WO2006080136A1 - Procede d'extraction, de purification et de clonage rapides d'un gene cible a l'aide d'adn bicatenaire circulaire ferme - Google Patents

Procede d'extraction, de purification et de clonage rapides d'un gene cible a l'aide d'adn bicatenaire circulaire ferme Download PDF

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WO2006080136A1
WO2006080136A1 PCT/JP2005/021603 JP2005021603W WO2006080136A1 WO 2006080136 A1 WO2006080136 A1 WO 2006080136A1 JP 2005021603 W JP2005021603 W JP 2005021603W WO 2006080136 A1 WO2006080136 A1 WO 2006080136A1
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dna
probe
sequence
stranded
target gene
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PCT/JP2005/021603
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Japanese (ja)
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Taira Enomoto
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Gene & Gene Technology Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the present invention relates to a method mainly for rapid extraction, separation and purification, detection, cloning, etc. of a target gene from a genomic gene library or cDNA library.
  • the hybridization method of the probe DNA bound to the solid particle and the target gene (Patent Document 1) or the probe bound to the solid particle
  • a method of extracting the target gene into a solid layer using a bridge nucleotide having a sequence complementary to both the DNA and the target gene (Patent Document 2), linking the biotin to one end of the oligonucleotide complementary to the target gene, and A method (Non-patent Document 1) for extracting a target gene from avidin-binding particles using a specific binding reaction between avidin and avidin has been developed and used.
  • Patent Document 1 Japanese Patent Laid-Open No. 8-89294
  • Patent Document 2 International Publication WO2004-101785
  • Patent Document 3 US Patent No. 5500356
  • Non-patent document 1 “Biomagnetic Techniques in Molecular Biology”, Dynal, 1995
  • oligonucleotide probe method bound to a solid layer particle "avidin 'biotin probe method” and "bridge nucleotide probe method”
  • shape of the probe for extracting the target gene is specified.
  • the shape of the DNA to be extracted is not specified.
  • DNA libraries such as the extracted plasmid (plasmid) cDNA library, plasmid genome DNA, and cosmid (cosmid) genomic library are characterized by the nature of their DNA: 1) super coiled DNA, It exists in the form of 2) open circle DNA and 3) linear DNA.
  • the target gene is isolated and extracted using DNA that is a mixture of these three DNA shapes without specifying the physical and physical shape of the target DNA, the properties of the DNA hybridization In the above three DNA forms, almost only 2) and 3) open circular DNA, linear single-stranded DNA derived from linear DNA or single-stranded circular DNA is separated and extracted.
  • the linear single-stranded DNA or single-stranded circular DNA isolated and extracted in this way is suitable for verifying the presence of the target gene. It is impossible or much more labor and time to introduce into a cell and clone the gene of interest from here.
  • Patent Document 3 the “gene cloning method based on double-stranded circular synthesis from single-stranded circular DNA” (Patent Document 3) requires a complicated in vitro enzyme synthesis process after extraction, which is complicated, inaccurate, and expensive. Cost is also a problem.
  • the base point is 0 to 7 bases upstream in the upstream region adjacent to the base sequence in the target gene corresponding to the base sequence complementary to the target gene in the probe DNA-binding particle or bridge nucleotide. 50-base single-stranded DNA (upstream blocking sequence; upstream
  • Blocking sequence, Up-BS and 20-50 base sequence single-stranded DNA (downstream blocking sequence, Down-BS) starting from 0-7 base downstream downstream Hybridization force between the denatured DNA sample containing the target gene and the probe DNA is hindered by the rehybridization of the DNA in the sample, resulting in a drastic decrease in the recovery efficiency of the target gene DNA.
  • clover DNA rapidly undergoes self-pairing (association) and closes again in a closed circle before hybridization with the probe DNA, and cannot be captured by the probe DNA particle, resulting in the target gene cloning. Yield reduction ⁇ There is a problem that causes a drastic decrease.
  • the present invention relates to DNA cleavage (nick) of any of the samples containing the gene of interest in the cDNA plasmid 'library or the genomic' plasmid 'library or the cosmid' library in the three existing forms. Rapid extraction and purification of target genes using completely closed double-stranded DNA without a site as a sample target.
  • the target gene DNA sequence is obtained by hybridizing the closed circular double-stranded DNA to the probe DNA complex particle containing a DNA sequence (probe DNA sequence) complementary to at least a part of the base sequence and a solid particle.
  • the closed circular double-stranded DNA in which DNA is incorporated is collected into solid-layer particles in a form that can be directly transferred to the host cell, eluted with an elution solution, and introduced into a host cell such as Escherichia coli (transformed).
  • a host cell such as Escherichia coli
  • the present invention is a novel cycle of 5 cycles or more in the range of 50 ° C to 70 ° C.
  • Single-stranded bridge nucleotide probe for probes prepared by the hybridization method DNA binding particle Single-stranded DNA sequence that binds to the probe Single-stranded DNA sequence that includes the binding sequence Complementary probe DNA binding particle force Complementary DNA sequence that is complementary to at least a part of the base sequence of the target gene ( Probe DNA sequence) and a single-stranded bridge nucleotide for probes comprising a single-stranded bridge nucleotide complementary binding sequence for probes of the probe DNA-binding particle,
  • the bridge nucleotide complementary binding sequence has a complementarity of 80% or more and is firmly complementary, or a single-stranded chain bound to a solid particle.
  • Probe DNA binding particle force consisting of partial column force Complementary sequence (Tm value A) complementary to at least part of the base sequence of the target gene and complementary to at least part of single-stranded DNA sequence part of probe DNA binding particle A sequence (Tm value B), and the difference between the two Tm values is at least 8 ° C or more (Tm value B—Tm value A> 8 ° C).
  • One or a single-stranded bridge nucleotide probe for probe DNA sharing particle consisting of a single-stranded DNA sequence part of the probe DNA-binding particle, or at least a part of the base sequence of the target gene
  • a nucleotide containing a complementary DNA sequence is formed by direct covalent bonding, and as a result, a probe single-stranded bridge nucleotide is directly bonded to a solid particle. Used as composite particles By the method to solve the above problems.
  • the present invention also provides, among the single-stranded bridged nucleotide probe DNA-binding particles for probes, a sequence complementary to a part of the base sequence of the target gene (Tm value A) and one of the probe DNA-binding particles.
  • Tm value A a sequence complementary to a part of the base sequence of the target gene
  • Tm value B a sequence complementary to at least part of the base sequence of the double-stranded DNA sequence
  • the present invention also provides the single-stranded DNA of the probe DNA-binding particle having a partial force of the single-stranded DNA sequence bound to the solid layer particle, among the single-stranded bridged nucleotide probe DNA covalent bond particles for the probe.
  • a DNA synthesizing enzyme a nucleotide containing a sequence (probe DNA sequence) complementary to at least a part of the base sequence of the target gene of the single-stranded bridge nucleotide is bound to the probe DNA.
  • the present invention relates to a 20-50 base sequence single-stranded DNA based on 0 to 7 bases upstream of the upstream region adjacent to the base sequence of the target gene corresponding to the probe DNA sequence in the probe DNA complex particle.
  • BS method the extraction, purification, and cloning method of the target gene performed in the presence of at least 1000 times the ratio of the number of molecules to the number of target gene molecules.
  • a closed circular double-stranded DNA sample containing a target gene is denatured in a 0.1N to 0.4N alkali solution at a low temperature of 0 ° C to 10 ° C for 20 minutes to 1 hour.
  • the above problem was solved by the extraction, purification, and cloning methods of the target gene.
  • the present invention also provides that a closed circular double-stranded DNA sample containing the gene of interest is treated in the presence of the blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately at 0 ° C.
  • the above problems were solved by a method of extracting, purifying, and cloning the target gene, which was rapidly cooled to ⁇ 4 ° C. and denatured.
  • the present invention relates to a closed circular double-stranded DNA sample containing a target gene as a probe DNA complex.
  • the above problems have been solved by the above-described extraction, purification, and cloning methods that hybridize with body particles at a low salt concentration (50 to 350 mM).
  • the present invention also provides a closed circular double-stranded DNA contained in a recovered product from a host cell containing the target gene concentrated by the extraction, purification, and cloning method of the present invention as a target DNA molecule. Extraction, purification, and cloning by the above-described method of the present invention. By the multi-site cloning method, the conventional method is almost impossible or requires a great deal of time and labor. One millionth (0.0001%), 10 million It enabled rapid and reliable cloning of a very small amount of a target gene that exists only with a probability of 1 / (0.00001%).
  • “in a form that can be directly transformed into a host cell” means a DNA form that can be introduced into a host and rapidly cleave the target gene DNA in large quantities, that is, at least fixed to a solid particle. Means that the shape of the closed circular DNA is maintained up to the point of time.
  • the single-stranded bridge nucleotide complementary binding sequence for a probe consists of a partial force of a single-stranded sequence bound to a solid particle as a probe DNA complex particle for recovering a target gene.
  • Probe DNA binding particle force For probes containing a DNA sequence (probe sequence) complementary to at least a part of the base sequence of the target gene and a sequence complementary to a part of the single-stranded sequence part of the probe DNA binding particle.
  • the term “complementation rate” refers to a sequence that has achieved complementary binding to the corresponding single-stranded bridge nucleotide for probes in the single-stranded bridge nucleotide complementary binding sequence for probes. It means the ratio to the entire probe single-stranded bridge nucleotide complementary binding sequence.
  • the closed circular double-stranded DNA sample containing the target gene DNA used in the present invention desirably contains 60% or more, particularly preferably 80% or more of the closed circular double-stranded DNA.
  • Linear single-stranded or double-stranded DNA open circular double-stranded with at least one nick If the DNA content exceeds 40%, it will be difficult to extract and purify the target gene in a closed circular double strand.
  • the DNA sample may be derived from any of cDNA plasmid 'library, genomic DNA library, cDNA or genomic' cosmid 'library. Any method may be used for preparing a DNA sample containing 60% or more of closed circular double-stranded DNA.
  • CsCl method gel cutting method, DNA adsorption particle membrane (Takara Bio mimi-prep kit)
  • DNA adsorption particle membrane Tekjet mimi-prep kit
  • the ratio of the closed circular double-stranded DNA exceeds 80%, it can be used as a sample for cloning the target gene DNA even if it is contaminated with RNA.
  • the probe DNA complex particle used in the present invention is not limited as long as it contains a DNA sequence (probe DNA sequence) complementary to at least a part of the sequence of the target gene bound to the solid particle.
  • a DNA sequence probe DNA sequence
  • the following two types are exemplified.
  • One has a sequence complementary to a part of the base sequence of the target gene (Tm value A) and a sequence complementary to at least a part of the single-stranded DNA sequence part of the probe DNA binding particle (Tm value B).
  • Tm value A a sequence complementary to a part of the base sequence of the target gene
  • Tm value B the difference between the two Tm values is at least 8 ° C (Tm value B—Tm value A> 8 ° C).
  • Probe single nucleotide bridge nucleotide and probe DNA binding particle hybridized probe nucleotide probe Using a DNA synthesizing enzyme such as DNA binding particle ( Figure 6) and the other, Taleno 'fragment' DNA 'polymerase (Klenow fragment DNA polymerase), probe the single-stranded DNA sequence portion of the probe DNA binding particle.
  • This is a bridged nucleotide probe DNA covalent particle for probes capable of forming any arbitrary DNA sequence by enzymatically linking nucleotides containing the DNA sequence (Fig. 7).
  • the Tm value refers to the temperature at which half of a DNA molecule having a single-stranded complementary base sequence of about 1 to 50 base pairs is complementary and hybridized! / .
  • probe D The NA-bonded particles are probed for the target gene bridge nucleotide probe, and the probe is bound to the probe-DNA-bound particle so that it does not dissociate in the extraction process such as hybridization with the DNA-bound particle and subsequent washing.
  • the single-stranded bridge nucleotide complementary binding sequence for complementary binding to the DNA sequence portion of the probe single-stranded bridge nucleotide prepared to bind to the DNA sequence in a complementary manner with a complementation ratio of 80% or more. It is preferable.
  • the complementary binding of the single-stranded DNA sequence portion bound to the surface of the probe DNA-binding particle and the sequence portion complementary to the single-stranded DNA sequence portion of the single-stranded bridge nucleotide for probe (Tm Since value B) is a hydrogen bond that is vulnerable to heat, low salt concentration, and pH fluctuations as an individual chemical bond type, it is complementary to at least part of the base sequence of the target gene in the single-stranded bridge nucleotide. It must have a DNA sequence that forms a value that is at least 8 ° C greater than the Tm value A of the sequence (probe DNA sequence).
  • the probe DNA sequence portion located on the surface of the bridging nucleotide probe DNA-binding particle for the probe is capable of being infinitely long in sequence and length.
  • Hybridization with a single-stranded bridging nucleotide To increase the efficiency of the determination as much as possible, and to minimize the occurrence of non-specific hybridization between the target gene and the single-stranded DNA sequence located on the surface of the DNA binding particle.
  • the nucleotide sequence is 10 base sequences or more and 100 base sequences or less.
  • the single-stranded DNA sequence portion of the probe DNA-binding particle that is necessary to create the bridge nucleotide probe DNA-binding particle for the probe that sufficiently satisfies “Tm value B—Tm value A> 8 ° C” is complementary binding. It is most desirable that the sequence has more dG or dC base sequences with three hydrogen-bonding arms when formed. Ideally, the probe in the single-stranded bridge nucleotide for the probe.
  • the single-stranded bridge nucleotide for a probe used in the present invention has a sequence complementary to at least a part of a single-stranded DNA sequence portion bound to a solid particle on one side, and at least a part of the base sequence of the target gene. And the other sequence (probe DNA sequence).
  • These complementary sequences do not necessarily have to be located at the ends, but are desirably located near the ends to minimize steric hindrance during hybrid formation.
  • the probe bridging nucleotide probe DNA covalently bonded particle is obtained by neutralizing the single-stranded DNA sequence portion of the probe DNA-binding particle and the single-stranded bridge nucleotide for the probe, and then synthesizing DNA such as talenow, fragment, DNA, polymerase, etc.
  • a nucleotide containing a sequence (probe DNA sequence) that is complementary to at least part of the DNA sequence of the target gene at the 3 'end of the DNA of the single-stranded DNA sequence of the probe DNA-binding particle is chemically directly Enzymatic synthesis' created by covalent linkage.
  • the nucleotide containing the probe DNA sequence is covalently bound to the surface of the solid particle, so that any temperature below 100 ° C, any salt concentration, 1N It is extremely resistant to (alternative) alkali and acid solutions, and is an optimal probe for gene DNA extraction 'purification' crawling via hybridization.
  • Probe bridge nucleotide probe Probe in the DNA covalent bond particle Single-stranded DNA sequence of the DNA-bonded particle The base sequence of the DNA sequence is also complementary to at least part of the base sequence of the target gene in the probe single-stranded bridge nucleotide Any base sequence of the probe sequence is effective.
  • the bridge nucleotide probe for the probe Probe of the DNA covalently bonded particle The base sequence of the DNA sequence is free The length is 5 to several hundred bases An array is preferred.
  • the solid particle to which the probe DNA sequence is bound is an organic polymer such as a latex bead in both the bridge nucleotide probe DNA binding particle for probe and the bridge nucleotide probe DNA covalent bond particle for probe.
  • organic polymer such as a latex bead in both the bridge nucleotide probe DNA binding particle for probe and the bridge nucleotide probe DNA covalent bond particle for probe.
  • Molecular particles and organic Polymer sheets particles that have a magnetic material inside and can be separated magnetically, metal particles such as gold, glass particles and plates, and colored particles or fluorescent particles may be used.
  • Complementary binding of a single-stranded bridging nucleotide for a probe having a difference between two Tm values used in the present invention of at least 8 ° C or more to a probe DNA-binding particle through a hydrogen bond is a cycle hybridizer. Use the Chillon method.
  • a sequence complementary to at least a part of the base sequence of the target gene (probe DNA sequence) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA binding particle A single-stranded bridge nucleotide for a probe having a cycle of 5 cycles or more within a temperature range of 50 ° C to 70 ° C in a single-stranded DNA sequence portion of a probe DNA-binding particle. Examples of hybrids:
  • Probes in single-stranded bridge nucleotides for probes Can form stronger complementary hydrogen bonds between sequences complementary to at least part of the single-stranded DNA sequence portion of the DNA-binding particle.
  • the DNA sample used in the present invention is simply used for the purpose of extraction and purification or detection of the target gene.
  • the extracted and purified target gene DNA can be immediately introduced (transfected) into Escherichia coli in physicochemical form, i.e., at least until it is recovered into solid particles. It is necessary to maintain the form of the strand DNA.
  • Closed circular double-stranded DNA can also be nicked by high-temperature, long-term treatment (over 2 minutes) such as 70 ° C or higher, or alkaline treatment at room temperature or high temperature, resulting in open circular double-stranded DNA or linear
  • high-temperature, long-term treatment such as 70 ° C or higher
  • alkaline treatment at room temperature or high temperature
  • closed circular double-stranded DNA is denatured slowly and mildly at 0 ° C (ice water) in a 0.1 to 0.4 N alkaline solution to thereby open circular double-stranded DNA.
  • a closed circular double-stranded DNA sample containing the gene of interest is also treated in the presence of the blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately.
  • the DNA was rapidly cooled to 0 ° C to 4 ° C and denatured to achieve single-stranded DNA in a closed circular double-stranded DNA.
  • This method makes it possible to single-stranded closed circular double-stranded DNA without nicks even at high temperature treatment, and more rapid (time required for all steps including high temperature treatment and cooling; 1-2 minutes ) Closed circular double stranded DNA—allows it to be stranded.
  • the double-leaf clover-type single-stranded DNA derived from the high-temperature treatment of the closed circular double-stranded DNA obtained in the present invention in the presence of the low-temperature alkaline denaturation / blocking sequence is Rapidly self-associates and returns to the original closed circular double-stranded DNA.
  • Adjacent upper / downstream region 20-100 bases single stranded DNA (upstream, downstream blocking sequence, Up-BS, Down-BS)
  • the efficiency can be further increased by using the BS method described in this specification together.
  • the BS method single-stranded DNA of 20 to several hundred base sequences (upstream) from 0 to 7 bases upstream and downstream of the upstream and downstream regions adjacent to the target sequence of the target gene.
  • Downstream blocking sequence, Up-BS, Down-BS force Dependent on the salt concentration and temperature of the double-leaf clover-type single-stranded DNA derived from low-temperature alkaline denaturation of the closed circular double-stranded DNA obtained in the present invention It has a physicochemical property that slows the rate of self-association.
  • Closed circular double-stranded DNA two-leaf clover-type single-stranded DNA denatured at low temperature with the salt concentration and temperature of the hybridization solution is the original closed Utilizes the property of slowing the speed of returning to circular double-stranded DNA, resulting in increased binding between the target DNA and the probe DNA immobilized on the solid layer.
  • the length of the base sequence of 20 to several hundred base sequences of single-stranded DNA in the upstream and downstream regions is effective by about 20 to 100 base sequences, but if it is too long, it itself becomes nonspecific binding Therefore, it is desirable to have a 20-50 base sequence.
  • the concentration of 20-50 base sequence single-stranded DNA in the adjacent upstream and downstream regions should be at least 1000 times the ratio of the number of BS molecules to the number of target gene molecules, usually at least lOuM or more. A sufficiently large increase in cloning can be obtained.
  • the salt concentration of the hybridization solution used in the present invention is a force of 50 to 350 mM, preferably 100 to 250 mM, which is possible even at 400 to 600 mM commonly used in ordinary Sarzanno and hybridization. It is desirable to be between.
  • the closed circular double-stranded DNA contained in the recovered product from the host cell containing the target gene is concentrated by the extraction, purification and cloning method of the present invention.
  • the target gene DNA that exists only in a very small amount can be rapidly and reliably cloned.
  • FIG. 1 is a diagram showing three types of DNA generated in the process of extracting a DNA sample.
  • FIG. 2 is a diagram showing the positional relationship between a target gene sequence and Up-BS and Down-BS sequences.
  • the base position is the position of the target gene base sequence complementary to the probe DNA of the target gene DNA, and 0 to 7 bases upstream of the adjacent upstream region of the target gene sequence complementary to the probe DNA. 20 to several hundred base sequence single-stranded DNA (upstream blocking sequence, Up-BS) and 20 to several hundred base sequence single-stranded DNA (downstream blocking sequence, Dn- BS) position and probe Position of DNA binding particles.
  • FIG. 3 is a diagram showing the results of analysis of DNA samples (pCY and pGBM) purified by the SDS Al-CsCl method.
  • Sample 1 shows the analysis results of the plasmid pCY DNA sample obtained by the SDS-Al force method (contamination of open circular (op) DNA in addition to closed circular (sp) DNA).
  • Sample 2 shows the analysis results of the plasmid pGBM DNA sample obtained by the SDS-Al force method (in addition to closed circular (sp) DNA, open circular (op) DNA contamination is observed).
  • Sample 3 shows the analysis result of the DNA sample of plasmid pCY obtained by SDS / Al force / CsCl method (consisting only of 98% or more of closed circular (sp) DNA).
  • Sample 4 shows the analysis result of the DNA sample of plasmid pGBM obtained by SDS ⁇ Al force ⁇ CsCl method (more than 98% is composed of closed circular (sp) DNA only).
  • Sample pCY-R shows the analysis result of non-RNA-removed DNA sample of plasmid pCY obtained by SDS-Al force method (in addition to closed circular (sp) DNA and open circular (op) DNA contamination. The amount is less than 10%, and a large amount of total RNA is seen).
  • Sample pGBM-R shows the analysis result of non-RNA-removed DNA sample of plasmid pGBM obtained by SDS-Al force method (in addition to closed circular (sp) DNA and open circular (op) DNA contamination. The amount is less than 10%, and a large amount of total RNA is seen).
  • FIG. 4 A diagram showing the results of analysis of a DNA sample (mouse brain cDNA plasmid library) purified by the SDS Al force method and SDS Al force method CsCl method.
  • Sample 5 shows the analysis results of mouse brain cDNA plasmid library obtained by SDS-alkali method (the ratio of open circle (op) is higher than closed circle (sp)).
  • Sample 6 shows the analysis result of mouse brain cDNA plasmid library obtained by SDS-alkali method (the ratio of closed circle (sp) is higher than open circle (op)).
  • Sample 7 shows the analysis results of mouse brain cDNA plasmid library obtained by SDS-alkaline. CsCl method (closed circular (sp) is 95% or more and open circular (op) is very small).
  • Sample 8 is a mouse brain cDNA plus replacement kit (Rule 26) obtained with the Takarabio Mini A prepDNA extraction kit. The analysis results of the Mid 'library are shown (the ratio of closed ring (sp) (approximately 80%) is higher than open ring (op) (approximately 20%)).
  • FIG. 5 A diagram showing the results of detection of a cilitestin cDNA fragment (270 base pairs) by PCR.
  • FIG. 6 is a diagram showing the relationship between Tm value A and Tm value B in a probe single-stranded bridge nucleotide.
  • the figure shows examples of DNA of DNA particles (Tm value B) and single-stranded bridged nucleotide (BN-CY) DNA (Tm value A) for pCY probes.
  • Tm value B DNA sequence on the DNA particle side (30mer); SEQ ID NO: 3 4 BN-CY DNA sequence; SEQ ID NO: 3 5)
  • FIG. 7 is a diagram showing a method for producing a bridge nucleotide DNA covalent bond particle for a probe.
  • Single-stranded bridge nucleotides for probes Enzymatic synthesis of DNA particles to the 3 'end of DNA particles Covalent synthetic synthesis DNA probes (ridge nucleotides DNA covalently bonded particles)
  • ridge nucleotides DNA covalently bonded particles One example of preparation is a probe for pCY Single-stranded bridge nucleotides (BN -CY) Take DNA as an example. Talenou fragment Bridging nucleotide (BN-CY-2) by DNA synthesizing enzyme such as DNA polymerase After synthesizing bridge nucleotide complementary to DNA, DNA is denatured and washed with 0.2N NaOH to form hydrogen bond single strand The probe nucleotide is removed to obtain a bridge nucleotide DNA covalent bond particle for probe.
  • FIG. 8 is a diagram showing the results of cloning of actin gene cDNA plasmid clones according to the method of the present invention.
  • Lane 1 shows the electrophoresis marker (Eh / Hind ⁇ )
  • Lane 2 shows the sample obtained by cleaving the actin gene clone 1 DNA cloned by BS method with restriction enzymes EcoRl and Notl
  • Lane 3 shows A sample obtained by PCR of DNAO.lng of actin gene clone 1 with a lactin primer is shown.
  • Lane 4 is a replacement paper for the BS method (Rule 26).
  • a sample obtained by cleaving the DNA of the actin gene clone 2 with restriction enzymes EcoRl and Notl is shown.
  • Lane 5 shows a sample obtained by PCR of the actin gene clone 2 DNAO.lng with the actin primer.
  • the black arrow shows the actin gene cDNA of about 1.26k base pairs cloned by the BS method of the present invention
  • the white arrow shows the PCR of the actin gene cDNA clone cloned by the BS method.
  • the 730 base pair PCR product obtained is shown, and the asterisk indicates the plasmid vector 5.3 k base pair DNA of two actin gene cDNA plasmid clones cloned by BS method.
  • FIG. 9 is a diagram showing the results of cloning an HPRT gene cDNA plasmid clone according to the method of the present invention.
  • Lane 1 shows an electrophoresis marker ( ⁇ / Hind IE)
  • Lane 2 shows a sample obtained by cleaving HPRT gene clone 1 DNA cloned by BS method with restriction enzymes EcoRl and Notl
  • Lane 3 Shows a sample obtained by PCR of DNAO.lng of HPRT gene clone 1 with actin primer
  • lane 4 shows a sample obtained by cleaving the DNA of HPRT gene clone 2 cloned by BS method with restriction enzymes EcoRl and Notl
  • lane 5 Shows a sample obtained by PCR of HPRT gene clone 2 DNAO.lng with actin primer
  • lane 6 shows a sample obtained by PCR using a crude extract extracted by the BS method with HPRT primer.
  • the black arrow indicates the HPRT gene cDNA of about 1.2k base pairs cloned by the BS method of the present invention
  • the white arrow indicates the HPRT gene cloned by the BS method.
  • a PCR product of 944 base pairs obtained by PCR of the DNA clone is shown.
  • the asterisk indicates the plasmid vector of two HPRT gene clones cloned by BS method. Show
  • FIG. 10 shows the results of cloning of HPRT gene cDNA plasmid by the method of the present invention.
  • HPRT gene cDNA plasmid clone obtained by cloning a closed circular mouse brain cDNA plasmid sample by the method of the present invention using a probe nucleotide DNA covalent bond particle is shown.
  • Lane 1 shows the migration target (Hind IE)
  • Lane 2 shows a sample of the cloned HPRT gene clone 1 DNA cut with restriction enzymes EcoRl and Notl
  • Lane 3 shows the HPRT gene clone 1
  • a sample of DNAO.l ng of PCR with aactin primer is shown
  • lane 4 is the replacement paper 03 ⁇ 4-rule 26
  • the cloned HPRT gene clone 2 DNA was cleaved with restriction enzymes EcoRl and Notl.
  • Lane 5 shows the HPRT gene clone 2 DNAO.lng PCR sample with actin
  • lane 6 is the cloning.
  • Lane 7 shows a sample obtained by cleaving HPRT gene clone 3 DNA with restriction enzymes EcoRl and Notl.Lane 7 shows a sample obtained by PCR with DNAO.lng of HPRT gene clone 3 using actin primer.
  • Lane 9 shows a sample obtained by cleaving HPRT gene clone 4 DNA with restriction enzymes EcoRl and Notl.Lane 9 shows a sample obtained by PCR of DNAO.lng of HPRT gene clone 4 with actin primer, and lane 10 shows A sample of the cloned HPRT gene clone 5 was cleaved with restriction enzymes EcoRl and Notl.
  • Lane 11 shows the HPRT gene clone 5 DNAO.lng shows the sample PCR in ⁇ Kuching primer.
  • the black arrow indicates the HPRT gene cDNA of approximately 1.2k base pairs cloned by the BS method of the present invention
  • the white arrow is obtained by PCR of the HPRT gene cDNA clone cloned by the BS method.
  • the 944 base pair PCR product is shown, and the asterisk indicates the 5.3 k base pair DNA of the four HPRT gene cDNA plasmid clones cloned by BS method.
  • the present invention in one example
  • Tm value A A sequence complementary to at least a part of the base sequence of the target gene and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (Tm value A) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (Tm value A) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (Tm value A) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (Tm value A) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (Tm value A) and a sequence complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA-binding particle (Tm value A
  • ⁇ for difference (Rule 26) a single-stranded bridge nucleotide for a probe having a m value B) and a difference between both Tm values of at least 8 ° C (Tm value B—Tm value A> 8 ° C) with the probe DNA-binding particle.
  • a nucleotide containing a sequence (probe DNA sequence) complementary to at least a part of the base sequence of the target gene is enzymatically synthesized in the probe DNA-binding particle.
  • upstream blocking sequence Up-BS
  • D own-BS base sequence single-stranded DNA
  • This example was performed using a purified mixed DNA sample solution of plasmid pGBM (3035 base pairs) forming blue colonies and plasmid pCY (3768 base pairs) incorporating 734 base pair cDNAs forming white colonies. It was.
  • Plasmid pGBM carries the 13-galactosidase gene and forms blue colonies on agar in the presence of 13-gal (5-bromo-4-chloro-3-indolyl-13-D-galactoside) To do.
  • plasmid pCY the mouse testis expressed gene Shiritesuchin (C yrit es tin) C D NA730 bp DNA the plasmid pGBM j8 - a incorporated into Garatatoshidaze gene site recombinant plasmid, forming a white colony on agar. Because of this difference, the model system has the advantage that the efficiency of the extracted 'purified' clawing can be tested by the colony type force formed on the agar.
  • pCY is derived from the mouse testis cDNA library by amplifying 1044- 1777 between citristin cDNA (approximately 2650 base pairs in size) by PCR, extracting it, and inserting it into the ⁇ -galatatosidase gene of pGBM. Produced.
  • Solution B (20mg lysosome / solution A) 0.5ml was added and left on ice for 10 minutes.
  • Solution D 29.4 g K'acetate and 11.5 ml acetic acid in 100 ml pure water was added to 4 ml, stirred gently, and allowed to stand on ice for 10 minutes.
  • FIG. 3 shows pCY and pGBM purified by SDS-alkali method-CsCl ultracentrifugation.
  • the SDS-alkali method sometimes yields DNA samples containing 90% or more of closed circular DNA, but it is not stable. If purified by the SDS-alkali method-CsCl ultracentrifugation method, more than 95% of the closed circular DNA is obtained. A sample containing is obtained.
  • samples consisting of 90% or more of closed circular DNA (sample pCY_R, sample pGB M-R) can be stably obtained.
  • FIG 4 shows the SDS-alkali method (samples 5 and 6), SDS 'alkali method .CsCl method (sample 7), SDS' alkali method ', DNA adsorption membrane method, and Takara Bio Co., Ltd. mini-prep kit.
  • the closed circular DNA is 20% or less, and the open circular DNA is 80% or more, and the target circular DNA that is the target DNA molecule of the present invention is considerably small.
  • the closed circular DNA was about 60%, exceeding 50% of the total DNA.
  • Sample 7 is a sample 6 purified by SDS 'alkali method CsCl method and further purified by CsCl ultracentrifugation, and the closed circular DNA exceeds 95%.
  • Sample 8 was purified by the SDS “alkali method” DNA adsorption membrane method using a Takara Bio Co., Ltd. mini-prep kit, and contains 80% or more of the closed circular DNA which is the target DNA of the present invention.
  • the probe DNA sequence for pCY extraction, the blocking sequences (Up-BS and Down_BS) located above and downstream of the corresponding target gene DNA sequence, and the position and sequence of the base sequence for PCR are shown below. Show.
  • pCY Insertion A bridge oligonucleotide sequence (BN-CY) composed of 30 bases 1286-1315 of the cDNA fragment and poly C30 base was synthesized and used as probe DNA for pCY extraction.
  • Oligo (dG) latex 'beads purchased from JSR Corporation were used.
  • the BN-CY probe hybridizes with the latex 'bead oligo (dG) at the poly C portion and is fixed to the bead solid layer.
  • Up-BS upstream blocking sequence
  • [0050] 2) Mix lul lOOOuM Up-BS and lul lOOOuM Down-BS, 1 to 100, 1 to 10000 mixed pCY and pGBM 2ul, distilled water 5ul, 0.4N NaOH 9ul, about 0 After denaturation ( single-stranded) by treatment at ° C (on ice) for 40 minutes, an equivalent amount (9 ul) of 0.4NHC1 was added for neutralization.
  • the purified closed circular pCY is cleaved with the restriction enzyme Sacl, and the closed circular pCY and the Sacl cleaved pCY are mixed at a ratio of 10: 0, 9.0: 1, 7.5: 2.5, 5.0: 5.0 to obtain linear DNA. This was used as a pCY for pCY: pGBM mixed sample.
  • (a): 1), 6), 11), 13) and 15) are the results of the conventional method without BS. 2 to 5), 7 to 10), 12), 14) and 16) are the results of the method of the present invention with BS added.
  • (B): ll) to 14) show the results using SDS'alkali purified sample 1 (pCY) and sample 2 (pGBM) shown in FIG. (C): sp) of l) to 10) indicates sample 3 (closed-ring super-coiled pCY>) in Fig. 1, and Sacl-cut pCY is a sample obtained by cleaving pCY of sample 3 with the restriction enzyme Sacl. Chain pCY is indicated.
  • the pGBM from 1) to 10) was purified in Fig. 1.
  • the pGBM of Sample 4 is shown.
  • FIG. 5 shows the results of colony PCR for generating 270 base pair DNA fragments by randomly selecting seven white and blue colonies obtained in the experiment of Table 1.
  • a 270 base pair PCR product of mouse testis-derived cilitestin gene cDNA inserted in pCY was detected only in white colonies.
  • Example 2 A sequence having a sequence complementary to a part of the base sequence of the target gene, while being complementary to at least a part of the base sequence of the single-stranded DNA sequence part of the probe DNA binding particle Probes prepared by a method of hybridizing a single-stranded bridge nucleotide for a probe having a cycle of 5 cycles or more within a temperature range of 50 ° C and 70 ° C to the single-stranded DNA sequence of the probe DNA-binding particle. Bridge nucleotide probe Cloning efficiency of target gene using DNA binding particles
  • Example 3 A sequence complementary to a part of the base sequence of the target gene (Tm value A) and a single-stranded probe of the probe DNA binding particle Complementary to at least a part of the base sequence of the DNA binding particle
  • Tm value A A sequence complementary to a part of the base sequence of the target gene
  • Tm value B a unique sequence for probes that have a unique sequence
  • Tm value B the difference between the two Tm values is at least 8 ° C
  • Tm value A of the DNA sequence complementary to a part of the base sequence of the target gene of the single-stranded bridge nucleotide and the DNA sequence complementary to the single-stranded DNA sequence part of the probe DNA binding particle
  • Tm value B The effect of the difference in Tm value (Tm value B) on the cloning efficiency of the target gene (here pCY) was examined by changing the Tm value B.
  • a bridge nucleotide having various Tm values B was prepared, and cCY cloning was carried out from the pCY / pGBM mixed DNA sample.
  • FIG. 6 shows an example of probe DNA-binding particles and single-stranded bridge nucleotides for probes.
  • BN-2 is 1286-13 of the inserted cDNA fragment of pCY. Shows a complementary DNA sequence (Tm value A) to 15 30 bases (inside the rectangle in Table 1).
  • Table 3 shows the cloning efficiency of pCY when the difference between Tm values (TmB-TmA) is changed to 0.0, 4.1, 8.2, 12.3, 20.5. Until the Tm value difference is 8.2, the cloning efficiency is 85% or more, but below that, even if the Tm value difference is TmB-TmA> 0, the cloning efficiency is considerably lowered. Under these conditions (difference in Tm value TmB-TmA ⁇ 8.0), effective extraction and cloning of target genes that are present in only 1 in 1000 or 1 in 10000 is difficult.
  • Target gene Having a sequence complementary to at least a part of the base sequence of DNA,
  • probe single-stranded bridge nucleotides for probes that have a sequence complementary to at least part of the base sequence of the single-stranded DNA sequence part of the DNA-binding particle DNA binding Produced by enzymatic chemical synthetic covalent binding to the particle
  • the target gene is cloned by the covalently bonded DNA particles.
  • the bridge nucleotide DNA covalently bonded particle is composed of the hybridization DNA of the probe single-stranded bridging nucleotide to the single-stranded DNA sequence part of the probe DNA-binding particle and the Talenow 'fragment' complementary DNA It was prepared by denaturation of double-stranded DNA in an alkaline solution such as NaOH. Dissociation, dissociation, and washing and removal of single-stranded bridged nucleotide DNA.
  • the bridge nucleotide probe for the probe The probe used for the preparation of the DNA covalent bond particle
  • the single-stranded DNA sequence portion of the DNA-binding particle can be any sequence, and the length of the base sequence Furthermore, the strength of about 3 base pairs that can be hybridized can be up to several hundred bases.
  • a single-stranded bridge nucleotide having a sequence that is at least partially complementary to the single-stranded DNA sequence portion of the probe DNA-binding particle and having a partial base sequence of the base sequence of the target gene is also present.
  • the single-strand synthetic complementary bridge nucleotide for the probe including the probe DNA sequence synthesized enzymatically based on the single-strand bridge nucleotide itself is used. It is bound to solid-phase beads and has properties that do not degrade even under alkaline denaturation conditions of double-stranded DNA. Because of this property, it is possible to simultaneously denature a closed circular double-stranded DNA sample containing the gene of interest and probe DNA complex particles (which can have a solid layer of any material that is resistant to alkali!). It is possible to make the cloning of the target gene easier, quicker and more accurate, such as being hardly affected by salt concentration and temperature during washing. It is a thing.
  • Table 4 shows the results of extraction, purification, and cloning of pCY from the pCY ⁇ pGBM mixed DNA sample force using the bridge nucleotide probe DNA covalently bonded particles for probes prepared in Fig. 7.
  • the results are obtained using a single-stranded BS of length.
  • a large enrichment rate can be seen.
  • the system of the present invention can be repeated in a short period of time (multi-cycle cloning method).
  • the target gene can be extracted, purified, and cloned easily, quickly and accurately.
  • the extraction method was the same as in Example 1 for the second round of multi-cycle cloning in 8) and 10).
  • PCY and pGBM used in the experiment are the DNA samples of Samples 3 and 4 in FIG.
  • an extraction amplification effect was seen at a length of 15 mer or more.
  • the pCY: pGBM ratio is 1/10000 with a high concentration ratio exceeding 50% (amplification rate exceeds 5000 times), and even with a ratio of 1/100000, BS concentration is 30% with lOOuM (amplification rate 30000 times).
  • the concentration of BS added to the reaction solution is the final concentration (corresponding to 3 of the above extraction operation) luM or more, and the effect of increasing the extraction efficiency can be seen.
  • Table 7 shows the effect of BS base distance from the upstream (5 'upstream) and downstream (3' downstream) base points of the target gene sequence complementary to the probe DNA sequence.
  • Table 7 Up-BS and Down-BS enrichment effect on pCY extraction (upstream (5 'upstream), downstream (3' downstream) of target gene sequence complementary to BS probe DM sequence Influence of base distance from
  • Table 8 shows the results of temperature conditions during alkaline denaturation of the closed circular double-stranded DNA containing the target gene (here pCY). Except for the alkali denaturation temperature conditions of the DNA samples, the same experimental procedure as in Example 1 was performed.
  • Table 8 shows the results. Alkaline denaturation of DNA samples at 0 ° C (on ice) yielded very high cloning efficiencies, while significant reductions in cloning efficiency were seen at 25 ° C (room temperature) and 37 ° C. Table 8 shows that BS is indispensable also in this example. Alkaline denaturation of the DNA sample was performed with 0.2N NaOH (final concentration) for 40 minutes in the same manner as in Example 1. At 10 minutes or 20 minutes denaturation, the yield of the extracted plasmid itself decreased even at a temperature of 1, different.
  • Table 8 Temperature conditions for DNA sample denaturation for pCY extraction
  • DNA sample pCY / pGBM BS in reaction solution Number of colonies obtained White colony Concentration rate Density temperature molecular ratio concentration (uM) White blue ratio (%) (times)
  • Table 9 examines the effect of salt concentration of the solution during hybridization of the denatured sample DNA to the BN-CY oligo (dG) latex bead complex on the efficiency of the extracted 'purified' clawing of pCY. It is a result. [0084] [Table 9] Table 9: Effect of hyperpredation solution salinity on BN-CY-oligo (dG) latex bead complex of denatured DM sample on the efficiency of extraction and purification of pCY Concentration PCY / pGBM BS in reaction solution obtained White: 3D 2 Concentration rate
  • the salt concentration was shown as the sum of the values calculated by multiplying the NaCl concentration by the pH adjustment Tris HC1 concentration of 0.6.
  • the mouse brain cDNA plasmid library used for the experiment was purchased from Takara Bio Inc. This is transfected into Escherichia coli, cultured in large quantities, and closed with various ratios of linear DNA and open circular DNA by the SDS-al strength method, CsCl ultracentrifugation method and DNA adsorption membrane method shown in Example 1.
  • a circular cDNA plasmid library was prepared and used for experiments.
  • Oligo (dG) latex 'beads purchased from JSR Corporation were used.
  • the BN-actin probe hybridizes with this latex bead oligo (dG) at the poly C moiety and is immobilized on the bead solid layer.
  • Up-BS upstream blocking sequence
  • Primer 2 5'-TAGGAGCCAGAGCAGTAATC-3 '(1045-1026)
  • Table 10 Extraction, purification, and cloning of cDM plasmid clone from a mouse brain cDNA plasmid library according to the present invention (purification without cloning, comparison with conventional methods) Actin used -BS Number of colonies obtained Actin primer PCR positive
  • HPRT hypoxanthine'guanine from mouse brain cDNA plasmid library
  • the mouse brain cDNA plasmid library used for the experiment was purchased from Takara Bio Inc. This is transferred to Escherichia coli, cultured in large quantities, and subjected to SDS-alkaline and CsCl ultracentrifugation, and a closed circular cDNA plasmid library that contains almost no linear or open circular DNA (from 95% or more closed circular DNA). And used for the experiment.
  • a bridge oligonucleotide sequence consisting of 30 bases of 899-870 of the cDNA fragment of HPRT gene and poly C30 base was synthesized and used as probe DNA for extraction of HPRT gene cDNA plasmid clone.
  • Oligo (dG) latex 'beads purchased from JSR Corporation were used.
  • the BN-HPRT probe hybridizes with the latex 'bead oligo (dG) at the poly C moiety and is immobilized on the bead solid layer.
  • Up-BS upstream blocking sequence
  • Primer 2 5'-GAGAGCTTCAGACTCGTCTA-3 '(1097-1078)
  • Table 11 HPRT gene cDNA from a mouse brain cDNA plasmid library according to the present invention
  • Experiments 1) and 3) are the results of a method that contains 40% or more of open circular DNA and does not contain BS.
  • the HPRT gene cDNA fragment consisting of 30 bases of 899-870 and poly C30 base strength is composed of the following sequence for the bridge oligonucleotide (BN— HPRT)
  • the HPRT gene from the mouse brain cDNA plasmid library by the same extraction procedure as in Example 4 was used. Extraction of the child cDNA clones were performed by 'purification' cloning. The concentration of BS for HPRT and the primer for PCR were the same as in Example 9.
  • Table 1 2 Extraction, purification and cloning of HPRT gene cDM plasmid clones from mouse brain cDNA plasmid library according to the method of the present invention (comparison with conventional method without BS)
  • HPRT-BS Number of obtained colonies HPRT primer PCR positive colonies
  • Experiment 1 is the result of the method of the present invention
  • Experiment 2 is the result of the conventional method not including BS.
  • Example 11 A closed circular double-stranded DNA sample containing a gene of interest is treated in the presence of a blocking sequence for 10 to 60 seconds under a temperature condition of 94 ° C to 98 ° C, and then immediately 0 Cloning of the target gene by rapid cooling to ° C to 4 ° C, denaturation, and partial single-strand cloning. Closed circular double-stranded DNA containing the target gene (pCY) is cloned in the presence of a blocking sequence.
  • Table 13 shows the results of cloning of the target gene when it was denatured by high-temperature treatment and rapid cooling. High-temperature denaturation of DNA samples ⁇ Except for conditions of rapid cooling in ice, the same experimental procedure as in Example 1 was performed.
  • a closed circular double-stranded DNA sample containing the gene of interest in a TE solution (10 mM Tris, ImM EDTA, pH 8.0) and BS are treated at a temperature range of 96 ° C to 98 ° C for 10 to 60 seconds, and immediately The DNA was denatured by quenching in ice water, partially unified, and hybridized with probe DNA complex particles. Although not shown in Table 13, this method also uses B s was essential.

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Abstract

L'invention se rapporte à un procédé permettant d'extraire, de purifier et de cloner rapidement un gène cible en utilisant comme cible un échantillon contenant un ADN bicaténaire circulaire entièrement fermé (ADN circulaire surtorsadé) exempt de tout site de clivage de l'ADN (cassure monocaténaire) choisi parmi les échantillons contenant le gène cible tels qu'une banque de plasmides à ADNc qui peut exister en trois états. Le procédé comprend l'hybridation d'une particule complexe d'ADN sonde comportant une séquence d'ADN sonde et une particule en couche solide avec l'ADN bicaténaire circulaire fermé, la récupération dans la particule en couche solide de l'ADN à double brin circulaire fermé résultant dans lequel est intégré la séquence d'ADN du gène cible, cette dernière étant sous une forme qui permet une transfection directe dans les cellules hôtes, son élution à l'aide d'une solution d'élution et son transfert dans une cellule hôte, ce qui permet d'extraire, de purifier et de cloner rapidement le gène cible.
PCT/JP2005/021603 2005-01-31 2005-11-24 Procede d'extraction, de purification et de clonage rapides d'un gene cible a l'aide d'adn bicatenaire circulaire ferme WO2006080136A1 (fr)

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JP2010051182A (ja) * 2008-08-26 2010-03-11 National Agriculture & Food Research Organization 環状二本鎖ｄｎａおよびそれを用いたｄｎａの増幅方法
GB2621159A (en) * 2022-08-04 2024-02-07 Wobble Genomics Ltd Methods of preparing processed nucleic acid samples and detecting nucleic acids and devices therefor

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CN104911179B (zh) * 2015-06-25 2017-09-19 深圳承启生物科技有限公司 一种提取dna的方法

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WO2004101785A1 (fr) * 2003-05-13 2004-11-25 Jsr Corporation Procede d'extraction d'un gene cible et particule a laquelle est lie de l'adn sonde
JP2004357701A (ja) * 2003-05-13 2004-12-24 Jsr Corp 目的遺伝子の抽出方法およびプローブｄｎａ結合粒子

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004101785A1 (fr) * 2003-05-13 2004-11-25 Jsr Corporation Procede d'extraction d'un gene cible et particule a laquelle est lie de l'adn sonde
JP2004357701A (ja) * 2003-05-13 2004-12-24 Jsr Corp 目的遺伝子の抽出方法およびプローブｄｎａ結合粒子

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010051182A (ja) * 2008-08-26 2010-03-11 National Agriculture & Food Research Organization 環状二本鎖ｄｎａおよびそれを用いたｄｎａの増幅方法
GB2621159A (en) * 2022-08-04 2024-02-07 Wobble Genomics Ltd Methods of preparing processed nucleic acid samples and detecting nucleic acids and devices therefor

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