WO2013024821A1 - Suspension pour réaction parallèle, procédé faisant appel à une réaction parallèle, et procédé de criblage - Google Patents

Suspension pour réaction parallèle, procédé faisant appel à une réaction parallèle, et procédé de criblage Download PDF

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WO2013024821A1
WO2013024821A1 PCT/JP2012/070531 JP2012070531W WO2013024821A1 WO 2013024821 A1 WO2013024821 A1 WO 2013024821A1 JP 2012070531 W JP2012070531 W JP 2012070531W WO 2013024821 A1 WO2013024821 A1 WO 2013024821A1
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dna
gel particles
protein
gel
reaction
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Japanese (ja)
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創作 市川
達之 山本
大輔 佐伯
慎治 杉浦
敏之 金森
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国立大学法人筑波大学
独立行政法人 産業技術総合研究所
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Priority to JP2013529010A priority Critical patent/JP6041154B2/ja
Publication of WO2013024821A1 publication Critical patent/WO2013024821A1/fr

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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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present invention relates to a suspension for simultaneously performing a plurality of chemical reactions and biochemical reactions in a large number of gel particles, a parallel reaction method using the suspension, and a screening method using the parallel reaction. .
  • Non-Patent Document 1 when cloning a cell that expresses the target protein, the cell is cultured from a concentration that allows one cell to enter one container by the limiting dilution method, and a clone that expresses the target protein is obtained. A method has been proposed.
  • Non-Patent Document 2 and Non-Patent Document 3 disclose that a library is prepared by randomly substituting some amino acids of a certain enzyme, and that an enzyme having industrially useful activity is searched from this library. Has been.
  • a reactor having a volume of about several hundred microliters to several milliliters such as a 96-well plate is generally used.
  • a cell suspension containing target cells is dispensed into a 96-well plate, and analytical reagents including antibodies are sequentially added to determine the amount of substance expressed on the cell surface or secreted from the cells.
  • analytical reagents including antibodies are sequentially added to determine the amount of substance expressed on the cell surface or secreted from the cells.
  • the enzyme is expressed in a reactor such as a 96-well plate. The enzyme activity is measured by adding a reaction substrate or the like.
  • Non-Patent Document 4 proposes making a minute reaction vessel using semiconductor processing technology, but the problem of dispensing operation has not been solved. Therefore, proposals that further reduce the reaction vessel and do not require a dispensing operation are proposed in Patent Documents 1, 2, and 3 and Non-Patent Documents 5, 6, 7, 8, and 9.
  • Patent Document 1 includes a content that induces a specific reaction by separately containing hydrophilic molecules and functional molecules such as enzymes in water droplets constituting a W / O emulsion and fusing these water droplets together. It is disclosed. This Patent Document 1 also discloses that DNA is contained.
  • Patent Document 2 a reactive substance is included in one charged vesicle, a substance that reacts with the reactive substance is included in the other charged vesicle, and the two vesicles are fused and fused. It is disclosed that a reaction is caused to occur in one vesicle.
  • Patent Document 3 a solid phase carrier to which a primer for PCR (polymerase chain reaction) is bound is contained in an aqueous phase (water droplet) of an emulsion, and a nucleic acid complex is formed on the solid phase carrier by causing PCR in the water droplet. It is disclosed that, after ligation, the emulsion is broken to recover the solid phase carrier-nucleic acid complex conjugate, and cell-free protein synthesis is performed using this solid phase carrier-nucleic acid complex.
  • Non-Patent Document 5 includes a primer for PCR (polymerase chain reaction) in an aqueous phase (water droplet) of an emulsion, and this water droplet is combined with a water droplet containing genomic DNA obtained by enzymatic degradation in a microfluidic device. Thereafter, a method of amplifying a specific portion in genomic DNA by performing PCR in a water droplet is disclosed.
  • Non-Patent Document 6 discloses a method in which an enzyme and a substrate for protein synthesis are contained in an aqueous phase (water droplet) of an emulsion and a protein synthesis reaction is performed in the water droplet.
  • Non-Patent Document 7 discloses a technique in which a target molecule is contained in a vesicle that is a closed vesicle composed of a phospholipid bilayer membrane, and these vesicles are moved and fused by a laser.
  • Non-Patent Document 8 discloses the contents of supplying a substrate into a gel particle through a polymer film on the surface of the gel particle in order to cause a protein synthesis reaction in the gel particle.
  • Non-Patent Document 9 discloses a method of performing PCR using gel particles as a reaction vessel.
  • the reaction vessel can be made smaller and a large number of parallel reactions can be performed.
  • a protein synthesis reaction is caused in a micro container such as gel particles to obtain a target protein, there is a problem that the protein is easily eluted from the micro reaction container such as gel particles.
  • Non-Patent Document 9 discloses that a primer DNA is contained in the agarose droplet of an emulsion in which agarose is a dispersed phase and an oil phase is a continuous phase, and PCR is performed in the agarose droplet. There is no disclosure of immobilizing synthesized proteins.
  • a method applied to multistage biochemical reactions is not disclosed.
  • a method of including DNA using a microfluidic device is used, but the processing of the microfluidic device is very complicated and expensive.
  • the suspension for parallel reaction according to the present invention has gel particles in which a substance recognizing DNA (deoxyribonucleic acid) and a protein having a specific tag sequence fixed is dispersed in a liquid. .
  • the DNA is preferably a single-stranded DNA having a length in the range of 15 to 50 bases.
  • enzymes such as protease, lipase, amylase, cellulase, and fluorescent proteins are considered.
  • the substance that recognizes the protein having the tag sequence is, for example, nitrilotriacetic acid (NTA), antibody, glutathione, or maltose.
  • agarose particularly low-melting point agarose is preferable as it does not inhibit PCR (FIG. 5).
  • Polyethylene glycol can be considered as a linker that binds the single-stranded DNA or NTA to the network structure of agarose.
  • one molecule of DNA is included in one reaction vessel (gel particle).
  • the DNA solution is subjected to limiting dilution and the size of the gel particles is set to 1 to 500 ⁇ m.
  • limiting dilution in the present invention is a concentration at which one DNA is included in one gel particle on average.
  • a substance that recognizes DNA and a protein having a specific tag sequence is immobilized in agarose gel particles, and the agarose gel particles are added at a concentration at which one average molecule of DNA is contained in the agarose gel particles.
  • the DNA immobilized in the gel particle is a single-stranded DNA having a length that functions as a nucleic acid amplification reaction primer in the range of 15 to 50 bases.
  • a DNA is amplified, and then a substance necessary for protein synthesis is introduced.
  • PCR Nucleic acid amplification reaction
  • PCR becomes high temperature (94 ° C) when heat-denatured to convert double-stranded DNA into single-stranded DNA, and protein synthase is inactivated, so after nucleic acid amplification reaction (PCR) is completed Introduce amino acids and enzymes necessary for protein synthesis.
  • the gel particles in order to prevent the reactants from moving between the gel particles during the reaction, the gel particles may be suspended in a liquid that does not mix with water in both nucleic acid amplification reaction and protein synthesis. is necessary.
  • a complicated and expensive microfluidic device can be obtained. Without using it, it becomes possible to carry out compartmentalized nucleic acid amplification reactions and protein synthesis reactions in individual gel particles.
  • the characteristics of proteins immobilized in individual gel particles are examined, and DNA encoding the target protein is selected.
  • This selection is performed, for example, by sorting gel particles that emit fluorescence.
  • Specific means for sorting include flow cytometry and micromanipulators.
  • the screening method according to the present invention comprises 15 to 50 base single-stranded DNA that can function as a nucleic acid amplification reaction primer, and nitrilotriacetic acid (NTA), an antibody, a substance that recognizes a protein having a specific tag sequence, Using gel particles fixed with either glutathione or maltose, the following steps 1 to 12 are used.
  • Process 1 Preparing a solution containing a plurality of types of DNA (DNA library) having a portion complementary to the DNA primer and a portion in which a gene encoding a protein is randomly mutated.
  • Process 2 A solution containing a plurality of types of DNA obtained in step 1 is diluted, a solution containing the diluted plurality of types of DNA, a solution containing a substance necessary for a nucleic acid amplification reaction, a suspension of gel particles, To introduce and immobilize DNA in gel particles with an average of 1 molecule or less and introduce substances necessary for nucleic acid amplification reaction into the gel particles.
  • Process 3 Re-suspending the gel particles in a solution that does not mix with water.
  • Process 4 Step 5 of amplifying DNA in the gel particle by nucleic acid amplification reaction Resuspending gel particles in an aqueous solution; Step 6 A step of mixing a solution containing substances necessary for synthesizing a protein and a suspension of gel particles.
  • Step 7 A step of suspending the gel particles, into which substances necessary for synthesizing the protein in Step 6 are introduced, in a solution that does not mix with water.
  • Process 8 A step of synthesizing a protein by a protein synthesis reaction in the gel particle.
  • Step 9 A step of resuspending gel particles, in which proteins are synthesized, in an aqueous solution.
  • Step 10 A step of mixing the gel particles and a reagent for evaluating the function of the protein.
  • Step 11 A step of separating gel particles containing a protein having a desired function.
  • Step 12 Decoding the DNA sequence contained in the sorted gel particle to obtain the amino acid sequence of the protein having the desired activity.
  • Another screening method includes 10-base or more single-stranded DNA capable of capturing DNA having a complementary sequence, nitrilotriacetic acid (NTA) as a substance recognizing a protein having a specific tag sequence, an antibody ,
  • NTA nitrilotriacetic acid
  • Process 1 Preparing a solution containing a plurality of types of DNA (DNA library) having a portion complementary to DNA immobilized on the gel particle and a portion in which a gene encoding a protein is randomly displaced.
  • Process 2 A solution containing a plurality of types of DNA obtained in step 1 was diluted, and gel particles were prepared using the diluted solution, so that DNA was fixed at an average concentration of 1 molecule or less in the gel particles.
  • Process 3 A step of mixing a solution containing a substance necessary for synthesizing a protein and a suspension of the gel particles.
  • Process 4 Resuspending the gel particles in a solution that does not mix with water.
  • Process 5 A step of synthesizing a protein in the gel particle by a protein synthesis reaction.
  • Step 6 Suspending the gel particles in an aqueous solution again.
  • Step 7 Mixing the gel particles with a reagent for evaluating the function of the protein.
  • Process 8 Step 9 of separating gel particles containing a protein having a target function A step of obtaining an amino acid sequence of a protein having a desired activity by decoding a DNA sequence contained in the sorted gel particles.
  • Another screening method includes 10-base or more single-stranded DNA capable of capturing DNA having a complementary sequence, nitrilotriacetic acid (NTA) as a substance recognizing a protein having a specific tag sequence, an antibody ,
  • NTA nitrilotriacetic acid
  • a protein screening method using gel particles in which either glutathione or maltose is immobilized comprising the following steps 1 to 9.
  • Process 1 Preparing a solution containing a plurality of types of DNAs (DNA libraries) having sites complementary to DNA immobilized on the gel particles and sites randomly displaced in a gene encoding a protein.
  • Process 2 Dilute the solution containing multiple types of DNA obtained in step 1, mix the diluted solution containing multiple types of DNA with a suspension of gel particles, and average the DNA in the gel particles.
  • Process 3 A step of mixing a solution containing a substance necessary for synthesizing a protein and a suspension of the gel particles.
  • Process 4 Suspending the gel particles again in a solution that does not mix with water.
  • Process 5 A step of synthesizing a protein in the gel particle by a protein synthesis reaction.
  • Step 6 Suspending the gel particles in an aqueous solution again.
  • Step 7 A step of mixing the gel particles and a reagent for evaluating the function of the protein.
  • Process 8 Step 9 of separating gel particles containing a protein having a target function A step of obtaining an amino acid sequence of a protein having a desired activity by decoding a DNA sequence contained in the sorted gel particles.
  • Another screening method includes 15 to 50 base single-stranded DNA that can function as a nucleic acid amplification reaction primer, and nitrilotriacetic acid (NTA) as a substance that recognizes a protein having a specific tag sequence,
  • NTA nitrilotriacetic acid
  • Process 1 A step of preparing a solution containing a plurality of types of DNAs (DNA libraries) having a site complementary to a nucleic acid amplification reaction primer and a site randomly displaced in a gene encoding a protein.
  • Process 2 Dilute the solution containing multiple types of DNA obtained in Step 1, mix the diluted multiple types of DNA solution with the solution containing the substances necessary for nucleic acid amplification reaction, and dilute and mix A step of preparing gel particles in which DNA is immobilized at a concentration of 1 molecule or less on average in the gel particles by preparing gel particles using a solution containing a plurality of types of DNA.
  • Process 3 Suspending the gel particles again in a solution that does not mix with water.
  • Process 4 A step of amplifying the DNA in the gel particles by a nucleic acid amplification reaction;
  • Process 5 Suspending the gel particles in an aqueous solution again.
  • Step 6 A step of mixing a solution containing a substance necessary for synthesizing a protein and a suspension of the gel particles.
  • Step 7 Suspending the gel particles again in a solution that does not mix with water.
  • Process 8 A step of synthesizing a protein in the gel particle by a protein synthesis reaction.
  • Step 9 Suspending the gel particles in an aqueous solution again.
  • Step 10 A step of mixing the gel particles and a reagent for evaluating the function of the protein.
  • Step 11 Process Step 12 for Sorting Gel Particles Containing Protein with Target Function A step of obtaining an amino acid sequence of a protein having a desired activity by decoding a DNA sequence contained in the sorted gel particles.
  • a diagram illustrating a suspension according to the present invention in which primer DNA and NTA are immobilized on agarose gel particles Chemical formula explaining immobilization of primer and NTA on agarose
  • Diagram explaining PCR The fluorescence photograph of the gel particle which amplified DNA using the DNA amplification reaction using the agarose gel particle suspension by which the primer DNA based on this invention was fix
  • the template DNA concentration is adjusted to 0 (A), 0.13 (B), 1.3 (C), 13 (D) molecules / gel particles.
  • a diagram showing the theoretical value and experimental observation of the existence probability of beads with advanced PCR Graph comparing PCR suitability of low melting point agarose and calcium alginate The figure explaining the protein synthesis reaction using the agarose gel particle suspension in which the NTA according to the present invention is immobilized.
  • Chemical formula explaining protein synthesis reaction using agarose gel particle suspension with NTA immobilized thereon according to the present invention Diagram illustrating protein synthesis in gel particles The figure explaining the two-step parallel reaction of DNA amplification reaction and protein synthesis reaction using the agarose gel particle suspension in which the primer DNA and NTA according to the present invention are immobilized.
  • FIG. 1 is a schematic diagram of gel particles in a suspension according to the present invention.
  • the gel particles are made of low melting point agarose and are dispersed in an aqueous solution or an organic solvent.
  • Low melting point agarose has a network structure by hydrogen bonding between polysaccharide chains, and either or both of DNA and a substance that recognizes (holds) a protein having a specific tag sequence is fixed to the network structure part. .
  • a single-stranded DNA having a length of 15 to 50 bases that functions as a nucleic acid amplification reaction primer as DNA is immobilized, and a substance that recognizes a protein having a specific tag sequence is NTA (Nitrilo III). Acetic acid) is fixed.
  • NTA Nitrilo III
  • Acetic acid is fixed.
  • a DNA amplification reaction using a gel particle suspension in which only DNA is immobilized will be described below.
  • a nucleic acid amplification reaction primer is immobilized on the network structure portion of the low melting point agarose in the state of FIG.
  • the gel particles are suspended in an aqueous solution containing the template DNA and a substance necessary for the amplification reaction, and these molecules are supplied into the gel particles by diffusion (FIG. 3b).
  • the gel particles are suspended in the oil phase to shield the mutual mass transfer between the gel particles (FIG. 3c), and a nucleic acid amplification reaction is performed by PCR (polymerase chain reaction).
  • the amplified DNA is captured by the primer DNA immobilized on the gel particle and immobilized on the gel particle (FIGS. 3d and 4).
  • PCR is heated (about 94 ° C.), and double-stranded DNA in gel particles is separated into single-stranded DNA by thermal denaturation.
  • annealing at 55 to 60 ° C. allows the single-stranded DNA to bind to the primer already immobilized on the network structure portion of the gel particle, and further, the primer is extended by an enzymatic reaction with DNA polymerase to form double-stranded DNA and To do.
  • DNA having the same base sequence is present in a high concentration in one gel particle as shown in FIG.
  • FIG. 6 shows a fluorescent photograph of gel particles on which the amplified DNA is immobilized. Green fluorescence indicates DNA amplified by PCR. Depending on the concentration of the template DNA, the proportion of gel particles with green fluorescence is increasing.
  • FIG. 7 shows a comparison between the theoretical value of the existence probability of the gel particle having fluorescence, that is, the gel particle having undergone the DNA amplification reaction, and the experimental observation value.
  • a clear correlation was confirmed between the concentration of the template DNA introduced into the gel particles and the abundance of the gel particles that had actually undergone PCR. Thereby, it was confirmed that the gel particles dispersed in the oil phase functioned as independent compartments, and the DNA amplification reactions were performed in parallel in the individual gel particles.
  • a protein synthesis reaction using a gel particle suspension in which only NTA is immobilized will be described below.
  • the gel particles are a DNA encoding an amino acid sequence and a cell-free protein synthesis reaction. These substances are suspended in an aqueous solution containing substances necessary for the preparation (amino acids, enzymes, ATP, etc.), and these molecules are supplied into the gel particles by diffusion (FIG. 9b). Subsequently, the gel particles are suspended in the oil phase to shield the mutual mass transfer between the gel particles (FIG. 9c), and the protein synthesis reaction is performed by incubation at 37 ° C. The synthesized protein is captured by NTA immobilized on the gel particles and immobilized on the gel particles (FIGS. 9d and 10).
  • RNA polymerase unwinds a part of the double helix of DNA to strip the base, and mRNA is transcribed using the stripped base as a template. Based on this, amino acids that require tRNA are collected to synthesize proteins with ribosomes. The synthesized protein binds to NTA.
  • the gel particles are suspended in an oil phase and subjected to an appropriate thermal cycle, whereby a nucleic acid amplification reaction is performed by PCR (polymerase chain reaction) in an independent compartment.
  • the amplified DNA is captured by the primer DNA immobilized on the gel particles and immobilized on the gel particles (FIGS. 12b and 13).
  • DNA having the same base sequence is present in a high concentration in one gel particle.
  • the gel particles that have been subjected to PCR are soaked with substances (amino acids, enzymes, ATP, etc.) necessary for protein synthesis, and cell-free protein synthesis is performed in the gel particles.
  • substances amino acids, enzymes, ATP, etc.
  • FIG. 14 shows a fluorescent photograph of gel particles on which proteins synthesized by two-stage parallel reaction of DNA amplification reaction and cell-free protein synthesis reaction are immobilized. Green fluorescence indicates the fluorescence of green fluorescent protein (GFP) synthesized by cell-free protein synthesis.
  • GFP green fluorescent protein
  • the DNA library is subjected to limiting dilution to produce gel particles.
  • the dilution concentration and the size of the gel particles are such that only one molecule of DNA is contained in each gel particle. In reality, the number of gel particles is so large that not all gel particles contain exactly one molecule of DNA, but some do not contain DNA or some contain gel particles. There is no problem in performing parallel reactions.
  • a nucleic acid amplification reaction is performed in each gel particle by the above-described PCR, and further, a substance necessary for protein synthesis is supplied into the gel particle and cell-free protein synthesis is performed in the gel particle.
  • fractionation is performed using flow cytometry such as FACS (registered trademark). Specifically, gel particles in which proteins are synthesized are sent one by one to the sheath flow, and laser light is irradiated in the middle of the flow path. Gel particles in which the protein of interest is synthesized are stained with a fluorescent antibody and distinguished from other gels, so that the gel particles are positively charged and negatively charged on the downstream side. It is separated and recovered.
  • flow cytometry such as FACS (registered trademark).
  • the evolutionary engineering method is a method that opposes the method of rationally modifying proteins.
  • the rational method is a method of identifying effective mutation sites for modification based on the three-dimensional structure of the protein by computer simulation, experimentally creating a candidate protein, and verifying it.
  • the evolutionary engineering method randomly mutates the target gene, screens the mutated enzyme library, selects the mutated enzyme with improved traits, and further mutates the mutated enzyme gene randomly. This is a method of selecting mutant enzymes having improved traits.
  • methods for introducing mutations into genes include Overlap Extension PCR method, error prone PCR method, saturation mutation method, DNA shuffling method and the like.
  • Primer3 was used for the primer design. (For left primer and right primer, see Fig. 5) left primer; CTTTAAGAAGGAGATATACCATGACACCAGAAATGCCTGTTCTG, right primer; TGATGATGAGAACCCCCCCCTTTCAGCCCCAGAGCG
  • each component of the PCR solution was used as the final concentration, 300nM left primer, 300nM right primer, 2.5mM MgCl 2, 250 ⁇ M
  • Each dNTP, 3U Expand High Fidelity Enzyme mix (Roche), and 1 ng pUT / phoA Vector were added, and the total amount was adjusted to 50 uL with PCR grade water.
  • PCR thermal cycle conditions were 94 ° C for 4 minutes, then (94 ° C for 1 minute, 60 ° C for 30 seconds, 72 ° C for 1 minute) 25 cycles, followed by heating at 72 ° C for 7 minutes. .
  • Takara Thermal cycler Dice Real Time PCR System was used for the thermal cycle.
  • Second PCR T7 promoter primer and T7 terminator primer included in TS 100 E. coli lin tempGenSet, His-tag kit were used as primers.
  • linear DNA can be prepared in such a manner that a T7 promoter region is added to the N-terminus of the Alkaline Phospatase coding region, a T7 terminator and a His-tag coding region are added to the C-terminus.
  • each component of the PCR solution is used as a final concentration, 480 nM T7 promoter primer, 480 nM T7 terminator primer, 2.5 mM MgCl 2, 250 uM
  • Each dNTP, 3U Expand High Fidelity Enzyme mix, 300 ng was added to become the first PCR product, and the total volume was adjusted to 50 uL with PCR grade water.
  • the conditions for the thermal cycle in PCR are the same as the conditions for the first PCR.
  • reaction solution after the first PCR and the second PCR was purified only to the target DNA fragment using High Pure PCR Product Purification Kit (Roche) and quantified using NanoNdrop (Thermo Fisher Scientific). Subsequently, agarose electrophoresis (4 wt% agarose, 100V, 30min, TAE buffer) was performed, and TAE buffer (Ph 8.0) 10000-fold diluted Sybr Green I (Roche) was observed and confirmed to produce the desired product ( 1st PCR product; 1373 bases, 2nd PCR product; 1763 bases).
  • First PCR 1 left primer
  • CGGCGTAGAGGATCGAGA right primer
  • TGATGCAGCNNNNNNNGACGTAGTCC First PCR 2 left primer
  • GGACTACGTCNNNNNNNGCTGCATCA right primer ACCCCTCAAGACCCGTTTAG
  • each component of the PCR solution was used as the final concentration , 300nM left primer , 300nM right primer, 2.5mM MgCl 2, 250uM
  • Each dNTP, 3U Expand High Fidelity Enzyme mix 10 ng wild type ERP-encoded linear DNA was added, and the total amount was adjusted to 50 uL with PCR grade water.
  • the thermal cycle conditions in PCR are the same as the PCR conditions in 1).
  • the molecular weight was confirmed by quantification and electrophoresis (First PCR 1 product; 435 bases, First PCR 2 product; 1241 bases).
  • the two types of DNA generated by the two different types of PCR have a common sequence at and around the mutation introduction site.
  • each component of the PCR solution is used as a final concentration, 300 nM left primer, 300 nM right primer, 2.5 mM MgCl 2, 250 ⁇ M
  • Each dNTP, 3U Expand High Fidelity Enzyme mix, 10 ng wild type ERP-encoded linear DNA was added, and the total amount was adjusted to 50 uL with PCR grade water.
  • the thermal cycle conditions in PCR are the same as the PCR conditions in 1). After purification of the product, the molecular weight was confirmed by quantification and electrophoresis (1,643 bases).
  • the gel particles were recovered from the PCR reaction solution, redispersed in the oil phase by pipetting, and subjected to thermal cycling. Thermal cycle conditions were 94 ° C for 4 minutes, followed by heating (94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute) for 25 cycles, followed by heating at 72 ° C for 7 minutes. Mineral oil in which span80 1 wt% and Sun Soft 818SK (Taiyou Kagaku) 4 wt% were dissolved was used for the oil phase. After the reaction, the gel beads were washed twice with 2 mL of hexane and then twice with 2 mL of water. Next, by washing with an aqueous nickel chloride solution, Ni 2+ was added to form a Ni-NTA complex. Excess nickel ions were sufficiently diluted by washing 8 times with 10 times the amount of water.
  • each component of the PCR solution is the final concentration , 300nM left primer , 300nM right primer, 2.5mM MgCl 2, 250uM
  • Each dNTP and 3U Expand High Fidelity Enzyme mix was included, and the total amount was adjusted to 50 uL with PCR grade water.
  • Thermal cycle conditions were 94 ° C for 4 minutes, followed by heating (94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute) for 25 cycles, followed by heating at 72 ° C for 7 minutes. After purification of the product, the molecular weight was confirmed by quantification and electrophoresis (1,643 bases).
  • each DNA sequence was analyzed using ABI 3500x1 genetic analyzer (Applied Biosystems). Only DNA having a sequence different from wild type ERP was expressed on a microplate using RTS 100 E. coli HY Kit (Roche). Thereafter, 1 mL of ELFR 97 Phosphatase Substrate (Invitrogen) 100-fold diluted solution (in pH 8.0) was added to each well, and the fluorescence intensity was read over time with a plate reader to calculate kcat and Km values.
  • ELFR 97 Phosphatase Substrate Invitrogen
  • the suspension according to the present invention searches for a cell having a large amount of secreted target substance from among a large number of cells, or searches for an enzyme having a target activity from a large number of randomly mutated candidate proteins. This can be used for parallel reactions.

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Abstract

L'objectif de cette invention est de pourvoir à une suspension pour une réaction parallèle, à une réaction parallèle utilisant la suspension, et à un procédé de criblage qui utilise ladite réaction parallèle. Pour ce faire, cette invention utilise des particules de gel à base d'un agarose à bas point de fusion ayant une structure rétiforme due aux liaisons hydrogène entre les chaînes polysaccharide, et un fragment d'ADN simple brin d'une longueur dans une plage de 15 à 50 bases, qui fonctionne comme une amorce pour une réaction d'amplification d'acide nucléique, et un acide nitrilotriacétique (NTA), à titre de substance qui reconnaît une protéine ayant une séquence d'étiquette spécifique, sont immobilisés dans une partie de ladite structure rétiforme.
PCT/JP2012/070531 2011-08-12 2012-08-10 Suspension pour réaction parallèle, procédé faisant appel à une réaction parallèle, et procédé de criblage WO2013024821A1 (fr)

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