Genomics applications for modified OLIGO nucleotides

• US 7,229,767 B2
• Filed: 09/26/2003
• Issued: 06/12/2007
• Est. Priority Date: 03/27/2001
• Status: Expired due to Fees

• Current Assignee: University of Delaware
• Original Assignee: University of Delaware
• Inventors: Kmiec, Eric B., Usher, Michael G., Gamper, Howard B., Rice, Michael C.
• Primary Examiner(s): Myers; Carla J.
• Assistant Examiner(s): BAUSCH, SARAE L
• Application Number:
  US10/672,735
• Publication Number:
  US 20050100921A1
• Time in Patent Office: 1,355 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone

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.

Claims

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.

2. The method or claim 1, wherein said second oligonucleotide comprises at least one modification selected from the group consisting of:

locked nucleic acid (LNA) monomer, 2′

-OMe monomer, peptide nucleic acid, and phosphorothioate linkage.

3. The method of claim 1, wherein said recombinase is E. coli RecA protein.

4. The method of claim 1, further comprising the subsequent step of deproteinizing said double-stranded nucleic acid.

5. A method for detecting the presence of a desired target sequence within a double-stranded nucleic acid, the method comprising:

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.

6. The method of claim 5, wherein said second oligonucleotide comprise at least one modification selected from the group consisting of:

locked nucleic acid (LNA) monomer, 2′

-OMe monomer, peptide nucleic acid, and phosphorothloate linkage.

7. The method of claim 5, wherein at least one of said oligonucleotides is detectably labeled.

8. The method of claim 5, further comprising the step, after said combining and adding and before detecting, of:

deproteinizing said nucleic acid sample.

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:

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.

10. The method of claim 9, wherein said second oligonucleotide comprises at least one modification selected from the group consisting of:

locked nucleic acid (LNA) monomer, 2′

-OMe monomer, peptide nucleic acid, and phosphorothioate linkage.

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:

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

then

discriminably detecting the species of first oilgonucieotldes present among stable D-loops, and thereafter determining the presence of at least two different target sequences.

12. The method of claim 11, wherein each of said first oligonucleotide species is discriminably labeled.

13. The method of claim 12, wherein each said first oligonucleotide specie is labeled with a different fluorophore, said fluorophores having distinguishable emission spectra.

14. The method of claim 11, wherein said double-stranded nucleic acids are selected from the group consisting of:

linear nucleic acids, relaxed closed circular DNA, supercoiled circular DNA, artificial chromosomes, SACs, YACs, nuclear chromosomal DNA, and organelle chromosomal DNA.

15. The method of claim 11, wherein said second oligonucleotide comprises at least one modification selected from the group consisting of:

locked nucleic acid (LNA) monomer, 2′

-OMe monomer, peptide nucleic acid, and phosphorothioate linkage.

16. The method of claim 11, further comprising the step, after said deproteinizing and before said discriminably detecting, of:

separating double D loop-containing nucleic acids from double-stranded nucleic acids lacking double D loops.

17. The method of claim 16, wherein said first oligonucleotide species, or said second oligonucleotide species, or both said first and second oligonucleotide species comprises a capture moiety, and said separating step is performed by specific binding to said capture moiety.

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.

19. The method of claim 18, wherein said step of forming double D loops comprises:

contacting said mixture of double-stranded nucleic acids first with said first, recombinase-bound oligonucleotide and thereafter with said second, recombinase-tree oligonucleotide.

20. The method of claim 18, further comprising the step, after forming double D loops and before purifying, of deproteinizing said double-stranded nucleic acids.

21. The method of claim 18, wherein said second oligonucleotide comprise at least one modification selected from the group consisting of:

locked nucleic acid (LNA) monomer, 240 -OMe monomer, peptide nucleic acid, and phosphorothioate linkage.

22. The method of claim 18, wherein said first oligonucleotide, said second oligonucleotide, or both said first and second oligonucleotides comprises a capture moiety, and said purifying step is performed by specific binding to said capture moiety.

23. 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 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.

24. The method of claim 23, wherein either or both of said oligonucleotide are methylated and said restriction enzyme target site is unmethylated.

25. The method of claim 23, wherein each of said oligonucleotides contains a mismatch to its respective target sequence strand.

26. The method of claim 23, wherein said second oligonucleotide comprises at least one modification selected from the group consisting of:

locked nucleic acid (LNA) monomer, 2′

-OMe monomer, peptide nucleic acid, and phosphorothioate linkage.

27. The method or claim 23, further comprising the step, after said forming of D loops and before digestion, of:

deproteinizing said nucleic acids.

28. A method of cleaving at or near a target sequence within a double-stranded nucleic acid, the method comprising:

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.

29. A method of cleaving at or near a target sequence within a double-stranded nucleic acid, the method comprising:

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.