least a region of complementarity therebetween;combining said first oligonucleotide with said double-stranded nucleic acid under conditions suitable for production of a double D-loop to form a mixture; adding the second oligonucleotide to the mixture; and detecting stabilized double D-loops having said oligonucleotides, said stable double D-loops signaling the presence of a desired target sequence. - View Dependent Claims (6, 7, 8)
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9. 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 form a mixture, adding a second oligonucleotide to the mixture, wherein said first oligonucleotide is bound by a recombinase, said second oligonucleotide is free of a recombinase, and said first and second oligonucleotides have at least a region of compiementarity 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 detecting stable double D-loops, said stable double D-loops signaling the presence of a desired target sequence. - View Dependent Claims (10)
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11. A method for detecting, in a sample of double-stranded nucleic acids suspected of having sequences that differ at a target, the presence of at least two different target sequences, the method comprising:
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forming double D-loops at said target by mixing a first oligonucleotide species with the sample of double-stranded nucleic acids to form a mixture, 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; adding at least one species of a second oligonucleotide, wherein each of said at least one second oligonucleotide species is free of 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 oilgonucieotldes present among stable D-loops, and thereafter determining the presence of at least two different target sequences. - View Dependent Claims (12, 13, 14, 15, 16, 17)
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18. 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:
- forming double D loops at said target by mixing a first oligonucleotide with the mixture of double-stranded nucleic acids, wherein said first oligonucleotide is bound by a recombinase, adding a second oligonucleotide, wherein the second oligonucleotide is free of 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
then purifying double-stranded nucleic acids having stable D loops. - View Dependent Claims (19, 20, 21, 22)
- forming double D loops at said target by mixing a first oligonucleotide with the mixture of double-stranded nucleic acids, wherein said first oligonucleotide is bound by a recombinase, adding a second oligonucleotide, wherein the second oligonucleotide is free of 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
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23. A method of protecting a restriction site target within double-stranded nucleic acids from cleavage during a restriction digest, comprising:
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forming double D-loops at said target by mixing a first oligonucleotide with the double-stranded nucleic acids to form a mixture, 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; adding a second oligonucleotide to the mixture, wherein said second oligonucleotide is free of 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; digesting said double-stranded nucleic acids with a restriction enzyme that recognizes said target sequence; and detecting resistance of said target sequence to restriction cleavage. - View Dependent Claims (24, 25, 26, 27)
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28. 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 by mixing a first oligonucleotide with the double-stranded nucleic acid to form a mixture, 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; adding a second oligonucleotide to the mixture, wherein said second oligonucleotide has at least a region that is substantially complementary in sequence to a second strand of said target and is free of recombinase; reacting said double-stranded nucleic acid with an enzyme that cleaves the double-stranded nucleic acid; and detecting cleavage at or near said target sequence.
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29. 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 by making a first oligonucleotide, with the double-stranded nucleic acid to form a mixture, 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; adding a second oligonucleotide to the mixture, wherein said second oligonucleotide is free of recombinase and has at least a region that is substantially complementary in sequence to a second strand of said target; reacting said double-stranded nucleic acid with an enzyme that cleaves at or near said double D-loop; and detecting cleavage at or near said target sequence.
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Specification