WO1994003639A1 - NON-ISOTOPIC DETECTION OF NUCLEIC ACID SEQUENCES USING AN RecA LABEL - Google Patents

NON-ISOTOPIC DETECTION OF NUCLEIC ACID SEQUENCES USING AN RecA LABEL Download PDF

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Publication number
WO1994003639A1
WO1994003639A1 PCT/US1993/007150 US9307150W WO9403639A1 WO 1994003639 A1 WO1994003639 A1 WO 1994003639A1 US 9307150 W US9307150 W US 9307150W WO 9403639 A1 WO9403639 A1 WO 9403639A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
reca
label
reca protein
probe
Prior art date
Application number
PCT/US1993/007150
Other languages
French (fr)
Inventor
Parke K. Flick
Original Assignee
United States Biochemical Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by United States Biochemical Corporation filed Critical United States Biochemical Corporation
Priority to AU47928/93A priority Critical patent/AU4792893A/en
Publication of WO1994003639A1 publication Critical patent/WO1994003639A1/en

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Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays

Definitions

  • This invention features use of RecA-coated single- stranded DNA probes for non-isotopic detection of target sequences of single-stranded DNA or RNA immobilized on a solid support.
  • Applicant describes the use of RecA coated single-stranded DNA which can specifically hybridize to single-stranded target sequences, and the use of the RecA as a label to allow specific detection of hybrids formed between the single-stranded DNA probe and the target nucleic acid.
  • the invention features a non- isotopic method for detection of a single-stranded nucleic acid by contacting a solid phase bonded with the nucleic acid with a single-stranded DNA probe bonded with a RecA protein, and detecting the presence of RecA protein bound with the probe-single-stranded nucleic acid hybrid complex.
  • the RecA protein is bonded to the single-stranded DNA probe in the presence of magnesium ions and ATP ⁇ s, and the RecA protein is covalently bonded with an enzyme label, or some other readily detectable label which can be bonded with the RecA protein.
  • the figure is a diagrammatic representation, of a method of the invention.
  • the present invention relates to the use of RecA-coated single-stranded DNA probes for the non- isotopic detection of target sequences of DNA or RNA immobilized on a solid support.
  • a probe DNA molecule for the target sequence of interest using standard methods.
  • This probe is then reacted with either RecA protein in the presence of 1-2 mM ATP ⁇ s or with an enzyme conjugate of RecA protein, such enzymes preferably being alkaline phosphatase or horseradish peroxidase.
  • conjugates may be prepared using standard methods in which both proteins are activated with appropriate reagents and coupled under controlled conditions such that the activities of both are retained.
  • an enzyme e.g.. alkaline phosphatase, or its equivalent by appropriate manipulation of coding sequences for the genes of the two proteins.
  • This protein could then be reacted with the single-stranded DNA in the presence of ATP ⁇ s to generate the coated probe.
  • the probe coated with RecA protein or the RecA- conjugate is allowed to react, in the presence of an appropriate buffer containing Mg++ and ATP ⁇ s, with denatured DNA or RNA immobilized on a solid support, preferably a nylon or nitrocellulose membrane.
  • the DNA or RNA contains the target seguence of interest along with other unrelated sequences.
  • the probe will bind to the target sequence by complementary base pairing, and excess probe is washed off the membrane.
  • the presence of the bound probe-RecA-target complex may be detected by reacting it with a labeled or unlabeled antibody against RecA, e.g. , an antibody conjugated to alkaline phosphatase or horseradish peroxidase, and then adding a substrate for alkaline phosphatase or horseradish peroxidase to generate either a color signal, or a chemiluminescent signal detectable with standard X-ray film as a dark band. If a conjugate of RecA protein or a RecA-enzyme fusion protein is used, one need only add a substrate for the enzyme and detect the color or chemiluminescent signal by standard methods, thereby avoiding the antibody step.

Abstract

Method to detect single-stranded nucleic acid by contacting a solid phase bonded with the nucleic acid with a single-stranded DNA probe including RecA protein, and detecting the presence of RecA protein bound with the probe-single-stranded nucleic acid hybrid complex.

Description

DESCRIPTION
Non-Isotopic Detection of Nucleic Acid Sequences
Using a RecA Label
Background of the Invention
Weinstock et al. , 76 Proc. Natl. Acad. Sci. USA 126,
1979, cEntee, 24 Biochemistry 4345, 1985, and Bryant and
Lehman, 82 Proc. Natl. Acad. Sci. USA 297, 1985, describe the mechanism of renaturation of complimentary DNA strands by the RecA protein of Escherichia coli .
Honigberg et al., 83 Proc. Natl. Acad. Sci. USA 9586, 1986, describe the ability of RecA protein to promote formation of base pairing between single-stranded DNA and duplex DNA. Radding et al. , U.S. Patent No. 4,888,27-4 and PCT Application WO 87/01730 described methods for use of this phenomenon for isolating specific duplex nucleic acids.
Summary of the Invention This invention features use of RecA-coated single- stranded DNA probes for non-isotopic detection of target sequences of single-stranded DNA or RNA immobilized on a solid support. Applicant describes the use of RecA coated single-stranded DNA which can specifically hybridize to single-stranded target sequences, and the use of the RecA as a label to allow specific detection of hybrids formed between the single-stranded DNA probe and the target nucleic acid.
Thus, in a first aspect the invention features a non- isotopic method for detection of a single-stranded nucleic acid by contacting a solid phase bonded with the nucleic acid with a single-stranded DNA probe bonded with a RecA protein, and detecting the presence of RecA protein bound with the probe-single-stranded nucleic acid hybrid complex. In preferred embodiments, the RecA protein is bonded to the single-stranded DNA probe in the presence of magnesium ions and ATPγs, and the RecA protein is covalently bonded with an enzyme label, or some other readily detectable label which can be bonded with the RecA protein.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments
The drawing will first briefly be described.
Drawing
The figure is a diagrammatic representation, of a method of the invention. Specifically, the present invention relates to the use of RecA-coated single-stranded DNA probes for the non- isotopic detection of target sequences of DNA or RNA immobilized on a solid support. To practice the inven¬ tion, one first obtains a probe DNA molecule for the target sequence of interest using standard methods. This probe is then reacted with either RecA protein in the presence of 1-2 mM ATPγs or with an enzyme conjugate of RecA protein, such enzymes preferably being alkaline phosphatase or horseradish peroxidase. These conjugates may be prepared using standard methods in which both proteins are activated with appropriate reagents and coupled under controlled conditions such that the activities of both are retained. Alternatively, one can isolate a fusion protein of RecA with an enzyme, e.g.. alkaline phosphatase, or its equivalent by appropriate manipulation of coding sequences for the genes of the two proteins. This protein could then be reacted with the single-stranded DNA in the presence of ATPγs to generate the coated probe. The probe coated with RecA protein or the RecA- conjugate is allowed to react, in the presence of an appropriate buffer containing Mg++ and ATPγs, with denatured DNA or RNA immobilized on a solid support, preferably a nylon or nitrocellulose membrane. The DNA or RNA contains the target seguence of interest along with other unrelated sequences.
The probe will bind to the target sequence by complementary base pairing, and excess probe is washed off the membrane. The presence of the bound probe-RecA-target complex may be detected by reacting it with a labeled or unlabeled antibody against RecA, e.g. , an antibody conjugated to alkaline phosphatase or horseradish peroxidase, and then adding a substrate for alkaline phosphatase or horseradish peroxidase to generate either a color signal, or a chemiluminescent signal detectable with standard X-ray film as a dark band. If a conjugate of RecA protein or a RecA-enzyme fusion protein is used, one need only add a substrate for the enzyme and detect the color or chemiluminescent signal by standard methods, thereby avoiding the antibody step.
Other embodiments are within the following claims.

Claims

Claims
1. A method for non-isotopic detection of a single- stranded nucleic acid, comprising contacting a solid phase bonded with said nucleic acid with a single-stranded DNA probe comprising RecA protein, and detecting the presence of RecA protein bound to said single-stranded nucleic acid.
2. The method of claim 1, wherein said contacting is in the presence of magnesium ions and ATPγs.
3. The method of claim 1, wherein said RecA protein is covalently bound to an enzyme.
PCT/US1993/007150 1992-08-04 1993-07-29 NON-ISOTOPIC DETECTION OF NUCLEIC ACID SEQUENCES USING AN RecA LABEL WO1994003639A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47928/93A AU4792893A (en) 1992-08-04 1993-07-29 Non-isotopic detection of nucleic acid sequences using an reca label

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US92484992A 1992-08-04 1992-08-04
US07/924,849 1992-08-04

Publications (1)

Publication Number Publication Date
WO1994003639A1 true WO1994003639A1 (en) 1994-02-17

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PCT/US1993/007150 WO1994003639A1 (en) 1992-08-04 1993-07-29 NON-ISOTOPIC DETECTION OF NUCLEIC ACID SEQUENCES USING AN RecA LABEL

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AU (1) AU4792893A (en)
WO (1) WO1994003639A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965361A (en) * 1993-12-28 1999-10-12 Daikin Industries, Ltd. In-situ hybridization method using RecA protein and RecA protein having marker or ligand for use in said method
US7229767B2 (en) 2001-03-27 2007-06-12 University Of Delaware Genomics applications for modified OLIGO nucleotides
US7468244B2 (en) 2001-09-27 2008-12-23 University Of Delaware Polymorphism detection and separation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985005685A1 (en) * 1984-06-01 1985-12-19 National Biomedical Research Foundation Catalyzed nucleic acid hybridization using enzymatic reagent
US4888274A (en) * 1985-09-18 1989-12-19 Yale University RecA nucleoprotein filament and methods
US5011770A (en) * 1987-09-04 1991-04-30 Molecular Devices, Inc. DNA detection method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1985005685A1 (en) * 1984-06-01 1985-12-19 National Biomedical Research Foundation Catalyzed nucleic acid hybridization using enzymatic reagent
US4888274A (en) * 1985-09-18 1989-12-19 Yale University RecA nucleoprotein filament and methods
US5011770A (en) * 1987-09-04 1991-04-30 Molecular Devices, Inc. DNA detection method

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965361A (en) * 1993-12-28 1999-10-12 Daikin Industries, Ltd. In-situ hybridization method using RecA protein and RecA protein having marker or ligand for use in said method
US7229767B2 (en) 2001-03-27 2007-06-12 University Of Delaware Genomics applications for modified OLIGO nucleotides
US7468244B2 (en) 2001-09-27 2008-12-23 University Of Delaware Polymorphism detection and separation

Also Published As

Publication number Publication date
AU4792893A (en) 1994-03-03

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