RecA nucleoprotein filament and methods

• US 4,888,274 A
• Filed: 01/20/1987
• Issued: 12/19/1989
• Est. Priority Date: 09/18/1985
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Welcher, Andrew A., Rigas, Basil, Radding, Charles M., Honigberg, Saul M., Ward, David C., Weissman, Sherman
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/005,470
• Time in Patent Office: 1,064 Days
• Field of Search: 435/6, 435/803, 435/810, 435/172.3, 436/501, 436/530, 436/808, 536/27, 935/28, 935/78, 530/367, 548/303
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

A stable, single-stranded nucleoprotein filament adapted to complex specifically and stably with a target duplex DNA having a selected base sequence. The filament is composed of a single-stranded DNA probe having a region of homology with the target base sequence, and RecA protein bound stably to the DNA probe by adenosine 5'"'"'-(γ-thio)triphosphate. The filament is useful in a novel system and method for enriching target duplex DNA which contains a region homologous to the probe sequence, for blocking selected restriction endonuclease sites in the target DNA, and in other DNA methodologies in which stable rapid triple-strand synaptic formation in duplex DNA can be exploited.

Claims

1. A method of separating target and non-target duplex DNA molecules on the basis of a selected base sequence which is unique to the target sequence, comprising

providing stable, single-stranded nucleoprotein filaments, each composed of a single-stranded DNA probe having a region of homology with the selected base sequence, and RecA protein molecules bound stably to the DNA probe, in the presence of adenosine 5'"'"'-(γ

-thio)triphosphate,

reacting the filaments with the target and non-target DNA molecules under conditions which promote rapid homologous alignment between the probe and homologous target base sequences in the reaction mixture, with formation of a stable filament/target complex,

contacting the reaction mixture with a solid support designed to selectively bind target molecules which are complexed with said filaments by such homologous alignment,

removing non-bound DNA molecules from the support, and

treating the support to release target DNA molecules which are selectively bound to the support through such complex formation.

2. The method of claim 1, wherein the filaments provided are formed by reacting the probe with RecA protein in the presence of adenosine 5'"'"'-(γ

-thio)triphosphate, at a concentration of at least about 0.5 mM, and Mg⁺⁺, at a concentration of between about 0.5 and 2 mM, and said reacting is carried out at a Mg⁺⁺ concentration of greater than about 4 mM.

3. The method of claim 1, for use in separating circular target DNA from a mixture of circular target and non-target molecules, wherein said providing includes reacting a such probe with adenosine 5'"'"'-(γ

-thio)triphosphate in the presence of up to 10-20 mM Mg⁺⁺, and an amount of RecA whose concentration is selected on the basis of the total amount of DNA in the reaction mixture, to give optimal or near-optimal complex formation by said reacting.

4. The method of claim 1, wherein the target DNA is a vector capable of transforming a suitable bacterial host, and containing a selectable marker by which host cells transformed with the target DNA can be distinguished from host cells transformed with non-target DNA, which further includes transforming such host cells with the released target DNA and selecting transformed cells containing the selectable marker.

5. The method of claim 1, wherein the filament contains biotin molecules which are derivatized to probe nucleotides through disulfide linkages, the reaction mixture is contacted with a solid support containing surface bound avidin, streptavidin, or an analog thereof, and said treated includes releasing bound complex material by washing the solid support with a mild reducing agent effective to cleave disulfide bonds.

6. The method of claim 1, wherein the filament contains covalently bound biotin which is also bound to avidin, streptavidin, the reaction mixture is contacted with a solid support containing surface bound iminodiacetic acid acid moieties in the presence of Cu⁺⁺, and said treating includes washing the solid support with a chelating agent effective to remove Cu⁺⁺ from support.

7. A method of forming a stable, single-stranded nucleoprotein filament effective to complex stably and specifically with a target duplex DNA having a selected base sequence, said method comprising

providing a single-stranded DNA probe having a region of homology with such selected base sequence, and

reacting the DNA probe with RecA protein in the presence of adenosine 5'"'"'-(γ

-thio)triphosphate at a Mg⁺⁺ concentration of less than about 10-20 mM.

8. The method of claim 7, wherein the Mg⁺⁺ concentration is between about 0.5-2 mM, and the concentration of adenosine 5'"'"'-(γ

-thio)triphosphate is at least about 0.5 mM.

9. A system for separating target and non-target duplex DNA molecules on the basis of a selected base sequence which is unique to the target sequence, comprising

stable, single-stranded nucleoprotein filaments, each composed of a single-stranded DNA probe having a region of homology with the selected base sequence, said filaments having a biotin/avidin complex formed by binding avidin, streptavidin, or an analog thereof with biotin, said complex derivatized to the probe nucleotides through disulfide linkages, and said filaments having RecA protein molecules bound stably to the DNA probe, in the presence of adenosine 5'"'"'-(γ

-thio)triphosphate, to promote rapid homologous alignment between the probe and homologous target base sequences, with formation of a stable filament/target complex, and

a solid support, designed to selectively bind target molecules, with surface-bound binding molecules effective to selectively bind biotin/avidin complexes to the support when the filaments are complexed by such homologous alignment to such target sequences, wherein said binding molecules are iminodiacetic acid moieties which are effective to bind to such biotin/avidin complexes in the presence of Cu⁺⁺.

10. A system for separating target and non-target duplex DNA molecules on the basis of a selected base sequence which is unique to the target sequence, comprising

stable, single-stranded nucleoprotein filaments, each composed of a single-stranded DNA probe having a region of homology with the selected base sequence, said filaments having biotin derivatized to the probe nucleotides through disulfide linkages and said filaments having RecA protein molecules bound stably to the DNA probe, in the presence of adenosine 5'"'"'-(γ

-thio)triphosphate, to promote rapid homologous alignment between the probe and homologous target base sequences, with formation of a stable filament/target complex, and

a solid support, designed to selectively bind target molecules, with surface-bound binding molecules effective to selectively bind biotin to the support when the filaments are complexed by such homologous alignment to such target sequences, wherein said binding molecules are avidin, streptavidin, or analogues thereof.

11. A stable, single-stranded nucleoprotein filament effective to complex specifically and stably with a target duplex DNA having a selected base sequence, in the absence of single-strand binding protein, said filament comprising

a single-stranded DNA probe having a region of homology with such selected base sequence, wherein said probe is derivatized with one or more ligands effective to bind specifically and with high affinity to binding molecules carried on a solid support, when the filament is complexed with target duplex DNA, and

RecA protein molecules bound stably to the DNA probe in the presence of adenosine 5'"'"'-(γ

-thio)triphosphate.

12. The filament of claim 11, wherein the ratio of RecA molecules to nucleotide residues in the probe is between about 1:

3 and 1;

6.

13. The filament of claim 11, wherein the RecA protein is derived from E. coli.

14. The filament of claim 11, wherein the ligands are biotin molecules derivatized to probe nucleotides for use with (a) a solid support containing surface attached avidin, streptavidin, or analogues thereof, where the biotin is linked to the nucleotides through disulfide linkages, and (b) a solid support having surface-bound iminodiacetic acid moieties, where the biotin is also complexed with avidin, streptavidin, or an analog thereof.