US4888274A — RecA nucleoprotein filament and methods

Claim 1

Independent Claim 151 words

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.

Claim 2

Independent Claim 151 words

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.

Claim 3

Dependent Claim 64 words

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.

Claim 4

Dependent Claim 75 words

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.

Claim 5

Dependent Claim 63 words

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.

Claim 6

Dependent Claim 61 words

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.

Claim 7

Dependent Claim 61 words

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.

Claim 8

Independent Claim 68 words

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.

Claim 9

Independent Claim 68 words

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.

Claim 10

Dependent Claim 27 words

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.

Claim 11

Independent Claim 173 words

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 ++ .

Claim 12

Independent Claim 173 words

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 ++ .

Claim 13

Independent Claim 143 words

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.

Claim 14

Independent Claim 143 words

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.

Claim 15

Dependent Claim 96 words

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.

Claim 16

Dependent Claim 96 words

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.

Claim 17

Dependent Claim 23 words

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.

Claim 18

Dependent Claim 14 words

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

Claim 19

Dependent Claim 64 words

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.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1992-12-18	1993-06-19	1993-12-19	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1996-12-18	1997-06-19	1997-12-19	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2000-12-18	2001-06-19	2001-12-19	$7700.00	Window Closed / Lapsed

RecA nucleoprotein filament and methods

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