WO2008062777A1 - Protéine de liaison d'adn monocaténaire modifiée et procédé d'amplification isotherme d'un acide nucléique utilisant la protéine - Google Patents

Protéine de liaison d'adn monocaténaire modifiée et procédé d'amplification isotherme d'un acide nucléique utilisant la protéine Download PDF

Info

Publication number
WO2008062777A1
WO2008062777A1 PCT/JP2007/072435 JP2007072435W WO2008062777A1 WO 2008062777 A1 WO2008062777 A1 WO 2008062777A1 JP 2007072435 W JP2007072435 W JP 2007072435W WO 2008062777 A1 WO2008062777 A1 WO 2008062777A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
nucleic acid
amplification
dna
modified
Prior art date
Application number
PCT/JP2007/072435
Other languages
English (en)
Japanese (ja)
Inventor
Yasushi Shigemori
Tsutomu Mikawa
Takehiko Shibata
Original Assignee
Aisin Seiki Kabushiki Kaisha
Riken
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aisin Seiki Kabushiki Kaisha, Riken filed Critical Aisin Seiki Kabushiki Kaisha
Publication of WO2008062777A1 publication Critical patent/WO2008062777A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • the present invention relates to a modified single-stranded DNA-binding protein and a method of using the same.
  • a modified single-stranded DNA-binding protein obtained by modifying a single-stranded DNA-binding protein derived from a highly thermophilic bacterium that can improve the amplification efficiency of a cocoon-type nucleic acid in an isothermal amplification system using a strand displacement polymerase, And its usage.
  • SDA strand displacement amplification
  • RCA rolling circle amplification
  • DNA is amplified using the action of a DNA polymerase (strand-displacing polymerase) that nicks a long nucleic acid with a restriction enzyme and sequentially replaces DNA fragments having this nick.
  • a DNA polymerase strand-displacing polymerase
  • RCA rolling circle amplification
  • hybridization is performed by the strand-displacing polymerase replacing the previous strand at the end of the extended strand synthesized from the primer annealed to the vertical nucleic acid. Therefore, in these methods, amplification of the target nucleic acid is performed isothermally and continuously, eliminating the need for thermal cycling.
  • the RCA method has various advantages as compared with the method using thermal cycling, since the amplification process of the vertical nucleic acid is simplified. For example, the production amount of the amplification product can be increased efficiently, the length of the vertical nucleic acid that can be effectively amplified is not limited, and the facility for performing thermal cycling is not required. It can be applied to various biological methods such as single nucleotide polymorphisms and simple amplification of vertical nucleic acids for sequence reactions. (For example, see Non-Patent Documents 1 and 2).
  • reaction temperature in isothermal amplification is usually about 30 to 60 ° C, so that primer dimers are likely to be formed. It is conceivable that DNA fragments are easily amplified. That is, primer dimers are formed even in the absence of saddle-type nucleic acid, and non-specific nucleic acids are amplified. DNA fragments that are non-specific to the vertical nucleic acid cause a reduction in amplification accuracy and background noise that hinders subsequent experiments. And since primers having random sequences are used for amplification, it has been difficult to control non-specific amplification.
  • the isothermal amplification method for vertical nucleic acids is expected as a highly versatile technique from the viewpoint of eliminating the need for thermal cycling as in PCR.
  • its use has been limited due to the problem of amplification accuracy due to amplification of nucleic acid non-specific to the above-described vertical nucleic acid.
  • SSB protein single-stranded DNA binding protein
  • SSB proteins have been isolated from a wide variety of sources, from bacteriophages to eukaryotes.
  • SSB protein has also been isolated from hyperthermophilic bacteria (see, for example, Non-Patent Document 5).
  • SSB protein has high non-sequence affinity for single-stranded DNA (hereinafter abbreviated as "ssDNA").
  • SSB protein for DNA replication, recombination, and biological genome repair is required.
  • the SSB protein specifically stimulates its cognate DNA polymerase, increasing the fidelity of DNA synthesis and improving DNA polymerase forwardness by destabilizing the helix. At the same time, it promotes binding to DNA polymerase and organizes and stabilizes the origin of replication. In other words, SSB protein is thought to act as a replication cofactor.
  • a method for amplifying a cocoon-type nucleic acid using a strand-displacement polymerase such as the RCA method relies on the strand-displacement ability of the strand-displacing polymerase that denatures the cocoon-type nucleic acid.
  • this strand displacement can be promoted by a replication cofactor or a strand displacement cofactor, it was thought that the presence of these could efficiently amplify a DNA fragment specific for a vertical nucleic acid.
  • a modified type having an amino acid sequence in which the 255th phenylalanin is substituted with proline for the amino acid sequence of a single-stranded DNA-binding protein derived from Thermus thermophilus was constructed by the present inventors. Furthermore, the present inventors have found and reported that such a modified single-stranded DNA binding protein improves the amplification efficiency of the truncated nucleic acid (see, for example, Non-Patent Document 6).
  • Non-Patent Document 3 using a chemically modified primer has the power that the primer dimer cannot be completely suppressed due to the strong enzyme activity of the strand displacement polymerase.
  • the method described in Non-Patent Document 4 for reducing the amount of the reaction solution also has a problem that the amount of nucleic acid synthesized is small and the practicality is poor.
  • the methods of Patent Documents 1 and 2 in which an SSB protein derived from butteriophagase such as Escherichia coli is problematic in terms of amplification efficiency and have not yet been put into practical use.
  • Non-Patent Document 1 Dean FB., Nelson JR., Giesler TL., Lasken RS., "Rapid amplification of plasmia and phage DNA using Phi 29 DNA polymerase and multipiy-primed rolled circle amplification.”, Genome Res. , Volume 11, Issue 6, pp. 1095-1099, June 2001
  • Non-Patent Document 2 Lizardi PM., Huang X., Zhu ⁇ ⁇ , Bray-Ward P., Thomas DC, Ward D C., 'Mutation detection and single-molecule counting using isothermal rolling-circle amplification. ", Nat. Genet., Vol. 19, No. 3, pp. 225-232, July 1998
  • Non-Patent Document 3 Brukner I., Paquin S., Belouchi M., Labuda D., Krajinovic M., " Sel f-priming arrest by modified random oligonucleotides facilitates the quality control of whole genome amplification., Anal. Biochem., J39, 2, 345-347, April 2005
  • Non-Patent Document 4 Hutchison CA., Smith HO., Pfannkoch C, Venter JC., "Cell-free cloning using phi29 DNA polymerase., Proc. Natl. Acad. Sci. USA., 102, 48, No. 17332-17336, 2005
  • Non-Patent Document 5 Dabrowski S., Olszewski M., Piatek R., Brillowska-Dabrowska A., o nopa G., ur J., "Identification and characterization or single-stranded-DNA-binding proteins from Thermus thermophilus "Microbiology, 148 (Pt 10), pp. 3307-3315, October 2002
  • Non-Patent Document 6 Inoue J., Shigemori ⁇ ⁇ , Mikawa ⁇ .," Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein. "Nucleic Acids Research, Vol. 34, No. 9, e69, May 2006
  • Patent Document 1 Japanese Patent Laid-Open No. 10-234389
  • Patent Document 2 Special Table 2002-525078
  • an object of the present invention is to establish a highly practical technique that can control non-specific amplification in the isothermal amplification reaction system that improves amplification accuracy and amplification efficiency.
  • the aim is to further improve the practicality by searching for a new protein that can suppress the width and contribute to specific amplification of the truncated nucleic acid.
  • the modified single-stranded DNA binding in which the amino acid in the C-terminal region is deleted in the amino acid sequence of the single-stranded DNA-binding protein derived from an extreme thermophile. Protein was constructed. Such a modified single-stranded DNA binding protein was added to an isothermal amplification system using a strand displacement polymerase. As a result, it was found that an amplification product specific to the vertical nucleic acid was obtained, and a highly accurate amplification product without non-specific amplification was obtained. The present invention has been completed based on these findings.
  • the present invention is as shown in the following [1] to [7].
  • [0018] An amino acid in which the carboxyl terminal region of a single-stranded DNA binding protein derived from a hyperthermophilic bacterium is deleted from the amino acid sequence of a single-stranded DNA binding protein derived from a hyperthermophilic bacterium A modified single-stranded DNA-binding protein consisting of a sequence and expressing a function that can contribute to the improvement of the amplification efficiency of a truncated nucleic acid in an isothermal amplification reaction system using a strand displacement polymerase.
  • the amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added, and the type of nucleic acid in an isothermal amplification reaction system using a strand displacement polymerase.
  • Protein expressing function that can contribute to improvement of amplification efficiency [4] A gene consisting of DNA encoding the modified single-stranded DNA-binding protein of [1] to [3] above.
  • a gene comprising the following DNA (a) or (b):
  • a modified SSB protein that expresses a function that can contribute to an improvement in the amplification efficiency of a vertical nucleic acid in an isothermal amplification reaction system using a strand displacement polymerase Can provide.
  • the modified SSB protein of the present invention can be added to a DNA isothermal amplification system using a strand displacement polymerase, a specific DNA fragment can be efficiently amplified. That is, non-specific amplification can be suppressed, and DNA fragments can be amplified without being affected by background noise, which can contribute to improvement in amplification efficiency. Therefore, the modified SSB protein provided by the present invention can be applied to various biological techniques that require nucleic acid amplification. Such effects cannot be achieved with SSB proteins derived from hyperthermophilic bacteria that do not have a modified site and other recombination-related proteins.
  • the modified SSB protein of the present invention has a property of high thermal stability because it is based on a protein derived from a highly thermophilic bacterium. Therefore, even in the production thereof, since the contaminating protein can be easily removed as an insoluble fraction by heat treatment, the preparation is easy. For example, even when producing by genetic engineering techniques, other proteins derived from the host can be easily removed. Therefore, there is an advantage that the degree of purification can be improved and a highly reliable protein can be produced.
  • a method for isothermal amplification of nucleic acid using a strand displacement polymerase comprising:
  • a method for isothermal amplification of nucleic acid wherein the amplification reaction is carried out by adding a modified single-stranded DNA-binding protein of any of the above [1] to [3].
  • nucleic acid amplification technique capable of efficiently amplifying a cage nucleic acid.
  • the strand-displacement polymerase to the isothermal amplification system of the nucleic acid of the modified SSB protein of the present invention, it is possible to efficiently amplify a DNA fragment specific to the vertical nucleic acid.
  • a DNA fragment specific to a vertical nucleic acid that is not affected by background noise can be amplified, which can contribute to improvement in amplification efficiency.
  • Such an effect cannot be achieved by adding an SSB protein derived from an extreme thermophile that does not have a modified site and other recombinant-related proteins.
  • the amplification method using the modified SSB protein of the present invention can be widely used for general biological techniques.
  • a useful method for preparing large amounts of DNA from a small amount of sample extracted from a small amount of microorganisms collected from the environment for genotyping or for preparing DNA for DNA sequencing As useful.
  • it can be used suitably for the RCA method, and it can be applied with the ability S to expand the application technology using the RCA method indefinitely.
  • the modified SSB protein of the present invention can be applied to a cloning system for a target cDNA clone as much as possible in a DNA library. This achieves enrichment or isolation of specific and efficient target cDNA clones from the DNA library. Specific and efficient cDNA cloning can greatly contribute to the field of analysis of gene expression, development, differentiation, etc. and production of useful substances!
  • the modified SSB protein of the present invention can be applied to a reverse transcription reaction system from RNA to DNA. This achieves a specific and efficient conversion of the desired target RNA to cDNA. Since conversion from RNA to cDNA is an indispensable technique in genetic engineering, its utility value is high, including gene expression detection and quantification, RNA structure analysis, and cDNA cloning.
  • Fig. 1 is an alignment diagram of the modified SSB protein of the present invention and an SSB protein derived from Thermus thermophilus,
  • FIG. 2 is a graph showing the results of Example 2 in which the binding ability of the modified SSB protein of the present invention to DNA was examined.
  • FIG. 3 shows the increase in the number of the truncated nucleic acids in the nucleic acid amplification reaction system of the modified SSB protein of the present invention It is an electrophoresis pattern showing the results of Example 3 that examined the effect on the width,
  • FIG. 4 is an electrophoretic pattern showing the results of Example 4 in which the influence of the modified SSB protein of the present invention on the amplification of the truncated nucleic acid in the nucleic acid amplification reaction system was examined.
  • FIG. 5 is an electrophoresis pattern showing the results of Example 5 in which the influence of the modified SSB protein of the present invention on the amplification of the truncated nucleic acid in the nucleic acid amplification reaction system was examined.
  • FIG. 6 is a diagram showing the deduced amino acid secondary structure of the modified SSB protein of the present invention.
  • the modified SSB protein of the present invention expresses a function that can contribute to the improvement of the amplification efficiency of a truncated nucleic acid in an isothermal amplification reaction system using a strand-displacement polymerase, and the carboxyl terminus of the wild-type SSB protein (hereinafter referred to as “the SBS protein”). , Which may be abbreviated as “C-terminal”) includes all modified types in which changes have occurred in the region. That is, the modified SSB protein of the present invention has an improved specificity for a truncated nucleic acid in an isothermal amplification reaction system using a strand-displacement polymerase as compared with the wild-type SSB protein.
  • the modified SSB protein of the present invention has a modified site in which a specific amino acid is modified in the C-terminal region of the wild-type SSB protein.
  • modified SSB protein of the present invention includes both modified forms.
  • the SSB protein that is the basis of the modified type is the amino acid sequence of the SSB protein isolated from nature, and the base sequence encoding the SSB protein, which is intentionally or unintentionally altered, Means that it has a modified site!
  • a preferred example of the SSB protein that is the basis for modification is a protein derived from a hyperthermophilic bacterium.
  • SSB proteins derived from hyperthermophilic bacteria derived from thermus thermophilus, Thermus aquaticus and the like are preferred.
  • sequence information of the SSB protein suitable as the basis of the modified form of the present invention the amino acid sequence of the SSB protein derived from Thermus' thermophilus is represented by SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the SSB protein is represented by This is shown in SEQ ID NO: 4 (GenBank: AJ564626). Ma .
  • Thermus amino acid sequence of SSB protein derived from SEQ ID No. 7 aquaticus shows the nucleotide sequence of the gene encoding the SSB protein in SEQ ID NO: 6 (GenB ank: AF276705) o
  • the known SSB protein can be used as a basis for modification.
  • SSB protein refers to a protein that may be the basis of the modification of the present invention and that does not have a modified site that has been intentionally or unintentionally modified. Therefore, the term “wild-type SSB protein” used to distinguish from the modified type of the present invention is used synonymously.
  • the C-terminal region of the SSB protein refers to a region on the C-terminal side of the SSB protein, although it does not have a strict boundary.
  • the SSB protein derived from Thermus thermophilus refers to the region consisting of the 230th proline region to the 263rd phenylalanin of the SSB protein.
  • an SSB protein derived from Thermus aquaticus refers to a region consisting of the vicinity of the 231st proline to the 264th phenylalanin of the SSB protein.
  • the modification means that a modification comprising at least one of deletion, substitution, and addition of one or more amino acids has occurred in the amino acid sequence of the protein that is the basis of the modification.
  • “Modification consisting of at least one of deletion, substitution and addition of one or more amino acids” means a known gene recombination technique, a point mutation introduction method, etc. for the gene encoding the protein that is the basis of the modification. Means that a sufficient number of amino acids that can be deleted, substituted or added are deleted, substituted or added. These combinations are also included.
  • the modified SSB protein of the present invention the SSB protein derived from a hyperthermophilic bacterium originally has a DNA binding ability changed! /, which can be suitably used. it can.
  • the C-terminal region of the hyperthermophilic bacterium-derived SSB protein is rich in glutamic acid and aspartic acid, which are acidic amino acid residues.
  • the isoelectric point (hereinafter abbreviated as “PI”) of the protein changes.
  • the PI value increases when an acidic amino acid residue is changed to a basic amino acid residue or a neutral amino acid residue. This suggests that the fluctuation of the isoelectric point may affect the function that can contribute to the enhancement of the amplification efficiency of the vertical nucleic acid in the isothermal amplification reaction system of the SSB protein. Therefore, preferred examples of the modified SSB protein of the present invention include those modified so that the isoelectric point of the SSB protein varies.
  • the C-terminal region of the hyperthermophile-derived SSB protein is deleted from the amino acid sequence of the hyperthermophile-derived SSB protein. Those having an amino acid sequence are exemplified.
  • a modified form in which the amino acid below the 230th proline of the SSB protein is deleted is exemplified. That is, a protein power S having an amino acid sequence consisting of amino acids 1 to 229 is preferably exemplified with respect to the amino acid sequence of the SSB protein derived from Thermus thermophilus.
  • the amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing, and the base sequence encoding it is shown in SEQ ID NO: 1 in the sequence listing.
  • Figure 1 shows an alignment diagram with the amino acid sequence of SSB protein derived from Thermos' thermophilus.
  • amino acid sequence of another SSB protein having high sequence similarity with the SSB protein derived from Thermus thermophilus is used as the basis for modification
  • a truncated protein cut at the same position is preferred. Illustrated. Specifically, a modified example in which the amino acid sequence below the 231st proline is deleted from the amino acid sequence of the SSB protein derived from Thermus aquaticus is preferably exemplified.
  • the amino acid sequence is represented by sequence recognition number 8 in the sequence listing. Show.
  • amino acids in the amino acid sequence shown in SEQ ID NO: 2 as long as it retains the function of improving the specificity to the truncated nucleic acid in the isothermal amplification system of the modified SSB protein
  • amino acid sequence in which is deleted, substituted or added are also included in the scope of the present invention.
  • the concept of deletion, substitution or addition is as described above.
  • the modified SSB protein of the present invention can be obtained by a known method.
  • the gene encoding the SSB protein derived from the extreme thermophile that is the basis of the modification is modified.
  • the obtained modified gene is used to transform a host cell, and the hyperthermophile SSB protein can be obtained from the culture of the transformant by using the force S.
  • the gene of the SSB protein derived from such extreme thermophile can be obtained using a known gene cloning technique. It can also be obtained by synthesizing by a conventional method based on gene information obtained by searching a known database such as GenBank. For example, DNA synthesis techniques such as the phosphoramidite method can be used. The modified gene thus obtained also forms part of the present invention.
  • a mutation introduction technique for preparing a modified protein known to those skilled in the art is not particularly limited.
  • a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using a PCR method, or a transposon insertion mutagenesis method can be used.
  • a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using a PCR method, or a transposon insertion mutagenesis method.
  • a known literature on mutagenesis of SSB protein derived from E. coli eg Chase JW et al., The Journal Biological Chemistry, 259 (2), 805-814, January 25, 1984. Can do.
  • a commercially available mutation introduction kit for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stra tagene) may be used. Also, once the amino acid sequence of the desired modified SSB protein is determined, an appropriate base sequence encoding it can be determined. Using DNA synthesis methods such as the conventional phosphoramidite method A gene encoding the modified SSB protein of the present invention can be synthesized.
  • a DNA consisting of the base sequence represented by sequence recognition number 1 is preferably exemplified. Such genes also form part of the present invention.
  • the DNA comprising the base sequence represented by SEQ ID NO: 1 A gene consisting of DNA consisting of a complementary base sequence and DNA hybridizing under stringent conditions is also included in the scope of the present invention.
  • the stringent condition means that DNA having identity of 60% or more, preferably 70%, more preferably 80% or more, particularly preferably 90% or more in the base sequence can hybridize.
  • the stringency can be suitably adjusted by those skilled in the art by appropriately changing the salt concentration and temperature during the hybridization reaction and washing.
  • the conditions for Southern hybridization described in Molecular Cloning: A Laboratory Manual 2nd edition (Sambrook et al., Co Id Spring Harbor Laboratory Press, 1989) can be mentioned.
  • a known host / expression vector system such as Escherichia coli can be used.
  • the modified SSB protein of the present invention is linked to an appropriate vector that can be stably incorporated, and introduced into a host such as Escherichia coli that can efficiently express the modified SSB protein of the present invention to produce a transformant.
  • the vector that can be used is not particularly limited as long as it can incorporate foreign DNA and can replicate autonomously in a host cell. Therefore, the vector contains at least one restriction enzyme site capable of inserting a gene encoding the modified SSB protein of the present invention. It contains an array.
  • plasmid vectors pEX system, pUC system, pBR system, etc.
  • phage vectors gtlO, gtl1, ⁇ ZAP, etc.
  • cosmid vectors virus vectors (vaccinia virus, baculovirus, etc.), etc. Is included.
  • the recombinant vector of the present invention is incorporated so that the gene encoding the modified SSB protein of the present invention can express its function. Therefore, other known base sequences necessary for expression may be included in the function of the gene. For example, a promoter arrangement IJ, a leader sequence, a signal arrangement IJ, a ribosome binding sequence and the like can be mentioned.
  • a promoter arrangement IJ for example, when the host is Escherichia coli, lac promoter, trp promoter and the like are preferably exemplified.
  • the present invention is not limited to this, and a known promoter sequence can be used.
  • the recombinant vector of the present invention can also include a marking sequence that can confer phenotypic selection in the host.
  • marking sequences include sequences encoding genes such as drug resistance and auxotrophy. Specific examples include kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, and the like.
  • the gene of the present invention is cleaved with an appropriate restriction enzyme, and the restriction enzyme site of an appropriate vector, or multicloning.
  • an appropriate restriction enzyme for example, a method of inserting into a site and linking can be used, but the method is not limited to this.
  • a known method such as a method using DNA ligase can be used. It is also possible to use a commercially available ligation kit such as DNA Ligation Kit (Takara-bio).
  • the host cell is not particularly limited as long as it is a host cell capable of efficiently expressing the modified SSB protein of the present invention.
  • Prokaryotes can be used preferably, and in particular, E. coli can be used.
  • Bacillus subtilis, Bacillus bacteria, Pseudomonas bacteria, etc. can also be used.
  • E. coli for example, E. coli DH5a, E. coli BL21, E. coli JM109 and the like can be used for IJ.
  • eukaryotic cells can be used without being limited to prokaryotes.
  • yeasts such as Saccharomyces cerevisiae
  • insect cells such as Sf9 cells
  • animal cells such as CHO cells and COS-7 cells, and the like
  • Transformation methods include the calcium chloride method, the electopore method, the ribosome fusion method, and the microinjection method.
  • a known method such as the cission method can be used.
  • the modified transformant of the present invention is expressed by inoculating the obtained transformant into an appropriate medium and culturing according to a conventional method.
  • the culture conditions may be selected in consideration of the nutrient physiological properties of the host cells.
  • the medium to be used is not particularly limited as long as it contains nutrients that can be assimilated by host cells and can efficiently express the protein in the transformant. Therefore, it does not matter whether it is a natural medium or a synthetic medium that is preferably a medium containing a carbon source, a nitrogen source and other essential nutrients necessary for the growth of host cells.
  • glucose, dextran, starch and the like as carbon sources, and nitrogen sources include ammonium salts, nitrates, amino acids, peptone, casein and the like.
  • nitrogen sources include ammonium salts, nitrates, amino acids, peptone, casein and the like.
  • inorganic salts, vitamins, antibiotics, and the like can be included as desired.
  • the host cell is Escherichia coli, LB medium, M9 medium, etc. can be suitably used.
  • the culture form is not particularly limited, but a liquid medium can be suitably used from the viewpoint of mass culture.
  • Selection of host cells that express the modified SSB protein of the present invention can be performed, for example, by the presence or absence of expression of a marking sequence.
  • a marking sequence for example, when a drug resistance gene is used as a marking sequence, it can be cultivated in a medium containing a drug corresponding to the drug resistance gene.
  • the modified SSB protein may be collected and purified from the thus obtained transformant culture according to, for example, a general method for purifying SSB protein derived from hyperthermophilic bacteria. Further, a technique according to a general protein isolation and purification method may be applied depending on the fraction in which the modified SSB protein of the present invention is present. Specifically, when the modified SSB protein of the present invention is produced outside the host cell, the culture solution is used as it is, or the host cell is removed by means such as centrifugation or filtration, and the culture supernatant is obtained. obtain. Subsequently, the modified SSB protein of the present invention can be isolated and purified by appropriately selecting a known protein purification method from the culture supernatant.
  • the modified SSB protein of the present invention can be transformed into a host cell.
  • the host cells When produced in vitro, the host cells are recovered by means of centrifugation, filtration or the like. Subsequently, the host cells are disrupted by an enzymatic disruption method such as lysozyme treatment or a physical disruption method such as sonication, freeze-thawing, and osmotic shock. After disruption, the solubilized fraction is collected by means such as centrifugation and filtration. The solubilized fraction thus obtained can be isolated and purified by treating in the same manner as in the case where it can be produced outside the cells.
  • the modified SSB protein of the present invention is based on the SSB protein derived from an extremely thermophilic bacterium, and thus is subjected to heat treatment in the isolation and purification step, which has high thermal stability. It is useful to use in combination.
  • the host cell and culture supernatant obtained from the culture contain various proteins derived from the host cell.
  • the host cell-derived contaminating protein is denatured and condensed and precipitated by heat treatment. Therefore, for example, when the host E. coli is disrupted and subjected to heat treatment, E. coli-derived proteins other than the SSB protein are thermally aggregated, and thus can be separated and removed by centrifugation or the like.
  • the SSB protein that is not heat-denatured can be separated from the E. coli-derived protein as a soluble fraction and purified using affinity chromatography or the like.
  • the SSB protein derived from an extreme thermophile, which is the basis for modification has heat stability, so its structure is stable at room temperature, and it has high stability against organic solvents. Therefore, the above purification process can be performed at room temperature.
  • the purified protein is a modified SSB protein having a modified site where a desired modification has occurred can be confirmed by a known amino acid analysis method. For example, an automatic amino acid determination method based on Edman degradation can be used. Also, whether or not the purified modified SSB protein is subjected to isothermal amplification reaction using a strand displacement polymerase and the specificity to the truncated nucleic acid is improved as compared with the SSB protein having no modified site. Can be done by checking. As a confirmation method, it can carry out by the method shown in the Example of this invention, for example.
  • the above-mentioned "function that can contribute to improving the amplification efficiency of a cage nucleic acid” means that nonspecific amplification unrelated to the cage nucleic acid is performed in an isothermal amplification reaction using a strand-displacing polymerase. This means that the nucleic acid is hardly recognized and can be amplified with high yield. And preferably, it means a function capable of improving the amplification efficiency of the vertical nucleic acid by 5 to 10 times.
  • the protein having the amino acid sequence represented by SEQ ID NO: 5 or 7 has substantially the same function that can contribute to the improvement of the amplification efficiency of the vertical nucleic acid in the isothermal amplification reaction system using strand displacement polymerase.
  • the present invention further provides a method for isothermal amplification of a truncated nucleic acid using a strand-displacement polymerase capable of isothermal amplification using the modified SSB protein of the present invention.
  • the amplification method of the present invention is to carry out an amplification reaction by adding the modified SSB protein of the present invention.
  • the isothermal amplification method using a strand displacement polymerase is a method of exponentially amplifying a nucleic acid by the strand displacement activity of the strand displacement polymerase under an isothermal condition that does not require heat denaturation by high heat.
  • the RCA method, the SDA method and the like are preferably exemplified, and in particular, it can be suitably applied to the RCA method.
  • the principle of the RCA method is as follows.
  • the isothermal amplification method of the ⁇ type nucleic acid by the RCA method is, for example, a method in which a strand displacement polymerase is used as a base of a plurality of random primers annealed to a circular DNA that is a ⁇ type nucleic acid under isothermal conditions. Extend the complementary strand. Then, even when the replication base point of another random primer is reached with the extension of the synthetic chain, the chain extension is continued (branching) while peeling off the other synthetic chain due to the chain replacement activity of the chain-substituted polymerase. At this time, the site where the random primer can be annealed is exposed in the peeled synthetic strand. In other words, this stripped synthetic strand that consists only of circular DNA can be used as a cocoon-shaped nucleic acid to form a new DNA synthetic strand, resulting in exponential amplification.
  • a random hexamer or the like can be preferably used.
  • a primer that specifically anneals at a set temperature to a target site of a vertical nucleic acid can be applied. This primer can be used alone or mixed with the random primer.
  • the primer is designed so as to amplify a desired region based on the target nucleic acid sequence, for example, by primer design support software.
  • the primer is designed to have a random sequence. Is done.
  • Primers designed in this way can be synthesized chemically. For example, it can be chemically synthesized by solid phase synthesis using a known phosphoramidite method. It is also possible to automatically synthesize a primer having a desired base sequence using a commercially available automatic nucleic acid synthesizer. After synthesis The primer is purified by a known method such as HPLC as necessary.
  • isothermal in isothermal amplification means that the reaction temperature is changed in each step of DNA denaturation, annealing, and chain extension as in the PCR method.
  • the temperature at which the amplification reaction is performed is preferably less than 60 ° C, more preferably less than 45 ° C, and even more preferably less than 37 ° C.
  • This temperature is appropriately determined depending on the applied chain-substituted polymerase.
  • a suitable temperature range for performing the amplification reaction is 25 to 42 ° C, preferably 30 to 37 ° C, more preferably 30 to 34. ° C.
  • a constant temperature chamber such as an incubator set to the constant temperature, the sample is placed for 4 to 24 hours, preferably 6 to 2 hours.
  • Phat29 phage-derived Phi29 DNA polymerase (US Patent No. 5,198,543 and US Patent No. 5,001, 050, Blanco et al.) Is preferably exemplified. However, it is not limited to this.
  • Bst large fragment DNA polymerase Exo (-) Bst (Aliotta et al., Genet. Anal.
  • a circular DNA is preferably exemplified as the cage nucleic acid, but is not limited to this, and linear DNA may be used. In the case of the RCA method, circular DNA is preferred from the viewpoint of amplification efficiency.
  • Single-stranded and double-stranded nucleic acids can be applied to the cage nucleic acid.
  • natural DNA As plasmid DNA 'eukaryotic and prokaryotic genomic DNA, and artificially prepared DNA molecules, various DNA molecules such as bacterial artificial chromosome (BAC) DNA' phagemid 'cosmids can serve as vertical nucleic acids.
  • synthetic DNAs such as oligonucleotides can also be used as cage nucleic acids.
  • the modified SSB protein of the present invention can be applied to the enrichment or isolation of their target cDNA clones using a DNA library.
  • the modified SSB protein of the present invention can be applied when an amplification reaction is performed using a partial sequence of a target cDNA desired to be concentrated or isolated as a primer and a DNA library as a saddle.
  • amplification reaction in addition to an isothermal amplification reaction system using a strand displacement polymerase, a normal PCR reaction system or the like can be used.
  • a normal PCR reaction system or the like can be used.
  • non-specific amplification unrelated to the target cDNA can be suppressed, and only the target cDNA can be specifically amplified. Therefore, by applying the modified SSB protein of the present invention to a cloning system for a target DNA clone from a DNA library, it is possible to concentrate and isolate the desired target cDNA clone specifically and efficiently. It becomes.
  • the DNA library a DNA library containing or expected to contain a target DNA region desired to be enriched or isolated is used.
  • the DNA library may be either a gene library or a cDNA library, but a cDNA library is particularly preferable.
  • the term “gene library” is used as a concept that means an aggregate of cloned DNA in which whole genome DNA of a specific single species is randomly incorporated into a vector.
  • a cDNA library was used as a concept that refers to an assembly of cDNA fragments prepared by converting mRNA from a specific tissue, cell, or organism into cDNA by reverse transcription and incorporating it into a vector.
  • the primer is usually designed to be complementary to a specific sequence of the target nucleic acid.
  • the target sequence to be amplified has a complementary base sequence at both ends thereof. Some sequences can be suitably used.
  • the primer design is well known and the target C
  • primer design support software Primers designed in this way can be chemically synthesized.
  • chemical synthesis can be performed by solid phase synthesis using a known phosphoramidite method, and a primer having a desired base sequence can be synthesized automatically using a commercially available automatic nucleic acid synthesizer.
  • the synthesized primer is purified by a known method such as HPLC as necessary.
  • the modified SSB protein of the present invention can be applied to a reverse transcription reaction from RNA to DNA.
  • the modified form of the present invention is used when cDNA is synthesized from RNA by reverse transcription using a random hexamer primer, oligo dT primer, and target gene-specific primer in the presence of reverse transcriptase.
  • SSB protein can be applied.
  • the present invention can also be applied to the amplification reaction using the synthesized cDNA as a saddle type.
  • synthesis of V and non-specific cDNA related to the target RNA can be suppressed, and cDNA can be specifically synthesized for the desired target RNA. Therefore, by applying the modified SSB protein of the present invention to a reverse transcription reaction system, cDNA for a desired target RNA can be synthesized specifically and efficiently.
  • the RNA is not particularly limited, such as mRNA, tRNA, rRNA, etc. in addition to total RNA. It is prepared from a cell or tissue expected to express or express a desired gene using a known method. For example, a guanidine / cesium TFA method, a lithium chloride / urea method, an AGPC method, or the like can be used.
  • a guanidine / cesium TFA method a lithium chloride / urea method, an AGPC method, or the like can be used.
  • the primer anneals to the truncated RNA under the applied reaction conditions, there is no particular limitation.
  • a random hexamer primer, an oligo dT primer, and a target gene specific primer can be used.
  • the target gene-specific primer has a base sequence complementary to a specific vertical RNA, and preferably the 3 ′ side of a primer used in a normal PCR reaction system is used. It is.
  • a modified SSB protein lacking the 230th proline or less amino acid in the amino acid sequence of the SSB protein derived from Thermus thermophilus (SEQ ID NO: 5) was prepared. That is, a modified SSB protein having an amino acid sequence consisting of amino acids 1 to 229 with respect to the amino acid sequence of SMB protein derived from Thermus thermophilus was prepared.
  • TthSSB-229 protein its amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing, and the base sequence of the gene encoding the protein is shown in SEQ ID NO: 1 in the sequence listing.
  • TthSSB-229 protein The gene encoding TthSSB-229 protein was cloned using the PCR method.
  • PCR was performed by using od DNA polymerase (Toyobo Co., Ltd.) with thermus' Samophilus genomic DNA (TaKaRa Code 3071) as a saddle type.
  • the primer contains Ndel and BamHI restriction sequences for cloning
  • Amplification products of the expected size were confirmed by amplification.
  • the obtained amplification product was purified, cleaved with restriction enzymes Ndel and BamHI for construction of the expression plasmid, and ligated to the plasmid pET17b. Subsequently, E. coli BL21 (DE3) strain (Novagen) or E. coli BL21 (DE 3) pLysS strain (Novagen) was transformed to obtain a TthSSB-229 protein expression clone.
  • IPTG isopropyl-b-D-thiogalatatopyranoside
  • the collected fraction was dialyzed in a buffer containing 20 mM Tris-HCl (H 7.5), 2 mM EDTA, and lOmM ⁇ -mercaptoethanol to adjust to a low salt concentration (0.6% or less).
  • the solution after adjustment was subjected to RESOURCEQ (6 ml: Amersham Biosciences) and equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, lOmM / 3-mercaptoethanol and lOOmM NaCl. Then, elution was performed with a NaCl linear gradient from 0.1 M to 1.0 M, and the fraction containing SSB protein was recovered.
  • the binding force of the modified SSB protein of the present invention to DNA was examined.
  • TthSSB-229 protein prepared in Example 1 was examined for its ability to bind to DNA.
  • a modified TthSSB protein with a known function that can contribute to the improvement of the amplification efficiency of a vertical nucleic acid in a nucleic acid amplification reaction system an SSB protein derived from a wild-type Thermos thermophilus (hereinafter referred to as “TthSSB-wt protein”) And wild-type E. coli For the SSB protein of the future (hereinafter referred to as “EcoSSB-wt protein”)!
  • TthSSB-255 protein the 255th phenylalanin in the SSB protein derived from wild-type Thermus thermophilus was replaced with proline (hereinafter referred to as "TthSSB-255 protein"). Is used).
  • the amino acid sequence is shown in SEQ ID NO: 3 in the sequence listing.
  • proteins are protein buffer (1.5M KC1, 50mM Tris_HCl (pH7.5), l.OmM
  • TthSSB-wt protein and TthSSB-255 protein used were those obtained by the method according to Example 1.
  • PCR amplification was performed using the following primer pair, and the gene encoding the TthSSB-wt protein was cloned.
  • PCR was performed using the following primer pairs: Amplification was performed and the gene encoding TthSSB-255 protein was cloned.
  • TthSSB-229 protein and TthSSB255 protein are more binding to ssDNA than TthSSB-wt protein and EcoSSB-wt protein, which are wild-type SSB proteins. Turned out to be weak.
  • a nucleic acid amplification reaction was performed in the presence of the TthSSB-229 protein prepared in Example 1 above, and the influence of the TthSSB-229 protein on the nucleic acid amplification system was examined.
  • nucleic acid amplification reaction was performed under the following conditions.
  • Example 2 the protein used was dissolved in a protein buffer.
  • As the protein buffer as in Example 2, 1.5M KC1, 50 mM Tris-HCl (p ⁇ 7.5), l.OmM EDTA, 0.5 mM DTT, 50% glycerol solution were used.
  • the nucleic acid amplification reaction was carried out using REPLI-g DNA Amplification kit (QIAGEN) according to the manufacturer's protocol under the conditions of! This was done by reacting for 18 hours. Note that human genomic DNA (Promega: Catalog No. G3041) was used as the vertical nucleic acid to be amplified. After amplification, each of the amplification reaction solutions was fractionated and subjected to 1.2% agarose gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide to visualize the nucleic acid band.
  • lanes 1 to 4 show the results of nucleic acid amplification reaction in the presence of saddle-shaped nucleic acid.Lane 1 performs nucleic acid amplification without adding any SSB protein and protein buffer. The results are shown.
  • Lane 2 shows the amplification result under the condition where only SSB protein is added and only protein buffer is added.
  • Lane 3 shows the amplification results under the condition where TthSSB-255 protein was added.
  • Lane 4 shows the amplification result under the condition where TthSSB-229 protein was added.
  • lanes 5 to 8 show the results of the nucleic acid amplification reaction in the absence of the cage nucleic acid. Lane 5 shows the result of nucleic acid amplification without adding any SSB protein or protein buffer.
  • Lane 6 shows the amplification result under the condition where no SSB protein was added and only the protein buffer was added.
  • Lane 7 shows the amplification results under the condition where TthSSB-255 protein was added.
  • Lane 8 shows the amplification results under the condition where TthSSB-229 protein was added.
  • TthSSB-229 protein and TthSSB-255 protein were added and nucleic acid amplification was performed, amplification of DNA fragments specific to the cocoon-type nucleic acid was confirmed (lane 3 in Fig. 3). Four).
  • the purity of the TthSSB-229 protein and TthSSB-255 protein used here is not constant. In particular, the purity of the TthSSB-229 protein is assumed to be lower than that of the TthSSB-255 protein.
  • the comparison was made taking into account differences in protein purification, etc., when TthSSB-229 protein was added, the production of the same level of amplification product as when TthSSB-255 was added could be confirmed.
  • the effect of improving the amplification efficiency of the vertical nucleic acid confirmed under the addition of TthSSB-229 protein and TthSSB-255 protein is an effect peculiar to these proteins. It has been found.
  • the expression of the function of improving the amplification efficiency of the truncated nucleic acids exhibited by the TthSSB-229 protein and the TthSSB-255 protein is related to the decrease in the DNA binding ability of these proteins. The knowledge that it is what is done is guided. Therefore, the modified SSB protein obtained by artificially reducing the DNA binding force obtained here exhibits an appropriate DNA binding force required as a replication cofactor in a nucleic acid amplification reaction system. As a result, it is understood that the reaction efficiency in the nucleic acid amplification reaction system is improved and erroneous amplification is suppressed. Further examination of the results obtained here was conducted in Examples 4 to 6.
  • Example 3 the effect of the modified SSB protein of the present invention on the nucleic acid amplification reaction system was examined.
  • Example 3 a nucleic acid amplification reaction was performed with the addition of TthSSB-229 protein, and the influence of the TthSSB-229 protein on the nucleic acid amplification system was examined. In this example, the optimum amplification time was examined.
  • nucleic acid amplification reaction was performed under the following conditions.
  • nucleic acid amplification reaction was carried out in the same manner as in Example 3 except that the amplification time was changed to 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours under the above conditions! went. After amplification, it was subjected to electrophoresis in the same manner as in Example 3.
  • FIG. 4 panel A shows the results of a nucleic acid amplification reaction in the presence of a cage nucleic acid.
  • Lanes 8-14 of Panenole A show the results of nucleic acid amplification reactions performed for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours, respectively, with the addition of TthSSB-wt protein.
  • Panels A lanes 15 to 21 show the results of nucleic acid amplification reactions carried out for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours with addition of TthSSB-255 protein, respectively.
  • Lanes 22-28 of Panenole A show the results of nucleic acid amplification reactions performed for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours, respectively, with the addition of TthSSB-229 protein.
  • panel B shows the result of nucleic acid amplification reaction in the absence of saddle-shaped nucleic acid.
  • the lanes in panel B;! -7 are controls, and show the results of nucleic acid amplification reactions performed for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours, respectively.
  • Panels B lanes 8 to 14 show the results of nucleic acid amplification reactions performed for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours with the addition of TthSSB-wt protein, respectively.
  • Panels B lanes 15 to 21 show the results of nucleic acid amplification reactions carried out for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours with the addition of TthSSB-255 protein, respectively.
  • Panels B, lanes 22-28 show the results of nucleic acid amplification reactions for 0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 hours with the addition of TthSSB-229 protein.
  • TthSSB-229 protein is equivalent to the addition of TthSSB-255. Production of amplified product was confirmed.
  • TthSSB-229 protein and TthSSB-255 protein No amplification product was observed when any of them was added (Panel B, lanes 15 to 21, lanes 22 to 28 in FIG. 4). The above results were consistent with the results of Example 3.
  • TthSSB-229 protein and TthSSB-255 protein can suppress nonspecific amplification of the vertical nucleic acid and achieve nucleic acid amplification specific to the vertical nucleic acid.
  • the TthSSB-229 protein can contribute to the effect of improving the amplification efficiency of the vertical nucleic acid in the nucleic acid amplification reaction system, like the TthSSB-255 protein.
  • Example 3 the improvement effect of the amplification efficiency of the vertical nucleic acid confirmed in the present Example under the addition of TthSSB-229 protein and Tth SSB-255 protein is It was strongly suggested that this was a protein-specific effect. On the other hand, it was found that TthSSB-wt might suppress the nuclear acid amplification reaction itself. Considering this result together with the result in Example 2, it is suggested that TthSSB-wt without modification strongly binds to DNA, and therefore the amplification efficiency of the truncated nucleic acid is reduced. It is.
  • the TthSSB-229 protein prepared in Example 1 was added to perform a nucleic acid amplification reaction, and the influence of the TthSSB-229 protein on the nucleic acid amplification system was examined. In this example, the optimum protein addition amount was particularly examined.
  • nucleic acid amplification reaction was performed under the following conditions.
  • TthSSB_wt protein 1.0, 2.0, 3.0, 4.0, 5.0
  • TthSSB-255 protein 1.0, 2.0, 3.0, 4.0, 5.0
  • TthSSB-229 protein 1.0, 2.0, 3.0, 4.0, 5.0
  • the nucleic acid amplification reaction was carried out using a TmpliPhi DNA Amplification kit (QIAGEN) according to the manufacturer's protocol under the conditions of! The reaction was carried out by volume for 0, 4, 8, 20 hours.
  • a nucleic acid to be amplified human genomic DNA was used as in Example 3. After amplification, the amplification reaction solution was subjected to electrophoresis in the same manner as in Example 3.
  • panel A shows the result of nucleic acid amplification reaction with an amplification time of 0 hour.
  • the lane of Panenole A;!-5 shows the result of having performed nucleic acid amplification reaction by adding 1.0, 2.0, 3.0, 4.0, and 5.0 g of TthSSB-wt protein, respectively.
  • Lanes 6-10 of Panenole A show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 g of TthSSB-255 protein added, respectively.
  • Lanes 11-15 of Panenole A show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 ⁇ g of TthSSB-229 protein added, respectively.
  • panel B shows the results of nucleic acid amplification reaction with an amplification time of 4 hours.
  • Panel B lanes;! To 5 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 g of TthSSB-wt protein added, respectively.
  • Panels B, lanes 6 to 10 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 g of TthSSB-255 protein added, respectively.
  • Panel B lanes 11-15 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 ⁇ g of TthSSB-229 protein added, respectively.
  • panel C shows the results of nucleic acid amplification reaction with an amplification time of 8 hours.
  • Panel C lanes;! To 5 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 g of TthSSB-wt protein added, respectively.
  • Panels C, Lanes 6 to 10 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0, and 5.0 g of TthSSB-255 protein added, respectively.
  • Panel C, lanes 11-15 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 ⁇ g of TthSSB-229 protein added, respectively.
  • panel D shows the results of nucleic acid amplification reaction with an amplification time of 20 hours.
  • Panels D, lanes 6 to 10 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 g of TthSSB-255 protein added, respectively.
  • Panel D lanes 11-15 show the results of nucleic acid amplification reactions with 1.0, 2.0, 3.0, 4.0 and 5.0 ⁇ g of TthSSB-229 protein added, respectively.
  • TthSSB-229 protein has a stronger DNA binding force than TthSSB-255 protein, so when the added amount is excessive, undesired binding to the vertical nucleic acid occurs. It is considered that the effect of improving the nucleic acid amplification efficiency disappears. However, it was found that this protein is a useful protein that exhibits the same effect of improving amplification efficiency as that of TthSSB-255 protein when added in an appropriate amount.
  • the amino acid secondary structure of the C-terminal region of the TthSSB-229 protein prepared in Example 1 was predicted.
  • the amino acid secondary structure was similarly predicted for the TthSSB-255 protein and the TthSSB-wt protein.
  • amino acid secondary structure of the protein was predicted by using a computer program (GENETYX-Win).
  • the spiral portion indicates the ⁇ helix structure
  • the broken line portion indicates the / 3 sheet structure.
  • the present invention provides a technique that can be used in various biochemical fields that require nucleic acid amplification.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une technique de contrôle d'une amplification non spécifique lors de l'amplification d'un acide nucléique par une réaction d'amplification isotherme dans le but d'améliorer la précision de l'amplification. L'invention concerne donc une protéine de liaison d'ADN monocaténaire modifiée comprenant une séquence d'acides aminés présentant la délétion d'une région carboxyle terminale dans la séquence d'acides aminés pour une protéine de liaison d'ADN monocaténaire dérivée d'une bactérie extrêmement thermophile et exerçant une fonction qui peut contribuer à l'amélioration de l'efficacité de l'amplification d'un acide nucléique modèle lors d'une réaction d'amplification isotherme en utilisant une polymérase déplaçant les brins. L'invention concerne également l'utilisation de la protéine.
PCT/JP2007/072435 2006-11-22 2007-11-20 Protéine de liaison d'adn monocaténaire modifiée et procédé d'amplification isotherme d'un acide nucléique utilisant la protéine WO2008062777A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2006315790A JP2008125464A (ja) 2006-11-22 2006-11-22 改変型一本鎖ｄｎａ結合タンパク質、及び該タンパク質を用いた核酸の等温増幅方法
JP2006-315790 2006-11-22

Publications (1)

Publication Number Publication Date
WO2008062777A1 true WO2008062777A1 (fr) 2008-05-29

Family

ID=39429707

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2007/072435 WO2008062777A1 (fr) 2006-11-22 2007-11-20 Protéine de liaison d'adn monocaténaire modifiée et procédé d'amplification isotherme d'un acide nucléique utilisant la protéine

Country Status (2)

Country Link
JP (1) JP2008125464A (fr)
WO (1) WO2008062777A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190120271A (ko) * 2017-02-24 2019-10-23 유니벨시테테트 이 트롬소 - 노르게스 알크티스키 유니벨시테트 단일-가닥 결합 단백질
CN113278642A (zh) * 2021-04-30 2021-08-20 上海交通大学 一种深海古菌单链dna结合蛋白ssb及其制备方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005333920A (ja) * 2004-05-28 2005-12-08 Aisin Seiki Co Ltd 等温増幅可能な鎖置換ｄｎａポリメラーゼを用いたテンプレートｄｎａ分子の増幅方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005333920A (ja) * 2004-05-28 2005-12-08 Aisin Seiki Co Ltd 等温増幅可能な鎖置換ｄｎａポリメラーゼを用いたテンプレートｄｎａ分子の増幅方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DABROWSKI S.: "Identification of characterization of single-stranded-DNA-binding proteins from Thermus thermophillus and Thermus aquaticus - new arrangement of binding domains", MICROBIOLOGY, vol. 148, no. 10, 2002, pages 3307 - 3315, XP002411103 *
INOUE J. ET AL.: "Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophillus SSB mutant protein", NUCLEIC ACIDS RES., vol. 34, no. 9, May 2006 (2006-05-01), pages E69 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20190120271A (ko) * 2017-02-24 2019-10-23 유니벨시테테트 이 트롬소 - 노르게스 알크티스키 유니벨시테트 단일-가닥 결합 단백질
JP2020508664A (ja) * 2017-02-24 2020-03-26 ウニベルシテテット イ トロムソ−ノルゲス アークティスク ウニベルシテット 一本鎖結合タンパク質
US11447812B2 (en) 2017-02-24 2022-09-20 Universitetet I Tromsø—Norges Arktiske Universitet Single-strand binding protein
JP7273717B2 (ja) 2017-02-24 2023-05-15 ウニベルシテテット イ トロムソ－ノルゲス アークティスク ウニベルシテット 一本鎖結合タンパク質
KR102636161B1 (ko) * 2017-02-24 2024-02-14 유니벨시테테트 이 트롬소 - 노르게스 알크티스키 유니벨시테트 단일-가닥 결합 단백질
CN113278642A (zh) * 2021-04-30 2021-08-20 上海交通大学 一种深海古菌单链dna结合蛋白ssb及其制备方法和应用

Also Published As

Publication number Publication date
JP2008125464A (ja) 2008-06-05

Similar Documents

Publication Publication Date Title
EP2388322A1 (fr) Preparation enzymatique contenant de l'adn polymerase thermostable, processus de production associe et procede de detection d'organisme d'analyte
JP2003510052A (ja) 改良されたポリヌクレオチド合成のための方法と組成物
JP6986286B2 (ja) 海洋生物ｄｎａポリメラーゼ
EP1754716B1 (fr) Protéine mutante de thermophile extrême liant l'ADN simple brin et son utilisation dans une méthode d'amplification isotherme d'acides nucléiques
Dąbrowski et al. Cloning, Overexpression, and Purification of the Recombinant His-Tagged SSB Protein ofEscherichia coliand Use in Polymerase Chain Reaction Amplification
CN114561374A (zh) 一种新型嗜热核酸内切酶突变体及其制备方法和应用
WO2013175815A1 (fr) Procédé pour supprimer des erreurs dans une réaction d'amplification d'acide nucléique
JP6387722B2 (ja) 改変型の耐熱性ＲｅｃＡタンパク質、該タンパク質をコードする核酸分子、該タンパク質を用いた核酸の増幅方法、及び核酸増幅用キット
WO2008062777A1 (fr) Protéine de liaison d'adn monocaténaire modifiée et procédé d'amplification isotherme d'un acide nucléique utilisant la protéine
US7981653B2 (en) Highly efficient hyperthermophilic DNA ligase
US6444429B1 (en) Gene coding for DNA ligase of hyperthermophilic bacteria Aquifex pyrophilus and protein expressed therefrom
CN114774399A (zh) 一种人工改造脱氨酶辅助的dna中5-羟甲基胞嘧啶修饰单碱基分辨率定位分析方法
US7902335B1 (en) Heat-stable recA mutant protein and a nucleic acid amplification method using the heat-stable recA mutant protein
CN111433373A (zh) Dna聚合酶
US20110053147A1 (en) Heat-resistant dna ligase with high reactivity
RU2809366C1 (ru) Способ получения большого фрагмента Bst-полимеразы (варианты)
US20230332118A1 (en) Dna polymerase and dna polymerase derived 3'-5'exonuclease
JP4714848B2 (ja) Ｄｎａポリメラーゼ変異体
US6399352B1 (en) Plant porphobilinogen synthase and fusion protein thereof
US20020119506A1 (en) Genes encoding UMP kinase, methods for purifying UMP kinase and methods of characterizing UMP kinase
JP5357445B2 (ja) 環状核酸の単離方法
CN117467640A (zh) 突变型Taq DNA聚合酶及其应用
WO2021064106A1 (fr) Adn polymérase et exonucléase 3' à 5' dérivée de l'adn polymérase
WO2024059581A2 (fr) Variants d'adn polymérase modifiés
WO2022271682A1 (fr) Polymérases pour amplification isotherme d'acides nucléiques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07832165

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07832165

Country of ref document: EP

Kind code of ref document: A1