DNA purification by triplex-affinity capture and affinity capture electrophoresis

• US 5,482,836 A
• Filed: 01/14/1993
• Issued: 01/09/1996
• Est. Priority Date: 01/14/1993
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ito, Takashi, Cantor, Charles R., Smith, Cassandra L.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/004,552
• Time in Patent Office: 1,090 Days
• Field of Search: 435/6, 435/81.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 935/1, 935/2, 935/4, 935/16, 935/19, 935/76, 935/77
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2537/119 Triple helix formation

Abstract

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

Claims

1. A method for purifying intact a particular double stranded DNA present in a sample comprising the steps of:

(a) contacting the sample with an oligonucleotide coupled either directly or indirectly to a first recognition molecule of a specific molecular recognition system to form a triple-helix between the particular double stranded DNA and the coupled oligonucleotide, said oligonucleotide being an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) contacting the reaction medium obtained in step (a) with a solid carrier to which is either directly or indirectly fixed a second recognition molecule of the molecular recognition system, the second recognition molecule specifically binding to the first recognition molecule;

(c) separating the reaction medium from the solid phase in step (b);

(d) separating the particular double stranded DNA from the oligonucleotide by treating the separated solid phase of step (c) with an alkaline reagent that breaks the bonds between the oligonucleotide and the particular double stranded DNA but conserves the double strandedness of the particular double stranded DNA, said reagent having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about;

8.5 if said oligonucleotide is an oligoribonucleotide; and

(e) recovering the particular double stranded DNA.

2. The method of claim 1 wherein the double stranded DNA comprises a plasmid DNA.

3. The method of claim 2 wherein the plasmid DNA is a single copy clone from a yeast genomic library.

4. The method of claim 1 wherein the double stranded DNA comprises a lambda DNA.

5. The method of claim 1 wherein the oligonucleotide is a pyrimidine rich oligonucleotide.

6. The method of claim 5 wherein the oligonucleotide comprises a homopyrimidine oligonucleotide.

7. The method of claim 6 wherein the homopyrimidine is biotinylated at the 5'"'"' end.

8. The method of claim 7 wherein the homopyrimidine comprises 5'"'"'-biotinylated CT₂ CT₄ CT₂ CT₃ CT₅ CT₂.

9. The method of claim 7 wherein the homopyrimidine comprises 5'"'"'-biotinylated (T-C)₁₀.

10. The method of claim 1 wherein the oligonucleotide includes the sequence (T-C)_n where n is at least 4.

11. The method of claim 1 wherein the oligonucleotide comprises a purine rich nucleotide.

12. The method of claim 1 wherein the molecular recognition system is selected from the group consisting of an antigen/antibody, an avidin/biotin, a streptavidin/biotin, a protein A/Ig and a lectin/carbohydrate system.

13. The method of claim 12 wherein the molecular recognition system comprises an antigen/antibody system wherein the antigen is digoxigenin and the antibody is anti-digoxigenin antibody.

14. The method of claim 1 wherein the solid carrier is selected from a group consisting of particles, beads or a porous matrix.

15. The method of claim 1 wherein the solid carrier is selected from a group consisting of a plastic, glass, agarose, cellulose, nitrocellulose, nylon, silicon or metal solid carrier.

16. The method of claim 1 wherein the solid carrier fixed with a second recognition molecule comprises streptavidin coated beads.

17. The method of claim 12 wherein the molecular recognition system comprises a streptavidin/biotin system, the coupled oligonucleotide comprises a biotinylated homopyrimidine and the solid carrier to which is fixed a second recognition molecule comprises a streptavidin coated magnetic solid phase.

18. A method for purifying intact a particular double stranded DNA in a sample comprising the steps of:

(a) contacting the sample with a biotinylated oligonucleotide under acidic conditions to form by means of Hoogsteen hydrogen bonding, a triple-helix between the particular DNA and the oligonucleotide, said oligonucleotide being an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) contacting the reaction medium obtained in step (a) with streptavidin coated magnetic beads to indirectly attach the triple-helix to the magnetic beads by means of biotin/streptavidin binding;

(c) separating the reaction medium from the magnetic beads;

(d) separating the particular double-stranded DNA from the oligonucleotide by incubating the magnetic beads of step (c) with a basic buffer that destablizes the Hoogsteen hydrogen bonds between the oligonucleotide and the particular double-stranded DNA but not the Watson-Crick bonds, said buffer having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oliogodeoxyribonucleotide or a pH that is no greater than about 8.5, if said oligonucleotide is an oligoribonucleotide; and

(e) recovering the particular double stranded DNA from step (d).

19. A method for the determination of a particular double stranded DNA in a sample comprising the steps of:

(a) contacting the sample with an oligonucleotide coupled either directly or indirectly to a first recognition molecule of a specific molecular recognition system to form a triple-helix between the particular double stranded DNA and the coupled oligonucleotide, said oligonucleotide being an oligodeoxyribonucleotide or an oligoribonucleotide and said particular DNA remaining intact during triple helix formation;

(b) contacting the reaction medium obtained in step (a) with a solid carrier to which is either directly or indirectly fixed a second recognition molecule of the molecular recognition system, the second recognition molecule specifically binding to the first recognition molecule;

(c) separating the reaction medium from the solid phase in step (b);

(d) separating intact the particular double stranded DNA from the oligonucleotide by treating the separated solid phase of step (c) with an alkaline reagent that breaks the bonds between the oligonucleotide and the particular double stranded DNA but conserves the double strandedness of the particular double stranded DNA, said reagent having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said oligonucleotide is an oligoribonucleotide;

(e) determining the DNA content of the eluate in step (d); and

(f) relating the determination of step (e) to a standard to determine the specificity of the particular double stranded DNA in the sample.

20. A method for isolating intact a particular double stranded DNA in a sample comprising the steps of:

(a) incubating a sample containing the DNA with an oligonucleotide for a time sufficient for the oligonucleotide to form a triple helix with the particular DNA, said oligonucleotide being coupled either directly or indirectly to a first recognition molecule of a specific molecular recognition system, wherein said oligonucleotide is an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) electrophoresing the reaction mixture obtained in step (a)in a gel containing a gel embedded solid carrier to which is either directly or indirectly fixed a second recognition molecule of the molecular recognition system, the second recognition molecule specifically binding to the first recognition molecule in the reaction mixture during electrophoresis;

(c) separating the solid phase in step (b) from the gel following electrophoresis;

(d) separating the particular double stranded DNA from the solid phase by treating the solid phase from (c) with a reagent that breaks the bonds between the DNA and the oligonucleotide but conserves the double strandedness of the particular double stranded DNA, said reagent having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said oligonucleotide is an oligoribonucleotide; and

(e) recovering the particular double stranded DNA intact.

21. The method of claim 20 wherein the oligonucleotide coupled to a first recognition molecule comprises a biotinylated homopyrimidine oligodeoxyribonucleotide and the solid phase to which is fixed a second recognition molecule comprises streptavidin coated beads.

22. A method for isolating intact a particular double stranded DNA in a sample comprising the steps of:

(a) incubating a sample containing the particular DNA with a biotinylated homopyrimidine under acidic conditions to form by means of Hoogsteen hydrogen bonding a triple-helix between the DNA and the homopyrimidine, said homopyrimidine being an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) electrophoresing the reaction mixture obtained in step (a) under acidic conditions in a gel containing a trap comprising immobilized streptavidin coated beads embedded in said gel, whereby the biotin coupled to the oligonucleotide-DNA triple-helix of step (a) binds to the streptavidin coated beads during electrophoresis;

(c) separating the resulting solid phase in step (b) from the gel following electrophoresis;

(d) separating the particular double stranded DNA from the solid phase by treating the solid phase from (c) with a basic buffer to break the Hoogsteen hydrogen bonds between the DNA and the oligonucleotide but not the Watson-Crick bonds between the double-stranded DNA, said buffer having a pH that is about 8.0 to about 10.0 if said homopyrimidine is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said homopyrimidine is an oligoribonucleotide; and

(e) recovering the particular double stranded DNA intact.

23. A method for determination of a particular double stranded DNA in a sample comprising the steps of:

(a) incubating a sample with an oligonucleotide for a time sufficient for the oligonucleotide to form a triple helix with the particular DNA intact, said oligonucleotide being coupled either directly or indirectly to a first recognition molecule of a specific molecular recognition system, wherein said oligonucleotide is an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) electrophoresing the reaction mixture obtained in step (a)in a gel containing a gel embedded solid carrier to which is either directly or indirectly fixed a second recognition molecule of the molecular recognition system under conditions for a time sufficient for the second recognition molecule to specifically bind to a first recognition molecule in the reaction mixture during electrophoresis;

(c) separating the solid phase in step (b) from the gel following electrophoresis;

(d) separating the particular DNA intact from the solid phase by treating the solid phase from (c) with a reaction medium that breaks the bonds between the DNA and the oligonucleotide, said medium having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said oligonucleotide is an oligoribonucleotide;

(e) determining the DNA content of the eluate in step (d); and

(f) relating the determination of step (e) to a standard to determine the specificity of the DNA in the sample.

24. A method for purifying intact a particular double stranded DNA in a sample comprising the steps of:

(a) contacting the sample with an oligonucleotide coupled either directly or indirectly to a first recognition molecule of a specific molecular recognition system and with a solid carrier to which is either directly or indirectly fixed a second recognition molecule of the molecular recognition system under conditions and for a time sufficient for the second recognition molecule to specifically bind to the first recognition molecule and for the coupled oligonucleotide and the DNA to form a triple helix, said oligonucleotide being an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) separating the reaction medium from the solid phase in step (a);

(c) separating the particular double stranded DNA from the oligonucleotide by treating the separated solid phase of step (b) with an alkaline reagent that breaks the bonds between the oligonucleotide and the particular double stranded DNA but conserves the double strandednes of the particular double stranded DNA, said reagent having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said oligonucleotide is an oligoribonucleotide; and

(d) recovering intact the particular double stranded DNA.

25. A method for purifying intact a particular double stranded DNA in a sample comprising the steps of:

(a) contacting the sample with a specific oligonucleotide backbone analog coupled either directly or indirectly to a first recognition molecule of a specific molecular recognition system to form a triple-helix between the particular DNA and the coupled oligonucleotide analog, said oligonucleotide being an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) contacting the reaction medium obtained in step (a) with a solid carrier to which is either directly or indirectly fixed a second recognition molecule of the molecular recognition system, the second recognition molecule specifically binding to the first recognition molecule;

(c) separating the reaction medium from the solid phase in step (b);

(d) separating the particular double stranded DNA from the oligonucleotide analog by treating the separated solid phase of step (c) with a reagent that breaks the bonds between the oligonucleotide backbone analog and the particular DNA but conserves the double strandedness of the particular double stranded DNA, said reagent having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said oligonucleotide is an oligoribonucleotide; and

(e) recovering the particular double stranded DNA intact.

26. The method of claim 25 wherein the oligonucleotide backbone analog is selected from the group consisting of polyamide nucleic acids and phosphotriesters.

27. A method for purifying intact a particular double stranded DNA in a sample comprising the steps of:

(a) forming an immobilized triple helix with the particular DNA by means of a biotinylated oligonucleotide and streptavidin coated beads, wherein said oligonucleotide is an oligodeoxyribonucleotide or an oligoribonucleotide;

(b) separating the particular double stranded DNA from the triple helix in (a) by treating the triple helix with an alkaline reagent that breaks Hoogsteen hydrogen triplex bonds but not Watson-Crick hydrogen duplex bonds, said reagent having a pH that is about 8.0 to about 10.0 if said oligonucleotide is an oligodeoxyribonucleotide or a pH that is no greater than about 8.5 if said oligonucleotide is an oligoribonucleotide; and

(c) recovering the particular double stranded DNA intact.