WO2000020558A1 - PROCEDE RELATIF A L'ELABORATION D'UN COMPLEXE SONDE TRES PERFORMANT A L'ACIDE NUCLEIQUE MONOCATENAIRE/RECOMBINASE DE TYPE RecA, ET UTILISATION DE CE COMPLEXE - Google Patents
PROCEDE RELATIF A L'ELABORATION D'UN COMPLEXE SONDE TRES PERFORMANT A L'ACIDE NUCLEIQUE MONOCATENAIRE/RECOMBINASE DE TYPE RecA, ET UTILISATION DE CE COMPLEXE Download PDFInfo
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- WO2000020558A1 WO2000020558A1 PCT/JP1999/005420 JP9905420W WO0020558A1 WO 2000020558 A1 WO2000020558 A1 WO 2000020558A1 JP 9905420 W JP9905420 W JP 9905420W WO 0020558 A1 WO0020558 A1 WO 0020558A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- the present invention provides a method for preparing a RecA-like recombinant enzyme (recombinase) / single-stranded nucleic acid probe complex (single-stranded nucleoprotein filament), and a RecA-like recombinant enzyme / single-stranded nucleic acid prepared by the method. It relates to the use of the lip-and-mouth complex.
- recombinase single-stranded nucleic acid probe complex
- RecA-like recombinant enzyme single-stranded nucleic acid prepared by the method. It relates to the use of the lip-and-mouth complex.
- RecA protein a recombinant enzyme derived from Escherichia coli (Takehiko Shibata, Cell Engineering, No. 4, 281-292, 1990).
- RecA protein performs homologous recombination between homologous single-stranded DNA and double-stranded DNA in vitro, resulting in a homologously paired triple-stranded DNA structure and other triple-stranded conjugated DNA. It is known to make molecules (joint DNA molecule) (B. Rigas et al., Proc. Atl. Acad. Sci. USA, 9591-9595, 1986. P. Hsieh et al.
- a double-stranded target nucleic acid molecule having only a trace amount in a sample can be synthesized by using a recombinant enzyme typified by RecA protein.
- a recombinant enzyme typified by RecA protein.
- RecA protein a higher-performance RecA-like recombinant enzyme (recombinase) / single-stranded nucleic acid probe (double-stranded target) suitable for the purpose is used. It is necessary to develop a method for preparing a single-stranded nucleoprotein filament (including a homoprobe having a sequence sufficiently complementary to the nucleic acid sequence) to further improve the reaction efficiency, yield, and specificity. Disclosure of the invention
- the present invention relates to a RecA-like recombinase / single-stranded nucleic acid probe (high homology having a sequence sufficiently complementary to a double-stranded target nucleic acid sequence) showing high reactivity and specificity to a double-stranded nucleic acid in a sample. And a double-stranded target nucleic acid sequence of the RecA-like recombinant enzyme / single-stranded nucleic acid probe complex prepared by the method.
- the task is to provide applications for evening targeting, enrichment, detection and / or isolation.
- ATPy S and a single-stranded nucleonucleotide filament complex which can form a stable complex effectively and specifically with a double-stranded target nucleic acid having a complementary sequence.
- Radding et al. Have already developed a method for preparing single-stranded DNA by reaction with RecA protein in the presence of low-concentration Mg 2+ and in the absence of SSB (single-strand binding protein) ( U.S. Patent 4,888,274). They state that it is preferably performed in the presence of ATP yS at a concentration of 0.5-2 mM, and that a minimum of 0.5 mM is required (page 7 of the patent and claim 8).
- one molecule of the nucleotide cofactor such as ATP binds to one molecule of the RecA protein.
- ATP is used as a cofactor
- the ATPase activity of the RecA protein itself is degraded to ADP, and the RecA protein ZADP complex is dissociated from single-stranded DNA.
- a large excess of ATP or an ATP regeneration system phosphocreatine + creatine kinase
- the cofactor When the cofactor is used, it is not decomposed into ADP to become a RecA protein / ADP complex and dissociated from single-stranded nucleic acid, and the affinity for RecA protein is very strong.
- the present inventors thought that it was not necessary to add excessively. From such a viewpoint, the present inventors consider that the hardly decomposable nucleotide cofactor such as ATPa S is not an absolute concentration in the reaction solution, but a single chain present during the reaction. It is necessary to discuss whether the number of nucleotide residues constituting the nucleic acid probe and the number of molecules of RecA protein are optimal for the presence of the hardly degradable nucleotide cofactor of about ⁇ molecule. Was.
- the number of ATPS molecules is 10 to 20,000 times the number of nucleotide residues constituting the single-stranded DNA, and about 30 to 60,000 times the number of molecules of RecA protein. Equivalent to double.
- the total DNA probe + 2 It is necessary to optimize the amount of RecA depending on the amount of the main-chain DN A). ”(Pages 9 to 10 of the patent), and 1.6 mM ATP yS, 8finol (100 ⁇ 1).
- refractory nucleotide cofactors such as AT Py S (at least 10 times the number of nucleotide residues in the single-stranded probe and 30 times the number of molecules in the RecA protein).
- Double ATP STP is used.
- the present inventors have found that when using a hardly degradable nucleotide cofactor that is not easily degraded by the nucleoside triphosphatase activity such as ATPase possessed by the RecA-like recombinant enzyme itself such as ATPaS, It is necessary to add a large excess like a law However, if there is a large excess of insoluble nucleotide cofactors such as ATPaS, the reaction efficiency, yield and specificity of RecA protein single-stranded nucleic acid probe double-stranded target nucleic acid complex formation will be adversely affected.
- the number of molecules of the nucleotide residues constituting the single-stranded nucleic acid probe and the number of times the number of molecules of the RecA protein that are hardly degradable nucleotide cofactors present during the reaction are considered.
- the number of molecules of the hardly decomposable nucleotide cofactor is more than 1/4 of the number of molecules of nucleotide residues constituting the single-stranded nucleic acid probe.
- RecA-like recombinase / single-stranded nucleic acid probe (double-stranded target nucleic acid sequence) under a condition of 10 times or less, preferably 5 times or less, more preferably 3 times or less the number of molecules of the recombinant enzyme
- a single-stranded nucleoprotein filament is prepared, and the complex is contacted with a sample containing a double-stranded target nucleic acid to produce RecA-like recombination. It has been found that an enzyme / single-stranded nucleic acid probe can form a double-stranded target nucleic acid complex extremely efficiently and specifically.
- the present inventors use a prepared RecA-like recombinase / single-stranded nucleic acid probe complex to allow a monovalent cathon to coexist when performing a homologous recombination reaction on a double-stranded target nucleic acid.
- a monovalent cathon to coexist when performing a homologous recombination reaction on a double-stranded target nucleic acid.
- the present invention relates to a RecA-like recombinase / single-stranded nucleic acid probe exhibiting high reactivity and specificity for a double-stranded target nucleic acid in a sample (a homoprobe having a sequence sufficiently complementary to a double-stranded target nucleic acid sequence). And a method for preparing the complex, and targeting, enrichment, detection and / or detection of the double-stranded target nucleic acid sequence of the RecA-like recombinant enzyme / single-stranded nucleic acid probe complex prepared by the method. Or in terms of its use for isolation,
- ADP 'A 1 F 4 - ATP, aluminum nitrate, and mixtures of sodium fluoride, or ADP, I Ayakasan secondary aluminum ⁇ arm, and fluoride diisocyanato mixture Riumu
- d AD ⁇ - a 1 F 4 - d ATP, aluminum nitrate, and mixtures of full Uz sodium or dADP, a mixture of aluminum nitrate, and full Uz of Natoriumu
- ADP 'B e F 3 — ATP, a mixture of beryllium sulfate and sodium fluoride, or a mixture of ADP, beryllium sulfate, and sodium fluoride
- d ADP ⁇ BeF 3 ⁇ d ATP, beryllium sulfate , And a mixture of sodium fluoride, or a mixture of dADP, beryllium sulfate, and sodium fluoride).
- RecA-like recombinase / single-stranded nucleus according to any of (1) to (9) A kit containing a RecA-like recombinant enzyme and a hardly degradable nucleotide cofactor for use in preparing an acid probe complex;
- a method for obtaining, enriching, detecting, and Z or isolating a double-stranded target nucleic acid in a sample comprising:
- a “homologous probe” refers to a single-stranded nucleic acid probe having sufficient complementarity with a double-stranded target nucleic acid sequence
- a “heterologous probe” refers to a two-stranded nucleic acid probe.
- the term "probe j" simply refers to a homoprobe alone or a mixture of a homoprobe and a heteroprobe.
- the “refractory nucleotide cofactor” refers to a nucleotide cofactor that is not easily degraded by nucleoside triphosphatase activity such as ATPase possessed by RecA-like recombinase itself.
- the “double-stranded target nucleic acid” refers to a double-stranded nucleic acid targeted by a homoprobe.
- the term “double-stranded nucleic acid” includes both a double-stranded target nucleic acid and a double-stranded nucleic acid not targeted by a homoprobe.
- the recombinant enzyme (reconiMnase) that can be used in the present invention refers to an Escherichia coli RecA protein that can catalyze a homologous pairing reaction and a Z or DNA strand exchange reaction in vitro.
- Equivalent RecA-like recombination enzyme A group of proteins that are isolated and purified from many prokaryotes and eukaryotes.
- the wild-type E. coli RecA protein T. Shibata et al., Methods in Enzymology, 100, 197, 1983
- its mutants eg RecA 803: M. Madiraju et al., Pro Natl. Acad. Sci. USA, 85, 6592
- RecA 441 H. Kawashima et al., Mol. Gen. Genet. 193, 288, 1984, etc.
- uvsX protein derived from T4 phage T. Yonesaki et al., Eur. J.
- RecA protein from Bacillus subtilis CLovett et al., J. Biol. Chem. ⁇ 260 ⁇ 3305, 1985
- Reel protein from smut fungus Ustilago
- a RecA-like protein from a thermotolerant bacterium such as Thernms aquaticus or Thermus thermophilus
- R. Kato et al., J. Biochem., 114, 926, 1993 RecA-like proteins from yeast, mouse, and human (A. Shinohara et al., Nature Genetics, 239, 1993).
- the RecA protein of Escherichia coli was used after purification from Escherichia coli by a conventional method (eg, S. Kuramitsu et al., J. Biochem., 1033, 1981. T. Shibata et al., Methods in Enzymology, Sato, 197, 1983). obtain.
- a commercially available RecA protein can be used.
- the double-stranded target nucleic acid used in the present invention includes DNA, cDNA and RNA (DNA / UNA hybrid, RNA region having a double-stranded structure, etc.), and its length, type, shape, etc. are particularly limited. However, it can be either cyclic (closed or 'open) or linear.
- the double-stranded target nucleic acid is a double-stranded target DNA.
- Body including parasites present in cells, such as bacteria, viruses, or organs such as nuclear mitochondria, chromosomes, or biological samples such as blood samples such as viruses or bacteria
- It may be contained in unfixed living cells or cell structures.
- double-stranded target nucleic acid optionally conventional manner RI (32 P, 35 S, etc.), fluorescent dyes (FITC, rhodamine, etc.), enzymatic labels (Pas one Okishida Ichize, Al force Li phosphatase, etc.) And various labels or ligands such as chemiluminescent agents (eg, acridinium esters), biotin-digoxigenin, etc. for detection and / or isolation.
- RI 32 P, 35 S, etc.
- fluorescent dyes FITC, rhodamine, etc.
- enzymatic labels Pas one Okishida Ichize, Al force Li phosphatase, etc.
- various labels or ligands such as chemiluminescent agents (eg, acridinium esters), biotin-digoxigenin, etc. for detection and / or isolation.
- the single-stranded nucleic acid probe used in the present invention is a single-stranded nucleic acid, and usually a single-stranded DNA is used.
- the shape is not particularly limited, and it may be cyclic or linear.
- the single-stranded nucleic acid probe sample may be only a single-stranded nucleic acid probe (homoprobe) having sufficient complementarity with the target sequence, or a single-stranded nucleic acid probe (heteroprobe having insufficient complementarity with the target sequence). Mixed with It may be a compound. It is known that specificity is improved by mixing heteroprobes (PCT / JP97 / 03019). It is necessary to optimize the amount of the single-stranded nucleic acid probe to be used according to the total amount of the sample nucleic acid and the amount of the double-stranded target nucleic acid contained therein.
- the “single-stranded nucleic acid probe (homoprobe) having sufficient complementarity with the target sequence” used in the present invention has a nucleotide sequence that is at least 70% or more of a part or the entire nucleotide sequence of the target sequence. It is a single-stranded nucleic acid containing a sequence having homology therein, and is usually a single-stranded DNA containing the sequence. Usually, a homoprobe is used to ensure that a base sequence-specific homologous recombination reaction (hybridization reaction by homologous pairing) between the double-stranded target DNA and the homoprobe is performed. ! It is preferable that a sequence having at least 90% or more homology with a part or the entire nucleotide sequence of the NA sequence is contained therein, and a sequence having 95% or more homology is contained therein Is more preferred.
- the homoprobe strand may also contain a terminal extension sequence that is not complementary to any of the DNA strands in the sample. If both strands of the double-stranded probe contain such terminal extension sequence portions, these extensions may be complementary to each other.
- probes can be prepared directly from a plasmid or cosmid or other vector having the sequence by a method for preparing a protein known in the art.
- the probe can be prepared by cutting out the probe portion from the enzyme with a restriction enzyme, isolating a specific restriction enzyme fragment by electrophoresis, or amplifying only the probe portion by PCR.
- the probe obtained in this way is usually double-stranded, but if necessary, it can be denatured to single-stranded or subcloned into a single-stranded vector such as M13 phage vector for use. I can do it.
- Oligonucleotide Single-stranded probes can be prepared by the synthetic method. When preparing long probes, they can be prepared by synthesizing subfragments of the probe and then joining the subfragments together.
- the sequence homologous to the target sequence to be possessed by the homoprobe has a length of at least 15 bases or more, preferably 25 to 2,000 bases, and a longer (2,000 bases or more) polynucleotide probe is also used. Can be done.
- homopropene may be prepared by a conventional method using RI ( 32 PS, etc.), a fluorescent dye (FITC, rhodamine, etc.), an enzyme label (veroxidase, alkaline phosphatase, etc.), a chemiluminescent agent (acrylic acid, etc.).
- RI 32 PS, etc.
- FITC fluorescent dye
- rhodamine rhodamine
- an enzyme label veroxidase, alkaline phosphatase, etc.
- chemiluminescent agent acrylic acid, etc.
- Various labels or ligands such as ditin ester), biotin and digoxigenin can be labeled for detection and detection or isolation.
- the “heteroprobe” used in the present invention refers to a nucleic acid probe that does not have sufficient complementarity with a target sequence, and is usually a single-stranded DNA. It is preferable that the sequence has low complementarity with a sequence other than the target sequence, for example, a sequence of a part of a vector into which the target sequence is inserted.
- the shape is not particularly limited, and may be cyclic or linear. It can also be prepared by denaturing a tethered probe to a double-stranded nucleic acid.
- single-stranded nucleic acid probes preferably single-stranded phage DNA such as M13 or 0X174, etc.
- virus pacteriophage Single-stranded DNA fragment derived from lambda phage virus pacteriophage Single-stranded DNA fragment derived from lambda phage
- single-stranded nucleic acid probe derived from a non-human eukaryote preferably single-stranded DNA fragment derived from salmon sperm or dicin sperm
- a synthetic DNA mixture of an appropriate length having a random sequence can be used as a heteroprobe.
- Heteroprobes usually do not have any labels or ligands.
- the length of the probe is at least 15 bases or more, preferably 30 to 10,000 bases. And more preferably 60 to 7,000 bases, longer polynucleotide probes (10,000 bases or more) can also be used.
- the preferred weight ratio of the two is about 1: 1 to about 1: 500. It is necessary to optimize the total amount of the homoprobe and the heteroprobe used and the weight ratio between them according to the total amount of the sample nucleic acid and the amount of the double-stranded target nucleic acid contained therein.
- the persistent nucleotide cofactor one that may be used in the present invention AT P 7 S, AD P ⁇ A 1 F 4 "(ATP - aluminum nitrate ⁇ hydrofluoric Kana preparative Riumu mixture or AD P-nitrate Aruminiumu-fluoride Kanaboku Riumu mixture), d AD P ⁇ A 1 F 4 one (d AT P 'aluminum nitrate sodium fluoride mixtures or d AD P' aluminum nitrate ⁇ fluoride Natoriumu mixture), ADP 'B e F 3 - ( ⁇ ⁇ beryllium sulfate ⁇ sodium fluoride mixture or ADP ⁇ beryllium sulfate ⁇ sodium fluoride mixture or dADP ⁇ BeFr (d ATP 'beryllium sulfate' sodium fluoride mixture or dADP ⁇ beryllium sulfate 'sodium fluoride (LP Moreau et al., J.
- the number of molecules of the hardly degradable nucleotide cofactor is 1/4 or more of the number of molecules of nucleotide residues constituting the single-stranded nucleic acid probe, and is preferably 10 times or less of the number of molecules of RecA-like recombinant enzyme, Is used to target a double-stranded target nucleic acid in a sample by reacting a single-stranded nucleic acid probe sample containing a homoprobe with a RecA-like recombinase under conditions of 5 times or less, more preferably 3 times or less.
- a RecA-like recombinant enzyme (recombinase) / single-stranded nucleoprotein filament suitable for enrichment, detection and / or isolation is prepared.
- ATPa S 3 to 4 times the amount of ATP A S may be allowed to coexist. That is, the homoprobe alone or a mixture of the homoprobe and the heteroprobe is usually heat-denatured at about 95 to 10 (TC for about 5 minutes to prepare a single-stranded nucleic acid probe sample, and is prepared for about 20 seconds to 1 minute. After cooling on ice, it is used for the binding reaction with the RecA protein and, if necessary, centrifuged at 0 to 4. C for about 5 to 20 seconds before the binding reaction with the RecA protein.
- the probe thus obtained can be stored in a freezer at -20 ° C, but preferably, is immediately added to the single-stranded probe in ice water with the hard-to-degrade nucleotide cofactor in the above-mentioned quantitative ratio.
- RecA protein is mixed with a standard RecA coating reaction solution [can be prepared so that the final concentration of each component in the reaction solution is in the following range; 1 to 100 mM (preferably 10 to 35 mM) Tris-HCl or acetic acid Buffer solution (pH about 7.5), 0.5-12.5mM (preferably 0.5-2mM) Magnesium chloride or magnesium acetate, O to 50m Sodium chloride or potassium chloride, or O to 100mM sodium acetate or acetate, 0 to ImM dithiothreitol, 0 to 100 1 EGTA, 0 to 50mM spermidine, 0 to 10% glycerol]
- the total amount of the mixed solution is preferably 100 ⁇ 1 or less, more preferably
- RecA protein is bound to the single-stranded nucleic acid probe sample to form a RecA 'protein / single-stranded nucleic acid probe complex (single-stranded nucleic acid protein complex).
- the above-mentioned coating reaction solution contains RecA protein in a ratio of at least one molecule to four nucleotide residues constituting at least a single-stranded nucleic acid probe, and preferably one molecule to three nucleotide residues. It must be added, and the total amount of RecA protein needs to be optimized according to the total amount of the nucleic acid sample containing the double-stranded target nucleic acid as well as the amount of the single-stranded nucleic acid probe used.
- SSB single-strand binding protein
- topoisomerase I or toboisomerase II.
- a single-stranded nucleic acid probe When mixing Tide cofactor-1 and RecA protein with a standard RecA coating reaction solution, simultaneously add the sample nucleic acid containing the double-stranded target nucleic acid, and mix the RecA protein / single-stranded nucleic acid probe complex.
- the formation and the homologous recombination reaction (homologous pairing) between the complex and the double-stranded target nucleic acid can be performed simultaneously. However, in this case, it is preferable to keep the concentration of 4 mM or more, or to make spermidine coexist.
- the homoprobe can also be used by preparing a labeled or liganded RecA protein homoprobe complex by binding to a RecA protein having various labels or ligands as described in W095 / 18236. . It is not preferable to prepare and use a complex with a RecA protein having such various labels or glands for a heterogeneous probe.
- the RecA protein Z single-stranded nucleic acid probe complex prepared according to the present invention as described above is subjected to the conditions under which the double-stranded target nucleic acid is not denatured, for example, under the temperature at which the double-stranded nucleic acid is denatured.
- the double-stranded target nucleic acid is added to the sample nucleic acid and reacted at 37 ° C. for 5 minutes to 24 hours, preferably 10 minutes to 2 hours under conditions suitable for homologous recombination (homologous pairing).
- a complex with RecA protein / single-stranded nucleic acid probe complex RecA protein / single-stranded nucleic acid probe / single-stranded target nucleic acid complex.
- the reaction conditions suitable for this homologous recombination reaction are almost the same as the conditions for the above RecA coating reaction, and can be carried out in the following reaction solution. That is, the components are prepared so that the final concentration of each component in the reaction solution is in the following range.
- a monovalent cation for example, sodium chloride of 150 rnM or less
- potassium chloride 250 mM or less sodium acetate or acetic acid coexistence allows the
- the type and concentration of the salt to be added depends on the type, shape (closed, open, linear) of the double-stranded target nucleic acid, and the length of the sequence homologous to the target sequence of the homoprobe. Needs optimization. For example, when the double-stranded target nucleic acid is a closed circular double-stranded DNA, the reaction is carried out by coexisting 25 to 150 mM sodium chloride or potassium chloride, or 50 to 250 mM sodium acetate or acetate stream. Not only the fidelity and specificity, but also the reaction efficiency and yield can be significantly improved.
- Complex formed with a double-stranded target nucleic acid (RecA protein / homoprobe having various knowledge or ligands / double-stranded target nucleic acid complex, or various labels or ligands
- Complex formed between a RecA protein / homoprobe / double-stranded target nucleic acid complex) and a double-stranded target nucleic acid having various labels or ligands (RecA The detection or isolation of protein Z homoprobe / double-stranded target nucleic acid complex having various labels or ligands) can be performed by known methods (US Patent No.
- Preferred methods for enriching, detecting and isolating or isolating the double-stranded target DNA include, for example, mixing a homoprobe labeled with bitin (biotin-labeled homoprobe) and an unlabeled heteroprobe at an appropriate ratio.
- a homoprobe labeled with bitin biotin-labeled homoprobe
- an unlabeled heteroprobe at an appropriate ratio.
- the single-stranded DNA probe sample at least one molecule of RecA protein for at least 4 nucleotide residues of the single-stranded DNA probe, and the nucleotide residues constituting at least the single-stranded DNA probe.
- RecA protein such as ATPy S Is reacted at 37 ° C in the presence of 0.5 to 2 mM Mg 2+ at pH 7.5 to prepare a RecA protein / single-stranded nucleic acid probe complex, and the complex is double-stranded.
- the formed biotin-labeled homoproveno double-stranded target DNA complex is combined with streptavidin-conjugated magnet beads (Dinal After washing the double-stranded nucleic acid and probe that were not captured, the biotin-labeled homoprobe / double-stranded target DNA complex bound to the beads contains sodium chloride.
- a method of releasing (eluting) the fraction containing the double-stranded target DNA by treating in a solution at room temperature to 85 ° C. for about 5 to 15 minutes. Further, the fraction containing the double-stranded target DNA thus recovered is inserted into an appropriate vector, and then transformed into an appropriate host cell. It is also possible to select cells and recover the target DNA from the transformed cells.
- the double-stranded target DNA is originally inserted into a transformable vector, it is not necessary to newly insert it into the vector, and the DNA can be used as it is for the transformation.
- a target gene is concentrated and / or isolated from a mixture of cDNAs and cDNAs.
- cDNA libraries or dienomic DNA libraries such as cosmid, Pl, BAC and YAC
- the RecA-like recombinant enzyme Z single-stranded nucleic acid loop complex prepared according to the present invention is an in situ hybridizer utilizing the RecA protein described in W093 / 05177 or W095 / 18236. It can also be used for gene therapy and transgenic methods by modifying genes or inhibiting transcription by in vivo gene targeting in living cells.
- the present invention also provides a kit for preparing a RecA-like recombinant enzyme Z single-stranded nucleic acid probe complex according to the present invention.
- This kit contains an appropriate amount of RecA-like recombinase and a refractory nucleotide cofactor. Further, for example, it may include a heteroprobe.
- the present invention further provides targeting, enrichment, detection and / or isolation of a double-stranded target nucleic acid in a sample nucleic acid comprising a RecA-like recombinant enzyme / single-stranded nucleic acid probe complex prepared according to the present invention.
- a kit to do this contains other components other than the single-stranded nucleic acid probe complex of RecA-like recombinant enzyme, such as a reaction stop solution, a method for washing unreacted double-stranded nucleic acid and single-stranded nucleic acid probe.
- a washing solution for example, a solid phase (for example, magnetic beads or the like) for capturing a complex of the double-stranded target nucleic acid and a homoprobe having a label or a ligand, and Z or an eluate may be included.
- a solid phase for example, magnetic beads or the like
- Z or an eluate may be included.
- Plasmid php53B full-length 6.6kb containing A sequence (R.Zakut-Houri et al., EMB0J -,, 1251, 1985 ), P UC18 is plasmid vector as double stranded non-target DN A annular the (2.7 kb)
- Each plasmid was purified from Escherichia coli containing these plasmids using the QIAGEN Plasmid Maxi Kit (QIAGEN GmbH).
- Salmon sperm-derived DNA (Sigma, Typelll) was fragmented by a conventional method (T. Maniatis et al., Molecular Cloning) and then heat-denatured to form a single-stranded DNA. .
- Example 1 A mixture of the double-stranded homoprobe with a biotinylated end and the hair probe prepared in Example 1 (3) was mixed with sterile water or TE buffer (10 mM Tris-HCl, lmM EDTA, pH 7.5). , And heat-treated in boiling water for 5 minutes in a microcentrifuge tube to denature the double-stranded probe.
- 1.0 / 1 10X coating buffer [300 mM _ Tris-HCl (pH 7.5 at 37 ° C), 20 mM MgCl 2N 1 ⁇ 2M DTT, 30% glycerol], 1.0 Add 1 ATPa S (Sigma) solution (prepared to various concentrations) and RecA protein (Boehringer-Mannheim), dilute with sterile water so that the total volume becomes 101, and 37 ° C For 12 minutes to prepare Rec-protein Z single-stranded DNA probe complex (single-stranded nucleoprotein filajnent) under various conditions.
- Single-stranded probe (homoprobe + heteroprobe) concentration (molar concentration of nucleotide residues constituting the probe.
- L M 350 g / l)
- ATPyS concentration ATPyS concentration
- RecA protein concentration ATPaS / nucleotide residue
- ATPA SZRecA protein molar ratio
- Example 1 1,01 in 10X reaction buffer [300mM Tris-HC1 (pH 7.5 at 37 ° C), 20mM MgCl 2 , 1 ⁇ 2M DTT, 30% glycerol], 1.0 ⁇ 1 in 80mM MgCl 2 solution, in Example 1 (1)
- the prepared double-stranded circular target DNA, php53B is 49 pg
- the double-stranded circular non-target DNA, pUC18 is l ⁇ g (molar ratio of target DNA to non-target DNA is 1: 50,000), and shown in Table 2.
- Transfer magnetic beads (manufactured by DYNAL) 201 coated with streptavidin into a microcentrifuge tube with a volume of 0.6 ⁇ 1 and use a magnetic bead separation rack (MAGNA-SEP) to make 100 ⁇ 1 30m Tris-HCK.
- the plate was washed twice with 50 mM NaCl (pH 7.5). After removing the washing solution, all of the above reaction solution (including the reaction stop solution) was added to the microcentrifuge tube containing the washed magnetic beads, mixed well, and left at room temperature for 15 minutes to capture. During this time, the mixture was stirred every 2-3 minutes.
- a 100-mL combi-tent cell prepared from E. coli JM109 according to the method of Nojima et al. (H. Inoue et al., Gene, 96, 23, 1990) was placed in a 1.5 ml microcentrifuge centrifuge tube, and collected in B. After adding 10 1 and mixing, the mixture was kept on ice for 30 minutes. Next, after keeping the temperature at 42 ° C for 30 seconds, it was returned to ice and cooled for 1-2 minutes.
- Table 2 shows the transformation results obtained under various reaction conditions.
- RecA protein prepared in the presence of 10 times or more of the number of molecules of the RecA protein or 30 times or more
- the yield and specificity of the double-stranded target DN DN were much higher than those obtained using the protein / single-stranded DNA probe complex ( ⁇ ⁇ 6-8, 18-20).
- the number of molecules of ATPas was reduced to 1/5 of the number of nucleotide residues constituting the single-stranded DNA probe (Nos. 9 and 10), the yield and specificity were reduced.
- the prepared RecA protein / single-stranded DNA probe complex is most suitable for isolation of double-stranded target DNA, and when the complex is used to isolate double-stranded target DNA, It has been found that very high yields and specificities can be obtained.
- the concentration of sodium chloride or chloride added during the homologous recombination reaction be 150 ⁇ or less. found.
- the concentration of sodium acetate and acetate stream was preferably 250 mM or less.
- the double-stranded target nucleic acid is a closed-chain double-stranded DNA
- sodium chloride or potassium chloride of about 25 to 150 mM, or sodium acetate or potassium acetate of about 50 to 250 mM is used.
- the homologous recombination reaction in the coexistence, it can be said that not only the accuracy (fidelity) and specificity of isolation of the double-stranded target DNA, but also the reaction efficiency and yield have been remarkably improved.
- RecA-like recombinase / single-stranded nucleic acid probe exhibiting high reactivity and specificity to a double-stranded target nucleic acid in a sample (having a sequence complementary to the double-stranded target nucleic acid sequence in seven minutes) (Including a homoprobe).
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Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002344468A CA2344468A1 (en) | 1998-10-01 | 1999-10-01 | Method for preparing high performance reca-like recombinase/single-stranded nucleic acid probe complex and utilization thereof |
AU60010/99A AU760583B2 (en) | 1998-10-01 | 1999-10-01 | Method for preparing high performance RecA-like recombinase/single-stranded nucleic acid probe complex and utilization thereof |
EP99970116A EP1118658A4 (en) | 1998-10-01 | 1999-10-01 | PROCESS FOR THE PREPARATION OF A HIGH-PERFORMANCE PROBE COMPLEX WITH SINGLE-STRING NUCLEIC ACID / RECOMBINASE OF RECA TYPE, AND USE OF THIS COMPLEX |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10/280380 | 1998-10-01 | ||
JP10280380A JP3065035B2 (ja) | 1998-10-01 | 1998-10-01 | 高性能なRecA様組換え酵素/1本鎖核酸プローブ複合体の調製方法及びその利用 |
Publications (1)
Publication Number | Publication Date |
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WO2000020558A1 true WO2000020558A1 (fr) | 2000-04-13 |
Family
ID=17624222
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/JP1999/005420 WO2000020558A1 (fr) | 1998-10-01 | 1999-10-01 | PROCEDE RELATIF A L'ELABORATION D'UN COMPLEXE SONDE TRES PERFORMANT A L'ACIDE NUCLEIQUE MONOCATENAIRE/RECOMBINASE DE TYPE RecA, ET UTILISATION DE CE COMPLEXE |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1118658A4 (ja) |
JP (1) | JP3065035B2 (ja) |
AU (1) | AU760583B2 (ja) |
CA (1) | CA2344468A1 (ja) |
WO (1) | WO2000020558A1 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CA2831140C (en) * | 2011-04-07 | 2017-11-07 | Alere San Diego, Inc. | Monitoring recombinase polymerase amplification mixtures |
CN110484600A (zh) * | 2019-09-06 | 2019-11-22 | 深圳市芯思微生物科技有限公司 | 基于RecA核蛋白丝的恒温核酸杂交液、杂交探针及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4888274A (en) * | 1985-09-18 | 1989-12-19 | Yale University | RecA nucleoprotein filament and methods |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0687738A4 (en) * | 1993-12-28 | 1999-05-19 | Daikin Ind Ltd | IN SITU HYBRIDIZATION METHOD USING RECA PROTEIN, WHICH HAS A MARKER OR LIGAND FOR USE IN THIS METHOD |
CA2263815A1 (en) * | 1996-08-29 | 1998-03-05 | Koji Kigawa | Methods for targeting, enriching, detecting and/or isolating target nucleic acid sequence using reca-like recombinase |
-
1998
- 1998-10-01 JP JP10280380A patent/JP3065035B2/ja not_active Expired - Fee Related
-
1999
- 1999-10-01 CA CA002344468A patent/CA2344468A1/en not_active Abandoned
- 1999-10-01 AU AU60010/99A patent/AU760583B2/en not_active Ceased
- 1999-10-01 WO PCT/JP1999/005420 patent/WO2000020558A1/ja not_active Application Discontinuation
- 1999-10-01 EP EP99970116A patent/EP1118658A4/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4888274A (en) * | 1985-09-18 | 1989-12-19 | Yale University | RecA nucleoprotein filament and methods |
Non-Patent Citations (3)
Title |
---|
MARTIN TEINTZE ET AL.: "RecA-assisted rapid enrichment of specific clones from model DNA libraries", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 211, no. 3, 26 June 1995 (1995-06-26), pages 804 - 811, XP002925238 * |
See also references of EP1118658A4 * |
STEPHEN C. KOWALCZYKOWSKI ET AL.: "DNA-strand exchange promot- ed by RecA protein in the absence of ATP: Implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions", PROC. NATL. ACAD. SCI. USA, vol. 92, no. 8, April 1995 (1995-04-01), pages 3478 - 3482, XP002925239 * |
Also Published As
Publication number | Publication date |
---|---|
EP1118658A1 (en) | 2001-07-25 |
CA2344468A1 (en) | 2000-04-13 |
JP3065035B2 (ja) | 2000-07-12 |
AU760583B2 (en) | 2003-05-15 |
JP2000106899A (ja) | 2000-04-18 |
AU6001099A (en) | 2000-04-26 |
EP1118658A4 (en) | 2004-03-10 |
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