JP5707705B2 - Novel nucleic acid synthesis composition - Google Patents

Description

The present invention relates to a novel composition and method useful for synthesizing DNA from a template nucleic acid using DNA polymerase mainly in the field of molecular biology, and particularly useful for polymerase chain reaction (PCR). The present invention relates to a nucleic acid synthesis composition and a nucleic acid synthesis method.

DNA synthesis using a template nucleic acid using DNA polymerase is used and applied to various methods such as sequencing and nucleic acid amplification in the field of molecular biology. In particular, nucleic acid amplification methods represented by PCR methods have already been put into practical use not only in the research field, but also in forensic fields such as genetic diagnosis and parentage testing, or in microbial tests in food and the environment.

The gene amplification method by the PCR method is performed by repeating 25 to 40 cycles of denaturation, annealing, and extension in the presence of a target nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and a DNA polymerase. The target nucleic acid region sandwiched between the primers is exponentially amplified.

Here, as a DNA polymerase used in the PCR method, a PolI type enzyme (belonging to "Family A") derived from an eubacteria and an α-type enzyme (belonging to "Family B") derived from Archaea (ultrahyperthermic archaeon) There is. In general, PolI-type enzymes such as Taq and Tth have a relatively high DNA synthesis rate, and the number of nucleotides synthesized before the DNA polymerase binds to the substrate DNA and leaves. On the other hand, it lacks 3′-5 ′ exonuclease activity, so that it is characterized by low accuracy during DNA synthesis and low thermal stability. In addition, since the base taken in by mistake cannot be removed, the amplification efficiency in the PCR reaction is generally low, and the length of the target that can be amplified is limited to about 5 kb.

On the other hand, as a method for compensating for these disadvantages in the Pol I type enzyme, a technique of adding a trace amount of α type enzyme to the Pol I type enzyme is known (Patent Document 1). Although this method improved the amplification efficiency in the PCR reaction and enabled amplification of a relatively long target, in terms of “accuracy” for accurately amplifying the target sequence, the PolI type enzyme alone was used. There is still a problem that it cannot be used for applications such as cloning that require accuracy. In contrast, α-type enzymes typified by KOD and Pfu have 3′-5 ′ exonuclease activity, and thus have high characteristics during DNA synthesis and are excellent in thermal stability. Further, KOD DNA polymerase is widely used as a highly accurate PCR enzyme because it is an α-type enzyme but has a high DNA synthesis rate and excellent processability.

On the other hand, however, in the case of α-type enzyme in particular, it has 3′-5 ′ exonuclease activity, so nonspecific amplification is likely to occur, and deamination of dCTP (2′-deoxycytidine 5′-triphosphate). Incorporation of dUTP (2′-Deoxyuridine, 5′-Triphosphate) generated by the reaction causes the reaction to stop there, resulting in a decrease in the amount of target DNA amplification or the inability to amplify long target DNA. was there.

For these disadvantages, the hot start method using an antibody that suppresses 3′-5 ′ exonuclease activity (Patent Document 2) and the improvement to a buffer composition that has the effect of suppressing nonspecific amplification. (Patent Document 3) reports that nonspecific amplification can be suppressed. Furthermore, as shown in Patent Document 4 and Non-Patent Document 1, the synthesis reaction is stopped by dUTP incorporation. By adding dUTPase (dUTP pyrophosphate) during the PCR reaction, dUTP is decomposed and the synthesis reaction is stopped. By doing so, it is known that the amplification efficiency can be increased or the target length that can be amplified can be improved.

Furthermore, not only α-type enzymes but also various additives have been studied and reported to be effective for the purpose of improving PCR performance. For example, as chemical additives, Tm lowering agents such as DMSO and betaine (trimethylglycine), and as protein additives, SSB (Single Strand Binding Protein), PCNA (Proliferating cell nuclear antigen), RFCS (RFCS) A replication factor C small subunit (RFC), a replication factor C large subunit (RFCL), and the like are known. These additives enhance the polymerase reaction by promoting the binding of template DNA and PCR primers, or template DNA and DNA polymerase.

However, even if these additives are added, the target DNA may not be amplified or a sufficient amount of amplification product may not be obtained depending on the length of the target DNA and the form of the sample. For example, it is impossible to accurately amplify a target having a length of more than 30 kb using human genomic DNA as a template, and it has not been possible with conventional techniques. Establishment of an excellent system for nucleic acid amplification is required.

US Pat. No. 5,436,149 Japanese Patent No. 3968606 Patent Publication 2003-144169 Special Table 2002-505572

Proc. Natl. Acd. Sci. USA 99,596-601 (2002) J. et al. Biol. Chem. 273, 6024-6029

As described above, although various related technologies have been reported for nucleic acid amplification, there is a limit to the DNA amplification efficiency and the target DNA length that can be amplified, and further improvement of DNA synthesis activity, that is, improvement of PCR performance. Is required.

In order to further improve the DNA synthesis activity of DNA polymerases, particularly KOD DNA polymerase and other DNA polymerases belonging to Family B, the present inventors have conducted extensive studies on the improvement of the composition thereof. As a result, betaine (trimethylglycine) ) In the presence of the present invention, it was found that PCR performance can be greatly improved by adding dUTTPase derived from archaea such as Sulfolobus isladicus rod-shaped virus (SIRV) and Thermococcus Kodakaraensis KOD1 strain. It came to.

That is, according to the present invention, the following is provided.
(1) A composition for nucleic acid synthesis, comprising an archaic DNA polymerase, a polypeptide having dUTPase activity, and betaine (trimethylglycine).
(2) The composition for nucleic acid synthesis according to (1), wherein the DNA polymerase is a DNA polymerase belonging to Family B.
(3) The composition for nucleic acid synthesis according to (2), wherein the DNA polymerase is KOD DNA polymerase.
(4) The composition for nucleic acid synthesis according to any one of (1) to (3), wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from archaea.
(5) The composition for nucleic acid synthesis according to (4), wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from SIRV (Sulfobus islandicus rod-shaped virus) or Thermococcus Kodakaraensis.
(6) The composition for nucleic acid synthesis according to (5), wherein the polypeptide having dUTPase activity is any of the following (a) to (c).
(A) polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (b) polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (c) deletion or insertion of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 A polypeptide comprising an amino acid sequence added or substituted and having dUTPase activity (7) A nucleic acid synthesis method using an archaea-derived DNA polymerase, wherein the polypeptide has dUTPase activity and betaine (trimethylglycine) A method for synthesizing nucleic acid, which is performed in the presence of
(8) The nucleic acid synthesis method according to (7), wherein the DNA polymerase is a DNA polymerase belonging to Family B.
(9) The nucleic acid synthesis method according to (8), wherein the DNA polymerase is KOD DNA polymerase.
(10) The nucleic acid synthesis method according to any one of (7) to (9), wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from archaea.
(11) The nucleic acid synthesis method according to (10), wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from SIRV (Sulfobus islandicus rod-shaped virus) or Thermococcus Kodakaraensis.
(12) The nucleic acid synthesis method according to (10), wherein the polypeptide having dUTPase activity is any one of the following (d) to (f).
(D) polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (e) polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (f) deletion or insertion of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 , A polypeptide comprising an added or substituted amino acid sequence and having dUTPase activity

According to the present invention, PCR performance can be dramatically improved in a nucleic acid synthesis method using a DNA polymerase, particularly in a PCR method. That is, the present invention makes it possible to increase the amount of PCR amplification or to amplify long fragments of DNA that have been considered difficult with conventional DNA polymerase compositions.

In particular, when a DNA polymerase belonging to family B such as KOD DNA polymerase is used, the amount of PCR product is increased while maintaining high accuracy of DNA amplification, that is, extremely low misbase incorporation (error) rate. Alternatively, the chain length of the target gene that can be amplified can be improved.

It is a figure which shows the result of having performed the agarose gel electrophoresis of the PCR product in Example 3. FIG. It is a figure which shows the result of having performed the agarose gel electrophoresis of the PCR product in Example 4.

Hereinafter, the present invention will be described in detail.
The first aspect of the present invention is a composition for nucleic acid synthesis containing a DNA polymerase, a polypeptide having dUTPase activity, and betaine.

The type of the DNA polymerase used in the present invention is not particularly limited, and any DNA polymerase is preferably derived from archaea (primordial fungus). Examples of archaea include Thermococcus genus, Pyrococcus genus, Sulfolobus genus, Aeropyrus genus, and the like. Some of the DNA polymerases derived from archaea have the activity of digesting the DNA strand from the end in the direction from the 3 ′ end to the 5 ′ end of the DNA and cutting it (3′-5 ′ exonuclease activity (DNA proofreading activity)), These are preferably used in order to synthesize DNA with high accuracy. These DNA polymerases further have a mechanism for detecting dUTP incorporation and stopping the elongation of the DNA strand by the polymerase reaction there.

In the archaic DNA polymerase, it is more preferable to use a DNA polymerase belonging to Family B. Family B (α type) DNA polymerases that can be purchased in the current market include KOD DNA polymerase (Toyobo), Pfu DNA polymerase (Stratagene, etc.), Pwo DNA polymerase (Roche Applied Science), Ultimate Examples thereof include DNA polymerase (Invitrogen) and PrimeSTAR DNA polymerase (Takara Bio). Among these, KOD DNA polymerase is more preferable, but the DNA polymerase used in the present invention is a DNA polymerase having a mechanism for detecting dUTP incorporation and stopping the elongation of the DNA strand by the polymerase reaction. Any of them can be applied and is not particularly limited.

The present invention is characterized by containing a polypeptide having dUTPase activity. The dUTPase activity refers to an enzyme activity that hydrolyzes a phosphate bond in dUTP. Due to the presence of the polypeptide having dUTPase activity, it is possible to prevent termination of the synthesis reaction caused by the incorporation of dUTP during the nucleic acid synthesis reaction.

The dUTPase activity is measured by measuring the amount of dUTP that decreases per unit time in the reaction of dUTP → dUMP + PPi (pyrophosphate) catalyzed by dUTPase, or measuring the amount of pyrophosphate produced. A method is mentioned.
In the present specification, the latter (quantification of pyrophosphate) was specifically performed as follows.
The dUTPase whose activity is to be measured is added to a dUTP solution (10 mM dUTP, 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl ₂ ), reacted at 80 ° C. for 1 hour, and then the amount of pyrophosphate produced is treated with pyrophosphate. Quantification is performed using a quantitative reagent (Pyrophosphatase reagent: Sigma). The activity is defined as “1 unit is the amount of enzyme that produces 0.1 μmol of pyrophosphate in a reaction at 80 ° C. for 1 hour”.

Any polypeptide can be used as long as it has dUTPase activity, but a polypeptide having dUTPase activity derived from archaea can be preferably used. Specifically, dUTPas derived from archaviruses such as Sulfolobus isladicus rod-shaped virus (SIRV) and dUTase derived from archaea such as Thermococcus Kodakaaraensis KOD1 strain and Pyrococcus furiosus.

More specifically, a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 is preferable as the dUTPase (SIRV-dUTPase) derived from Sulfolobus isladicus rod-shaped virus. Alternatively, the amino acid sequence shown in SEQ ID NO: 1 is preferably composed of an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted, and has dUTPase activity. That is, the polypeptide may be one obtained by modifying the amino acid sequence shown in SEQ ID NO: 1 by introducing a mutation into a specific site by a known technical method. Viewed from another aspect, it preferably has 70% or more homology with the amino acid sequence set forth in SEQ ID NO: 1. More preferably, those having a homology of 80% or more, more preferably 90% or more, and most preferably 95% or more of homology.

Moreover, as dUTPase (KOD-dUTPase) derived from Thermococcus Kodakaraensis KOD1, a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 is preferable. Alternatively, the amino acid sequence shown in SEQ ID NO: 2 is preferably composed of an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted, and has dUTPase activity. That is, the polypeptide may be one obtained by modifying the amino acid sequence shown in SEQ ID NO: 2 by introducing a mutation into a specific site by a known technical method. Viewed from another aspect, the amino acid sequence described in SEQ ID NO: 2 preferably has a homology of 70% or more. Further, those having a homology of 80% or more, more preferably 90% or more, and most preferably 95% or more are preferred.

The amount of dUTPase to be added is the final concentration in the nucleic acid synthesis reaction composition, and the lower limit is preferably 0.001 ng / μl, more preferably 0.005 ng / μl, more preferably 0.01 ng / μl. . A preferable concentration as the upper limit is 10 ng / μl, more preferably 6 ng / μl, and more preferably 4 ng / μl. If it is less than the lower limit, the effect of improving the performance of nucleic acid synthesis is not so much seen.

In the present invention, betaine is further used. Betaine used in the present invention is a general term for compounds in which three methyl groups are attached to the amino group of an amino acid. In the present invention, betaine is used as a name indicating trimethylglycine (molecular formula: C ₅ H ₁₁ NO ₂ ). It is done. Betaine is contained in plants of the genus Rhododendron, sugar beet, mushrooms, sugar beets, shrimps, crabs and the like. The amount of betaine (trimethylglycine) to be added is the final concentration in the nucleic acid synthesis reaction composition solution. The lower limit is preferably 0.1M, more preferably 0.5M, and more preferably 1M. A preferable concentration as the upper limit is 5M, more preferably 3M, and more preferably 2M. If it is less than the lower limit, the effect of improving the performance of the nucleic acid synthesis reaction is not seen, and even if it is more than the upper limit, the effect of improving the performance of the nucleic acid synthesis that is more outstanding is not seen. In addition, betaine is difficult to dissolve at 5M or more.

By using both a polypeptide having dUTPase activity and betaine in combination, the amplification efficiency of DNA and the size of the target that can be amplified are dramatically improved by the synergistic effect. In particular, it is preferable to use a combination of dUTPase and betaine in the above preferred ranges.

The composition for nucleic acid synthesis in the present invention includes at least the following components.
(1) DNA polymerase (2) Betaine (3) SIRV-dUTPase or KOD-dUTPase
Or at least the following structures are included.
(4) DNA polymerase (5) Betaine (6) SIRV-dUTPase and KOD-dUTPase
Furthermore, the following configuration may be included.
(7) DNA polymerase (8) betaine (9) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a deletion of 1 to several amino acids in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, A polypeptide comprising an inserted, added, or substituted amino acid sequence and having dUTPase activity may further comprise a composition for nucleic acid synthesis comprising at least the following components.
(10) DNA polymerase (11) Betaine (12) Polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, wherein 1 to several amino acids are deleted, A polypeptide comprising an inserted, added, or substituted amino acid sequence and having dUTPase activity

  The composition for nucleic acid synthesis in the present invention may refer to a stock solution (DNA polymerase storage solution) containing DNA polymerase or a reaction solution for nucleic acid synthesis reaction.

  When the nucleic acid synthesis composition is a stock solution containing DNA polymerase, a buffer can be further added. In the case of a configuration containing a reaction buffer, it is preferably contained as a stock solution concentrated to a concentration of about 2 to 20 times, preferably about 2 to 10 times. When this composition for nucleic acid synthesis is used for a nucleic acid synthesis reaction, a reaction buffer or four types of deoxyribonucleoside triphosphates (dNTPs; a mixture of dATP, dCTP, dTTP, and dGTP) as required before the nucleic acid synthesis reaction Furthermore, a nucleic acid synthesis reaction can be performed by adding a metal ion, an inorganic salt, or the like. Alternatively, these optional components can be mixed in advance, and further necessary components can be added before the nucleic acid synthesis reaction to prepare a reaction solution. These types and concentrations can be appropriately set by those skilled in the art.

When the composition for nucleic acid synthesis is used as a reaction solution for nucleic acid synthesis reaction, the composition for nucleic acid synthesis further comprises, if necessary, a reaction buffer for DNA polymerase and four types of deoxyribonucleoside triphosphates as substrates. (DNTPs; a mixture of dATP, dCTP, dTTP and dGTP) and the like. Examples of the reaction buffer include Tris-HCl buffer, Tris-sulfate buffer, Tricine buffer, and the like, and Tris-sulfate buffer is particularly preferable. As a density | concentration, 10-200 mM is preferable and 20-100 mM is more preferable. As pH, the range of 7.0-9.5 is preferable and the range of 7.5-9.0 is more preferable. The reaction buffer preferably contains Mg ²⁺ at a concentration of 1.0 to 5 mM, preferably 1.5 to 2.5 mM. Further, it may contain a salt such as KCl. Moreover, surfactant and other stabilizers may be included as needed.

When the composition for nucleic acid synthesis is a reaction solution for nucleic acid synthesis reaction, the polypeptide having DNA polymerase, betaine, dUTPase activity may be configured in a premixed form, or each It may be in a separate tube and mixed before the reaction to prepare a composition for nucleic acid synthesis.

The nucleic acid synthesis reaction in the present invention refers to a reaction in which a nucleic acid having a complementary sequence is synthesized in a sequence-dependent manner with respect to a template nucleic acid. Specifically, the polymerase chain reaction (PCR) method, the Loop-Mediated Isotropic Amplification (LAMP) method, the Transcribation Reverse Transcribable Reaction Reaction (TRC) method, and the Nucleic Acid Sequential ACID method. Amplification of Nucleic acids (ICAN) method and the like are exemplified. Of these, the PCR method is preferably used.

The composition for nucleic acid synthesis of the present invention has a remarkable effect in that a long region of DNA that could not be synthesized and amplified so far is synthesized very accurately. For example, when a PCR reaction is carried out with a composition for nucleic acid synthesis containing a polypeptide having dUTPase activity and betaine and containing KOD DNA polymerase, the limit of the fragment length capable of amplifying DNA is 6-7 kbp. When only betaine is added and a PCR reaction is performed with a nucleic acid synthesis composition containing KOD DNA polymerase, it is difficult to synthesize DNA of about 8.5 kbp or more. On the other hand, when only a polypeptide having dUTPase activity is added, according to Patent Document 4, only a polypeptide having dUTPase activity derived from Pyrococcus fliosus is used, and DNA polymerase belonging to family B (Pwo DNA polymerase or Pfu DNA polymerase or Pfu) is used. The length of the DNA fragment whose amplification was confirmed by DNA polymerase was around 6.2 kbp. As a result of the PCR reaction performed by the present inventors by adding a polypeptide having dUTPase activity, it was possible to amplify 6.2 kbp or more, but it was not possible to reliably amplify DNA fragments of 10 kbp or more. It was. The difficulty of DNA synthesis becomes exponentially difficult as the chain length of the region to be amplified increases.

However, when the present invention is used, amplification is possible even at around 32 kbp. Moreover, since the DNA polymerase derived from archaea, especially the DNA polymerase belonging to family B, has a proofreading activity, the error rate due to incorporation of a wrong base is 0.001 to 0.01%, compared with the DNA polymerase belonging to family A. Show high accuracy. Therefore, the present invention has a very remarkable effect in that a long fragment of DNA can be accurately amplified.

  The second aspect of the present invention is a nucleic acid synthesis method characterized in that nucleic acid synthesis is performed using a DNA polymerase in the presence of a polypeptide having dUTPase activity and betaine (trimethylglycine).

  The preferable range of the concentration of the polypeptide having dUTPase activity is preferably 0.001 ng / μl, more preferably 0.005 ng / μl, still more preferably 0.01 ng / μl as the lower limit in the nucleic acid synthesis reaction solution. is there. The upper limit is preferably 10 ng / μl, more preferably 6 ng / μl, still more preferably 4 ng / μl. If it is less than the lower limit, the effect of improving the performance of nucleic acid synthesis is not so much seen.

  The preferred range of the concentration of betaine is preferably 0.1M, more preferably 0.5M, and even more preferably 1M as the lower limit in the nucleic acid synthesis reaction solution. The upper limit is preferably 5M, more preferably 3M, and even more preferably 2M. If it is less than the lower limit, the effect of improving the performance of nucleic acid synthesis is not so much seen, and even if it is more than the upper limit, the effect of improving the performance of nucleic acid synthesis that is more conspicuous is not seen.

This nucleic acid synthesis method is further carried out in the presence of a reaction buffer for DNA polymerase, a substrate of four types of deoxyribonucleoside triphosphates (dNTPs; a mixture of dATP, dCTP, dTTP and dGTP), if necessary. Examples of the reaction buffer include Tris-HCl buffer, Tris-sulfate buffer, Tricine buffer, and the like, and Tris-sulfate buffer is particularly preferable. As a density | concentration, 10-200 mM is preferable and 20-100 mM is more preferable. As pH, the range of 7.0-9.5 is preferable and the range of 7.5-9.0 is more preferable. The reaction buffer preferably contains Mg ²⁺ at a concentration of 1.0 to 5 mM, preferably 1.5 to 2.5 mM. Further, it may contain a salt such as KCl. Moreover, it can carry out by adding surfactant and another stabilizer as needed.

  Examples of the nucleic acid synthesis method include a PCR method, a LAMP method, a TRC method, a NASBA method, and an ICAN method, and a nucleic acid synthesis method by the PCR method is preferable.

Those skilled in the art can appropriately set the temperature and time of each step of the denaturation reaction, annealing reaction, extension reaction, and the number of reaction cycles in the PCR method. The present invention is also effective for RT-PCR and real-time PCR using the PCR method.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Cloning of dUTPase (SIRV-dUTPase) derived from Sulfolobus isladicus rod-shaped virus (SIRV) and acquisition of the dUTPase The gene encoding dUTPase (SIRV-dut) was cloned by the following method. It has been reported that the base sequence of SIRV-dut is composed of an open reading frame of 474 base pairs (Non-patent Document 2). Therefore, we synthesized a DNA fragment containing the target sequence by PCR using 16 sets of artificial synthetic primers, linked DNA fragments by nested PCR using this DNA fragment as a template, It came to acquire DNA chain length.

Specifically, cloning of the SIRV-dut gene was performed by nested PCR using 32 synthetic oligonucleotides (SEQ ID NOs: 3 to 34) and KOD-Plus- (manufactured by Toyobo Co., Ltd.). First, [1] SIRVdut f1 and r16 (sequence numbers 3 and 34), [2] SIRVdut f2 and r15 (sequence numbers 4 and 33), [3] SIRVdut f3 and r14 (sequence numbers 5 and 32), [ 4] SIRVdut f4 and r13 (SEQ ID NOS: 6 and 31), [5] SIRVdut f5 and r12 (SEQ ID NOS: 7 and 30), [6] SIRVdut f6 and r11 (SEQ ID NOS: 8 and 29), [7] SIRVdut f7 r10 (SEQ ID NO: 9 and 28), [8] SIRVdut f8 and r9 (SEQ ID NO: 10 and 27), [9] SIRVdut f9 and r8 (SEQ ID NO: 11 and 26), [10] SIRVdut f10 and r7 (SEQ ID NO: 12) And 25), [11] SIRVdut f11 and r6 (SEQ ID NOS: 13 and 24), [12] SIRVdut f12 r5 (SEQ ID NO: 14 and 23), [13] SIRVdut f13 and r4 (SEQ ID NO: 15 and 22), [14] SIRVdut f14 and r3 (SEQ ID NO: 16 and 21), [15] SIRVdut f15 and r2 (SEQ ID NO: 17 And 20), 16 primer sets of [16] SIRVdut f16 and r1 (SEQ ID NOs: 18 and 19) were annealed, and then an extension reaction was performed to form 16 double-stranded DNAs of 45 base pairs (DNA fragment) [1] to [16], [16] only 34 base pairs).

Next, (a) primer: SIRVdut f1 and r15, template: DNA fragment [1] and [2], (b) primer: SIRVdut f3 and r13, template: DNA fragment [3] and [4], (c) Primer: SIRVdut f5 and r11, DNA fragment [5] and [6], (d) Primer: SIRVdut f7 and r9, Template: DNA fragment [7] and [8], (e) Primer: SIRVdut f9 and r7, Template : DNA fragment [9] and [10], (f) Primer: SIRVdut f11 and r5, DNA fragment [11] and [12], (g) Primer: SIRVdut f13 and r3, Template: DNA fragment [13] and [ 14], (h) Primer: SIRVdut f15 and r1, Template: DNA fragment [15] and [16], these 8 sets of primers, DN PCR was carried out using fragments to form a double-stranded DNA8 75-bp (DNA fragment (a) ~ (h), (h) only 64 base pairs).

[17] Primer: SIRVdut f1 and r13, Template: DNA fragments (a) and (b), [18] Primer: SIRVdut f5 and r9, Template: DNA fragments (c) and (d), [19] Primer : SIRVdut f9 and r5, template: DNA fragment (e) and (f), [20] primer: SIRVdut f13 and r1, template: DNA fragment (g) and (h), using these 4 sets of primer and DNA fragment PCR was carried out to form four 135-base-pair double-stranded DNAs (DNA fragments (a) to (h) and (h) only 124 base pairs). Further, (i) primer: SIRVdut f1 and r9, template: DNA fragment [17] and [18], (j) primer: SIRVdut f9 and r1, template: DNA fragment [19] and [20], these two sets PCR was carried out using the primers and DNA fragments, and two double-stranded DNAs of 255 base pairs and 244 base pairs were formed (DNA fragments (i) and (j)). Finally, PCR was performed using SIRVduts f1 and r1 as primers and DNA fragments (i) and (j) as templates to form a double-stranded DNA of 478 base pairs.

Next, in order to construct an expression plasmid vector, PCR was performed using SIRVdut Nde1 and SIRVdut Xba1 (SEQ ID NOs: 36 and 37) as primers and the previous 478 base pair double-stranded DNA as a template, and the amplified DNA fragments were restricted. After digestion with enzymes NdeI and XbaI, the plasmid was ligated to pET11c plasmid (manufactured by Novagen) previously digested with NdeI / NheI. This cloned plasmid was confirmed to have no mutation introduced into the base sequence of the SIRV-dut gene by DNA sequencing analysis. This plasmid was named pDUT (SIRV).

Using E. pDUT (SIRV) After transforming E. coli BL21 (DE3) (Stratagene), the cells were cultured in LB medium at 37 ° C. for about 2 hours. In order to confirm the growth state of the bacterial cells, after confirming that OD660 was 0.5 to 1.0, 1 mM IPTG was added, and expression induction was further performed at 37 ° C. for about 5 hours. The cells were collected from the obtained culture solution, suspended in a cell disruption buffer (30 mM Tris-HCl, pH 8, 30 mM NaCl, 0.1 mM EDTA) and subjected to ultrasonic disruption. This crushed suspension is heated (80 ° C., 30 minutes), and the microbial cell fragments are separated by centrifugation, and then added with 50 mM KCl and 0.2% polyethyleneimine at a final concentration at room temperature. The nucleic acid removal treatment was performed with stirring for 30 minutes. After adding ammonium sulfate to this denucleic acid treatment solution to precipitate 0.5 to precipitate the target protein, purification buffer 1 (50 mM Tris-HCl, pH 8, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, (5% glycerol). This salting-out redissolved solution was desalted with a G-25 column, adsorbed on heparin chromatography, and the target protein was eluted and fractionated with a gradient of 200 to 1000 mM based on the purification buffer 1. Fractions eluted in the vicinity of the expected molecular weight (18 kD) of SIRV-dUTPase by SDS-PAGE are pooled, and buffer 2 (50 mM Tris-HCl, pH 8, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT) Dialyzed with 10% glycerol, 0.001% Tween®-20, 0.001% Nonidet P-40). Next, a sufficient amount of final shape buffer (50 mM Tris-HCl, pH 8, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.001% Tween®-20, 0.001% Nonidet P-40) was dialyzed several times to obtain a purified SIRV-dUTPase preparation for evaluation.

Cloning of dUTPase (KOD-dUTPase) from Thermococcus Kodakaraensis KOD1 strain and acquisition of the dUTPase For dUTTPase derived from Thermococcus Kodakaraensis KOD1 strain (hereinafter referred to as “KOD-dut”). That is, referring to the nucleotide sequence of KOD-dut (database name: GenBank, Accession No. NC — 006624) published in a public database, PCR primers for cloning: KODdut f1 and r1 (SEQ ID NOs: 38 and 39) were synthesized. did. It should be noted that an NdeI restriction enzyme site was added to the 5 ′ end of KODdut f1 in order to facilitate transfer of the amplified fragment. PCR was performed using these primers and KOD-Plus- (manufactured by Toyobo Co., Ltd.), using genomic DNA derived from Thermococcus Kodakaraensis KOD1 as a template. Next, the obtained amplification product was subjected to TA cloning into a vector pTA2 (manufactured by Toyobo Co., Ltd.) using TARGET Clone-Plus- (manufactured by Toyobo Co., Ltd.). The obtained plasmid (pTA2-KOD-dut) was confirmed for its nucleotide sequence by DNA sequencing to confirm that the target KOD-dut gene was cloned.

Next, in order to prepare an expression plasmid vector, the KOD-dut gene was excised from pTA2-KOD-dut with restriction enzymes NdeI and BamHI and subcloned into pET14b plasmid (manufactured by Novagen) previously cleaved with NdeI / BamHI. did. This plasmid was named pDUT (KOD). Using this pDUT (KOD), E.I. After transforming E. coli BL21 (DE3), the enzyme was purified by the same purification method as in Example 1 to obtain a purified sample of KOD-dUTPase.

Nucleic acid amplification effect by dUTPase and betaine Using SIRV-dUTPase obtained in Example 1 and KOD-dUTPase obtained in Example 2, various strands using human genomic DNA (200 ng / 50 μl) as templates PCR was performed targeting the long gene region, and the effect of adding dUTPase and betaine to the PCR reaction was evaluated. As targets, 3.6 kbp, 8.5 kbp, 17.5 kbp, 32 kbp of the HBg (human β-globin) gene region and 24 kbp of the tPA (human tissue plasminogen activator) gene region were used. PCR was performed using KOD Plus Ver.2 (Toyobo Co., Ltd.) with the reaction composition shown in Table 1 and the primer set and PCR cycle conditions shown in Table 2.

  As a comparative example, the same test was performed when no additive was added, only SIRV-dUTPase was added, only KOD-dUTPase was added, and only 1.5 M betaine was added.

PCR was performed using a thermal cycler (GeneAmp (registered trademark) PCR System 9700; Applied Biosystems) and maintained at 4 ° C. after completion of the PCR reaction. Thereafter, 5 μl of the PCR product was subjected to 1% agarose electrophoresis to confirm whether the target of interest was amplified. These results are shown in Table 3 and FIG.

As can be seen from Table 3 and FIG. 1, 8.5 kbp nucleic acid could not be amplified in the composition without additives. Furthermore, under the condition where dUTPase or betaine is added alone, 8.5 kbp can be reliably amplified, but longer targets such as 17.5 kbp and 24 kbp cannot be amplified at all, or weak amplification. Only was seen.

On the other hand, it was confirmed that even when dUTPase and betaine were added at the same time, not only amplification of 17.5 kbp and 24 kbp but also amplification product of 32 kbp could be obtained. In addition, as can be seen from the 8.5 kbp amplification, when dUTPase and betaine coexist, the band of the amplification product is very strong compared to the case where each is added alone, and the amplification efficiency is greatly improved. It was shown that. As described above, it was confirmed that the dUTPase and betaine were allowed to act simultaneously to dramatically improve the amplification efficiency of the DNA and the size of the target that can be amplified. An archaea-derived DNA polymerase, particularly a DNA polymerase belonging to family B, exhibits 3'-5 'exonuclease activity and proofreading activity, and thus exhibits extremely high accuracy in DNA synthesis. It has been shown that the present invention has an effect that is not found in the prior art in terms of accurately synthesizing long fragments of DNA.

Reconfirmation of nucleic acid amplification effect of long nucleic acid fragment by SIRV-dUTPase and KOD-dUTPase again using KOD-dUTPase obtained in Example 2 and SIRV-dUTPase obtained in Example 1 Under the same conditions as in Example 3, PCR was performed using human genomic DNA as a template and the HBg gene (32 kbp) as a target.

After the PCR reaction, 5 μl of the PCR product was subjected to 1% agarose gel electrophoresis. The results are shown in FIG. Even in the case of using KOD-dUTPase, as in the case of using SIRV-dUTPase, amplification of 32 kbp, which is not observed with betaine alone, is possible, and the size that can be amplified reliably by coexistence with dUTPase and betaine. It can be confirmed that it can be improved. In addition, since there is almost no difference in the effect between KOD-dUTPase and SIRV-dUTPase, this effect of significantly improving the PCR performance due to the coexistence of dUTPase and betaine is not limited to these two types of dUTPase, It is speculated that the same effect can be obtained with other dUTPases, especially dUTPase having heat resistance derived from archaea.

Since the present invention is superior in amplification efficiency compared to conventionally known nucleic acid synthesis methods and exhibits good amplification even with respect to the amplification of long-sized target DNAs, gene biology as well as in the field of molecular biology research. It is expected to be used in a wide range of fields such as forensic medicine, food and environmental microbiology.

Claims (12)

A composition for nucleic acid synthesis, comprising only an archaic DNA polymerase as a DNA polymerase, a polypeptide having dUTPase activity, and betaine (trimethylglycine).   The composition for nucleic acid synthesis according to claim 1, wherein the DNA polymerase is a DNA polymerase belonging to Family B.   The composition for nucleic acid synthesis according to claim 2, wherein the DNA polymerase is KOD DNA polymerase.   The composition for nucleic acid synthesis according to any one of claims 1 to 3, wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from archaea.   The composition for nucleic acid synthesis according to claim 4, wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from SIRV (Sulfobus islandicus rod-shaped virus) or derived from Thermococcus Kodakaraensis. The composition for nucleic acid synthesis according to claim 5, wherein the polypeptide having dUTPase activity is any of the following (a) to (c).
(A) polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (b) polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (c) deletion or insertion of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 , A polypeptide comprising an added or substituted amino acid sequence and having dUTPase activity A method of synthesizing nucleic acid using only archaic DNA polymerase as a DNA polymerase, wherein the method is performed in the presence of a polypeptide having dUTPase activity and betaine (trimethylglycine). The nucleic acid synthesis method according to claim 7, wherein the DNA polymerase is a DNA polymerase belonging to Family B. The nucleic acid synthesis method according to claim 8, wherein the DNA polymerase is KOD DNA polymerase.   The nucleic acid synthesis method according to any one of claims 7 to 9, wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from archaea. The method for synthesizing nucleic acid according to claim 10, wherein the polypeptide having dUTPase activity is a polypeptide having dUTPase activity derived from SIRV (Sulfobus islandicus rod-shaped virus) or derived from Thermococcus Kodakaraensis. The method for synthesizing nucleic acid according to claim 11, wherein the polypeptide having dUTPase activity is any of the following (d) to (f).
(D) polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (e) polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 (f) deletion or insertion of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 , A polypeptide comprising an added or substituted amino acid sequence and having dUTPase activity