WO2016013620A1 - DNA AMPLIFICATION METHOD USING DEAD-box RNA HELICASE DERIVED FROM THERMOPHILIC ARCHAEA OR VARIANT THEREOF, AND DEAD-box RNA HELICASE DERIVED FROM THERMOPHILIC ARCHAEA AND VARIANT THEREOF TO BE USED FOR DNA AMPLIFICATION METHOD - Google Patents

DNA AMPLIFICATION METHOD USING DEAD-box RNA HELICASE DERIVED FROM THERMOPHILIC ARCHAEA OR VARIANT THEREOF, AND DEAD-box RNA HELICASE DERIVED FROM THERMOPHILIC ARCHAEA AND VARIANT THEREOF TO BE USED FOR DNA AMPLIFICATION METHOD Download PDF

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WO2016013620A1
WO2016013620A1 PCT/JP2015/070980 JP2015070980W WO2016013620A1 WO 2016013620 A1 WO2016013620 A1 WO 2016013620A1 JP 2015070980 W JP2015070980 W JP 2015070980W WO 2016013620 A1 WO2016013620 A1 WO 2016013620A1
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dead
variant
rna helicase
derived
pcr
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藤原　伸介
綾子 藤原
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学校法人関西学院
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  • the present invention relates to a DNA amplification method using a thermophilic archaea-derived DEAD-box type RNA helicase and a variant thereof, and a thermophilic archaea-derived DEAD-box type RNA helicase and a variant thereof used for the method.
  • a DNA amplification method by polymerase chain reaction (PCR) is widely used.
  • the PCR method has a problem that a sequence different from a desired DNA sequence is erroneously amplified.
  • PCR is carried out using two pairs of primer sets that target both ends of the base sequence to be amplified and have complementary sequences thereto.
  • Non-specific amplification occurs when the primer misanneals with a base sequence other than the target.
  • misannealing is likely to occur when the target base sequence is a palindromic sequence or has a high GC content.
  • the primer dimer is introduced between the preparation of the PCR reaction solution containing the DNA polymerase and the primer and the start of PCR.
  • side reactions such as degradation of the primer proceed. This is known to cause nonspecific amplification.
  • Non-Patent Documents 1 to 3 Conventionally, as a means for suppressing such misamplification, a primer design has been devised or an annealing temperature has been improved. In addition, when using a DNA polymerase having proofreading activity, these activities are suppressed at room temperature by binding two kinds of neutralizing antibodies against the polymerase activity and the proofreading activity to the DNA polymerase. It has also been performed to suppress side reactions that can cause amplification (Non-Patent Documents 1 to 3).
  • An object of the present invention is to provide means for suppressing nonspecific amplification in PCR.
  • thermophilic archaia-derived DEAD-box RNA helicase The inventors of the present invention have intensively studied to solve the above problems and completed the present invention. Specifically, the present inventors have found that nonspecific amplification can be suppressed by performing PCR in the presence of a thermophilic archaia-derived DEAD-box RNA helicase. The present inventors have conducted further studies and have found that such effects are derived from heat resistance and helicase activity among the functions and properties of thermophilic archaia-derived DEAD-box RNA helicase.
  • Item 1 A DNA amplification method by polymerase chain reaction, A method comprising a step of performing a polymerase chain reaction in the presence of a thermophilic archaea-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
  • Item 2. Item 8. The method according to Item 1, wherein the thermophilic archaea-derived DEAD-box RNA helicase comprises the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7.
  • Item 3. Item 3.
  • Item 2 wherein the variant comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 1 to 8.
  • Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the variant exhibits helicase activity at 90 ° C.
  • Item 5. The method according to any one of Items 1 to 4, wherein the variant exhibits unwinding activity of a double-stranded nucleic acid at 50 ° C and ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C.
  • Item 6. Item 6.
  • thermophilic archaea-derived DEAD-box type RNA helicase The method according to any one of Items 1 to 5, wherein the step is a polymerase chain reaction in the presence of 20 to 150 nM of the thermophilic archaea-derived DEAD-box type RNA helicase.
  • Item 7. The method according to any one of Items 1 to 6, wherein the step is a step of performing a polymerase chain reaction in the presence of magnesium ions.
  • FIG. 1 is a drawing schematically showing an action mechanism in which non-specific proliferation in PCR is suppressed by the action of a thermophilic archaea-derived DEAD-box type RNA helicase or a variant thereof used in the present invention. It is drawing which shows the measurement result of the ATPase activity of an Example. It is drawing which shows the evaluation result of the influence on PCR amplification by SF2 helicase addition of an Example. Is a view showing verification results of PCR efficiency due to the high concentration MgSO 4 addition Example. It is drawing which shows the result of the nonspecific band disappearance test by TK0566 addition of an Example.
  • Thermophilic archaea-derived DEAD-box RNA helicase has a characteristic motif of Asp-Glu-Ala-Asp and exhibits RNA helicase activity.
  • thermophilic archaia-derived DEAD-box RNA helicase used in the present invention is derived from a thermophilic archaea, and thus exhibits helicase activity even in a high-temperature environment. Although it does not specifically limit, Preferably helicase activity is shown in 90 degreeC.
  • the thermophilic archaea-derived DEAD-box RNA helicase used in the present invention is preferably derived from a hyperthermophilic archaea.
  • thermophilic archaic DEAD-box RNA helicase used in the present invention are not particularly limited.
  • it is derived from Thermococcus kodakarensis consisting of the amino acid sequence represented by SEQ ID NO: 1. TK0566.
  • TK0566 ortholog can be used instead of TK0566.
  • the TK0566 ortholog is a DEAD-box type RNA helicase and has the same function as TK0566.
  • TK0566 ortholog is considered to be an archaea-specific functional molecule from the results of Bootstrap analysis of the phylogenetic tree.
  • it is important that the RNA helicase used is not inactivated by PCR. Therefore, in the present invention, a DEAD-box RNA helicase derived from a thermophilic archaea can be used.
  • TK0566 orthologs have been found to be present in particular at the gates of Euryarchaota and Clenarchaeota. It is known that the TK0566 ortholog belonging to the Yuriakiota gate shows 77% coverage and 43% minimum (identity) by BLAST (registered trademark) analysis as compared with TK0566. Further, it has been found that the TK0566 ortholog belonging to the Clenarchiota Gate shows a cover rate of 40% and a minimum value (identity) of 27% by BLAST (registered trademark) analysis as compared with TK0566. Therefore, in the present invention, a DEAD-box type RNA helicase derived from a thermophilic bacterium belonging to the urea chiota gate and the clenarchiota gate can be used.
  • thermophile belonging to the Yuriakiota gate is not particularly limited. Examples include those belonging to the genus Methanococcus, the genus Methanococcus, and the like.
  • DEAD-box RNA helicase derived from thermophilic bacteria belonging to the genus Thermococcus include, but are not limited to, those derived from Thermococcus kodakarensis (SEQ ID NO: 1) and the like.
  • thermophilic DEAD-box RNA helicase belonging to the genus Pyrococcus include, but are not limited to, those derived from Pyrococcus furiosus (SEQ ID NO: 2) and the like.
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanothermus include, but are not limited to, those derived from Methanothermus fervidus (SEQ ID NO: 3) and the like.
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanothermobacter include, but are not particularly limited to, those derived from Methanothermobacter marburgens (SEQ ID NO: 4).
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanococcus include, but are not particularly limited to, those derived from Methanococcus voltae (SEQ ID NO: 5).
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanocadococcus include, but are not particularly limited to, those derived from Methanocardococcus jannaschii (SEQ ID NO: 6).
  • thermophilic bacterium belonging to the Clenarchiota is not particularly limited, and examples thereof include those belonging to the genus Pyrobaculum and the genus Sulfolobus.
  • thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Pyrobaculum include, but are not limited to, those derived from Pyrobaculum califontis (SEQ ID NO: 7) and the like. .
  • DEAD-box RNA helicases derived from thermophiles belonging to the genus Sulfolobus include, but are not limited to, those derived from Sulfolobus tokodaii (SEQ ID NO: 8) and the like.
  • thermophilic archaic-derived DEAD-box RNA helicase variant which has heat resistance and helicase activity, is also used. it can.
  • the above-mentioned variant is not particularly limited, but preferably exhibits helicase activity at 90 ° C.
  • the above-mentioned variant is not particularly limited, but preferably exhibits the unwinding activity of the double-stranded nucleic acid at 50 ° C. and the ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C.
  • the unwinding activity of the double-stranded nucleic acid is measured as follows.
  • a radiolabeled substrate is used for measuring the unwinding activity (Table 1).
  • 50 pmol of 63merRNA (ssRNA63) was reacted with [ ⁇ - 32 P] ATP (6000 Ci / mmol) (ParkinElmer, Boston, USA) and T4 polynucleotide kinase (Takara bio) at 37 ° C. for 30 minutes and labeled for 32 minutes. To do. Unreacted ⁇ -ATP is removed using Microspin G-25 (GE Healthcare). 20 pmol of ssRNA33, ssRNA48-5 ′, and ssRNA48-3 ′ were added to 10 pmol of labeled ssRNA63, respectively, and treated at 95 ° C.
  • both-end protruding dsRNA, 5 ′ protruding dsRNA, and 3 ′ protruding dsRNA are prepared. This is subjected to Microspin S-400HR to remove RNA that has not been annealed. The obtained dsRNA and unannealed ssRNA are confirmed using a 7% non-denaturing acrylamide gel. The unwinding activity is measured using this ssRNA and the prepared dsRNA as a substrate.
  • the rewinding activity is measured as follows. 10 ⁇ L of a reaction solution containing 32 P-labeled nucleic acid substrate, 0.2 ⁇ M purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of RNase inhibitor, 50 mM HEPES (pH 7.6) is reacted at 50 ° C. for 30 minutes. Immediately afterwards, stop the reaction by transferring to ice. The reaction product is applied to a 7% acrylamide gel (NATIVE-PAGE) (Table 2). After drying the gel, the radioactivity of the reaction product is detected by BAS-2500 (FUJIFILM) and exposed to X-ray film.
  • ATPase activity in the presence of double-stranded nucleic acid is measured as follows.
  • ATPase activity is measured by measuring the amount of free phosphate released when ATP is converted to ADP.
  • ssRNA63 SEQ ID NO: 9
  • the total amount of the reaction solution is 10 ⁇ L, and contains 5 nM nucleic acid substrate, 0.2 ⁇ M purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of Ribonuclease inhibitor (Humanplacenta) (Takarabio), 50 mM HEPES (pH 7.6). This is reacted at 90 ° C. for 30 minutes and immediately transferred to ice to stop the reaction.
  • the amount of free phosphoric acid in the reaction solution is measured using BIOMOLGREEN TM (BIOMOL), and Ab 530 is measured using Multiskan Spectrum (ThermoLabsystems).
  • the variant is not particularly limited, and consists of an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, or added in TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8. It may be a thing.
  • the variant is preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, in TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8, Most preferably, it consists of an amino acid sequence in which one to several amino acids are deleted, substituted or added.
  • the modified body may alternatively be composed of an amino acid sequence having a homology of 80% or more with TK0566 or TK0566 ortholog comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 8.
  • Said variant is a TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, more preferably an amino acid sequence having a homology of 90% or more, more preferably a homology Consists of 95% or more amino acid sequence.
  • TK0566 ortholog is SF2 helicase, and its structural analysis has already been done. It can be roughly divided into an N-terminal region, a helicase core and a C-terminal region from the N-terminal side, and the helicase core has a structure in which domain 1 and domain 2 are connected in this order from the N-terminal side. The DEAD motif is present in domain 1.
  • higher-order structural analysis has also been performed.
  • the modified body can be prepared. Specifically, the above-mentioned variant is introduced by introducing amino acid alterations in other regions as necessary while preserving the regions important for maintaining heat resistance and helicase activity or minimizing the degree of modification. Should be created.
  • homology is determined by comparing two optimally aligned sequences.
  • a method for aligning and comparing the sequences for example, an algorithm of Basic Local Alignment Search Tool (BLAST) can be used.
  • BLAST Basic Local Alignment Search Tool
  • homology in the present invention refers to “identity” in BLAST.
  • the homology may be calculated using another algorithm.
  • DNA amplification method by PCR comprises: It is a method characterized in that PCR is carried out in the presence of a thermophilic archaic-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
  • Nonspecific amplification can be suppressed by the action of a thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof.
  • Such effects are derived from heat resistance and helicase activity among the functions and properties of thermophilic archaea-derived DEAD-box RNA helicase.
  • thermophilic archaia-derived DEAD-box RNA helicase or a variant thereof does not lose its helicase activity in PCR due to its heat resistance.
  • thermophilic archaia-derived DEAD-box RNA helicase or a modified product thereof was obtained by non-specifically annealing the primer to the nucleic acid due to its helicase activity, as schematically shown in FIG. Unwind the nucleic acid portion of the strand. This function suppresses nonspecific DNA growth.
  • PCR is not particularly limited and can be performed under a wide range of conditions.
  • thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof it is preferable to perform PCR in the presence of 20 to 150 nM thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof.
  • PCR is performed in the presence of 20 nM or more thermophilic archaic DEAD-box type RNA helicase or a variant thereof, non-specific amplification can be effectively suppressed.
  • thermophilic archaic-derived DEAD-box type RNA helicase of 150 nM or less or a modified form thereof the amount of specifically amplified DNA is improved.
  • the polymerase chain reaction is preferable to perform the polymerase chain reaction in the presence of magnesium ions in terms of the effects of the present invention.
  • the polymerase chain reaction is more preferably carried out in the presence of 0.5 mM to 3 mM magnesium ions, although not particularly limited.
  • magnesium ions are not particularly limited to the reaction system, but for example, magnesium ions may be added as magnesium sulfate.
  • composition used for DNA amplification method by PCR contains a thermophilic archaea-derived DEAD-box type RNA helicase, or a variant thereof having heat resistance and helicase activity.
  • composition of the present invention further includes, for example, a buffer such as Tris-HCl, a salt such as KCl, a redox agent such as dithiothreitol, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), glycerol, It may contain at least one selected from the group consisting of surfactants such as Tween-20 and NP-40.
  • a buffer such as Tris-HCl
  • a salt such as KCl
  • a redox agent such as dithiothreitol
  • a chelating agent such as ethylenediaminetetraacetic acid (EDTA), glycerol
  • TK0566 Using the total DNA of T. kodakarensis as a template, the TK0566 gene was amplified using primers (TK0566EX-F (SEQ ID NO: 10), TK0566EX-R (SEQ ID NO: 11)). The obtained DNA fragment containing the TK0566 gene was cleaved with NdeI and EcoRI and inserted into pET28a. The constructed pET-TK0566 plasmid was used to transform E. coli BL21-Codon-Plus (DE3) -RIL.
  • IPTG ⁇ -D-1-thiogalactopyranoside
  • the LB medium contains 20 ⁇ g / mL kanamycin and 30 ⁇ g / mL chloramphenicol.
  • the cultured cells were collected, suspended in 10 mL of buffer D: 20 mM Tris-HCl, 500 mM NaCl, 0.1% Triton X-100, pH 7.9, and sonicated.
  • the disrupted solution was centrifuged at 8000 g for 10 minutes, and the supernatant was heat-treated at 80 ° C. for 15 minutes. Then, the supernatant was collected again by centrifugation at 8000 g for 10 minutes. Since His6 tag was added to the N-terminal side of expressed TK0566, it was purified using a Ni column.
  • the column was equilibrated with buffer D, and the supernatant was applied thereto. Then, after washing with buffer E: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.1% Triton X-100, pH 7.9, buffer F: 20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.1 Elute with% Triton X-100, pH 7.9. The eluted fraction was dialyzed against buffer D.
  • ATPase activity was measured by measuring the amount of free phosphate released when ATP was converted to ADP.
  • ssRNA63 63mer RNA
  • the total volume of the reaction solution is 10 ⁇ L, and 5 nM nucleic acid substrate, 0.2 ⁇ M purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of Ribonuclease inhibitor (Human placenta) (Takara bio), 50 mM HEPES (pH 7.6) Including. This was reacted at 50 ° C. to 110 ° C.
  • FIG. 2 shows the measurement results.
  • 16S rDNA was amplified by PCR using primers (TKr05-Fw (SEQ ID NO: 12) and TKr05-Rv (SEQ ID NO: 13)). Further, the toxA gene was amplified using the total DNA genome of P. aeruginosa as a template and using primers (PA1148-Fw (SEQ ID NO: 14), PA1148-Rv (SEQ ID NO: 15)). The obtained amplified fragment was gel-extracted and purified using Nucleo Spin Gel and PCR Clean-up (Takara). The distributor of PAO1 strain genomic DNA is NBRC, NITE, Kisarazu, Japan (NBRC Number: 106052G).
  • FIG. 3 shows the results of PCR under the following PCR reaction composition (Table 3) and PCR conditions (Table 4) in order to verify whether the purified SF2 helicase affects PCR amplification. As shown in FIG. 3, when TK0566 was added, the effect of reducing PCR misamplification was observed.
  • TK0566 can suppress primer misannealing.
  • TK0566 may have increased the specificity of PCR amplification by specifically interacting with KOD polymerase.
  • KOD polymerase thermophilus
  • ALDH2 is an enzyme that oxidizes aldehydes produced by the metabolism of ethanol, and the lack of activity is due to the point mutation E487K of the ALDH2 gene.
  • genes from combinations of alleles normal homozygotes (NN type), heterozygotes (NM type), and mutant homozygotes (MM type). Sensitivity to alcohol can be determined by examining which of these genotypes are present.
  • the PCR reaction composition and conditions are shown below, and the results are shown in FIG.

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Abstract

The present invention addresses the problem of providing a means for inhibiting non-specific amplification in a PCR. As a means for solving this problem, provided is a DNA amplification method through a polymerase chain reaction, characterized in that the polymerase chain reaction is performed in the presence of DEAD-box RNA helicase derived from a thermophilic archaea or a variant thereof that has thermophilic properties and helicase activity.

Description

好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ及びその改変体を用いるＤＮＡ増幅方法、並びにその方法のために使用する好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ及びその改変体DNA amplification method using thermophilic archaia-derived DEAD-box RNA helicase and its variants, and thermophilic archaea-derived DEAD-box RNA helicase and its variants
　本発明は、好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ及びその改変体を用いるＤＮＡ増幅方法、並びにその方法のために使用する好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ及びその改変体に関する。 The present invention relates to a DNA amplification method using a thermophilic archaea-derived DEAD-box type RNA helicase and a variant thereof, and a thermophilic archaea-derived DEAD-box type RNA helicase and a variant thereof used for the method.
　ポリメラーゼ連鎖反応（ＰＣＲ）によるＤＮＡ増幅方法が幅広く利用されている。しかしながら、ＰＣＲ法においては、所望のＤＮＡ配列とは異なる配列が誤って増幅されてしまう問題がある。 A DNA amplification method by polymerase chain reaction (PCR) is widely used. However, the PCR method has a problem that a sequence different from a desired DNA sequence is erroneously amplified.
　ＰＣＲは、増幅しようとする塩基配列の両端部分を標的とし、これらに相補的な配列を有する二対のプライマーセットを用いて行われる。プライマーが標的とは別の塩基配列とミスアニールすることにより、非特異的な増幅が起こる。一般に、標的塩基配列が、回文配列となっていたり、ＧＣ含量が高かったりする場合にミスアニールが起こりやすい。 PCR is carried out using two pairs of primer sets that target both ends of the base sequence to be amplified and have complementary sequences thereto. Non-specific amplification occurs when the primer misanneals with a base sequence other than the target. In general, misannealing is likely to occur when the target base sequence is a palindromic sequence or has a high GC content.
　また、特に３’→５’エキソヌクレアーゼ活性（校正活性）を有するＤＮＡポリメラーゼを用いる場合は、そのＤＮＡポリメラーゼやプライマーを含むＰＣＲ反応溶液を調製してからＰＣＲを開始するまでの間にプライマーダイマーが生成し、その状態でポリメラーゼ活性及びエキソヌクレアーゼ活性が発現するとプライマーが分解するなどの副反応が進行する。このことが非特異的増幅の原因になることが知られている。 In particular, when a DNA polymerase having 3 ′ → 5 ′ exonuclease activity (proofreading activity) is used, the primer dimer is introduced between the preparation of the PCR reaction solution containing the DNA polymerase and the primer and the start of PCR. When the polymerase activity and exonuclease activity are expressed in this state, side reactions such as degradation of the primer proceed. This is known to cause nonspecific amplification.
　従来、このような誤増幅を抑制する手段として、プライマー設計を工夫したり、アニーリング温度を向上させたりすること等が行われてきた。また、校正活性を有するＤＮＡポリメラーゼを使用する場合は、ポリメラーゼ活性及び校正活性それぞれに対する二種類の中和抗体をＤＮＡポリメラーゼに結合させておくことにより常温下でこれらの活性を抑制し、非特異的増幅の原因となりうる副反応を抑制することも行われてきた（非特許文献１～３）。 Conventionally, as a means for suppressing such misamplification, a primer design has been devised or an annealing temperature has been improved. In addition, when using a DNA polymerase having proofreading activity, these activities are suppressed at room temperature by binding two kinds of neutralizing antibodies against the polymerase activity and the proofreading activity to the DNA polymerase. It has also been performed to suppress side reactions that can cause amplification (Non-Patent Documents 1 to 3).
　本発明は、ＰＣＲにおいて、非特異的な増幅を抑制する手段を提供することを課題とする。 An object of the present invention is to provide means for suppressing nonspecific amplification in PCR.
　本発明者らは、上記課題を解決するべく鋭意検討を重ね、本発明を完成させた。具体的には、本発明者らは、好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの存在下でＰＣＲを行うことにより、非特異的な増幅を抑制することができることを見出した。本発明者らは、さらなる検討を重ね、かかる作用効果は、好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの機能及び特性のうち、耐熱性及びヘリカーゼ活性に由来するものであることを突き止めた。 The inventors of the present invention have intensively studied to solve the above problems and completed the present invention. Specifically, the present inventors have found that nonspecific amplification can be suppressed by performing PCR in the presence of a thermophilic archaia-derived DEAD-box RNA helicase. The present inventors have conducted further studies and have found that such effects are derived from heat resistance and helicase activity among the functions and properties of thermophilic archaia-derived DEAD-box RNA helicase.
　本発明はかかる知見に基づきさらに検討を重ねた結果完成されたものであり、下記に掲げるものである。
項１．
ポリメラーゼ連鎖反応によるＤＮＡ増幅方法であって、
好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ、又は
耐熱性及びヘリカーゼ活性を有するその改変体
の存在下でポリメラーゼ連鎖反応を行う工程を含む方法。
項２．
前記好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼが、配列番号１～７のいずれかで表されるアミノ酸配列からなる、項１に記載の方法。
項３．
前記改変体が、配列番号１～８のいずれかで表されるアミノ酸配列と相同性が８０％以上のアミノ酸配列からなる、項２に記載の方法。
項４．
前記改変体が、９０℃においてヘリカーゼ活性を示す、項１～３のいずれか一項に記載の方法。
項５．
前記改変体が、５０℃において二本鎖核酸の巻き戻し活性を示し、かつ９０℃において二本鎖核酸の存在下でＡＴＰａｓｅ活性を示す、項１～４のいずれか一項に記載の方法。
項６．
前記工程が、２０～１５０ｎＭの前記好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの存在下でポリメラーゼ連鎖反応を行う工程である、項１～５のいずれか一項に記載の方法。
項７．
前記工程が、マグネシウムイオンの存在下でポリメラーゼ連鎖反応を行う工程である、項１～６のいずれか一項に記載の方法。
項８．
前記工程が、０．５ｍＭ～３ｍＭのマグネシウムイオンの存在下でポリメラーゼ連鎖反応を行う工程である、項７に記載の方法。
項９．
ポリメラーゼ連鎖反応によるＤＮＡ増幅方法のために用いられる、
好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ、又は
耐熱性及びヘリカーゼ活性を有するその改変体
を含有する、組成物。 The present invention has been completed as a result of further studies based on such knowledge, and is described below.
Item 1.
A DNA amplification method by polymerase chain reaction,
A method comprising a step of performing a polymerase chain reaction in the presence of a thermophilic archaea-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
Item 2.
Item 8. The method according to Item 1, wherein the thermophilic archaea-derived DEAD-box RNA helicase comprises the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7.
Item 3.
Item 3. The method according to Item 2, wherein the variant comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 1 to 8.
Item 4.
Item 4. The method according to any one of Items 1 to 3, wherein the variant exhibits helicase activity at 90 ° C.
Item 5.
Item 5. The method according to any one of Items 1 to 4, wherein the variant exhibits unwinding activity of a double-stranded nucleic acid at 50 ° C and ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C.
Item 6.
Item 6. The method according to any one of Items 1 to 5, wherein the step is a polymerase chain reaction in the presence of 20 to 150 nM of the thermophilic archaea-derived DEAD-box type RNA helicase.
Item 7.
Item 7. The method according to any one of Items 1 to 6, wherein the step is a step of performing a polymerase chain reaction in the presence of magnesium ions.
Item 8.
Item 8. The method according to Item 7, wherein the step is a step of performing a polymerase chain reaction in the presence of 0.5 mM to 3 mM magnesium ions.
Item 9.
Used for DNA amplification method by polymerase chain reaction,
A composition comprising a thermophilic archaea-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
　本発明を利用することにより、非特異的な増幅を抑制しつつ目的配列を増幅することができる。 By utilizing the present invention, it is possible to amplify the target sequence while suppressing nonspecific amplification.
本発明において使用する好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体のはたらきによって、ＰＣＲにおける非特異的な増殖が抑制される作用機序を模式的に表した図面である。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing schematically showing an action mechanism in which non-specific proliferation in PCR is suppressed by the action of a thermophilic archaea-derived DEAD-box type RNA helicase or a variant thereof used in the present invention. 実施例のATPase活性の測定結果を示す図面である。It is drawing which shows the measurement result of the ATPase activity of an Example. 実施例のSF2ヘリカーゼ添加によるPCR増幅への影響の評価結果を示す図面である。It is drawing which shows the evaluation result of the influence on PCR amplification by SF2 helicase addition of an Example. 実施例の高濃度MgSO4添加によるPCR効率の検証結果を示す図面である。Is a view showing verification results of PCR efficiency due to the high concentration MgSO 4 addition Example. 実施例のTK0566添加による非特異バンド消失試験の結果を示す図面である。It is drawing which shows the result of the nonspecific band disappearance test by TK0566 addition of an Example. 実施例のTK0566よるPCR増幅特異性の検証結果を示す図面である。It is drawing which shows the verification result of PCR amplification specificity by TK0566 of an Example. 実施例のTK0566よるPCR増幅特異性の検証結果を示す図面である。It is drawing which shows the verification result of PCR amplification specificity by TK0566 of an Example. 高GC含量鋳型に対するTK0566添加による PCR増幅への影響を検証した実施例の結果を示す図面である。It is drawing which shows the result of the Example which verified the influence on the PCR amplification by addition of TK0566 with respect to a high GC content template. Tth ポリメラーゼ又はTaq ポリメラーゼを用いた時のTK0566添加効果を検証した実施例の結果を示す図面である。It is drawing which shows the result of the Example which verified the TK0566 addition effect at the time of using Tth * polymerase or Taq * polymerase. 実施例のARMS法を用いたALDH2遺伝子型の検出結果を示す図面である。It is drawing which shows the detection result of ALDH2 genotype using ARMS method of an Example.
　１．好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ
　ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼは、Ａｓｐ－Ｇｌｕ－Ａｌａ－Ａｓｐという特徴的なモチーフを有し、ＲＮＡヘリカーゼ活性を示す。 1. Thermophilic archaea-derived DEAD-box RNA helicase DEAD-box RNA helicase has a characteristic motif of Asp-Glu-Ala-Asp and exhibits RNA helicase activity.
　本発明で使用する好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼは、好熱性アーキア由来であるため、高温環境下においてもヘリカーゼ活性を示す。特に限定されないが、好ましくは９０℃においてヘリカーゼ活性を示すものである。本発明で使用する好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼは、好ましくは超好熱性アーキア由来である。 The thermophilic archaia-derived DEAD-box RNA helicase used in the present invention is derived from a thermophilic archaea, and thus exhibits helicase activity even in a high-temperature environment. Although it does not specifically limit, Preferably helicase activity is shown in 90 degreeC. The thermophilic archaea-derived DEAD-box RNA helicase used in the present invention is preferably derived from a hyperthermophilic archaea.
　本発明で使用する好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、例えば、配列番号１で表されるアミノ酸配列からなる、サーモコッカス・コダカレンシス（Ｔｈｅｒｍｏｃｏｃｃｕｓ　ｋｏｄａｋａｒｅｎｓｉｓ）由来のＴＫ０５６６が挙げられる。 Specific examples of the thermophilic archaic DEAD-box RNA helicase used in the present invention are not particularly limited. For example, it is derived from Thermococcus kodakarensis consisting of the amino acid sequence represented by SEQ ID NO: 1. TK0566.
　本発明では、ＴＫ０５６６の替わりに、ＴＫ０５６６オルソログも使用することができる。ＴＫ０５６６オルソログは、ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼであり、ＴＫ０５６６と同様の機能を有している。 In the present invention, a TK0566 ortholog can be used instead of TK0566. The TK0566 ortholog is a DEAD-box type RNA helicase and has the same function as TK0566.
　ＴＫ０５６６オルソログは、系統樹のＢｏｏｔｓｔｒａｐ解析の結果から、アーキア特有の機能分子であると考えられる。また、本発明においては、使用するＲＮＡへリカーゼがＰＣＲによっては失活しないことが重要である。したがって、本発明では好熱性アーキア由来のＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼを使用することができる。 TK0566 ortholog is considered to be an archaea-specific functional molecule from the results of Bootstrap analysis of the phylogenetic tree. In the present invention, it is important that the RNA helicase used is not inactivated by PCR. Therefore, in the present invention, a DEAD-box RNA helicase derived from a thermophilic archaea can be used.
　ＴＫ０５６６オルソログは、特に、ユリアーキオータ門（Ｅｕｒｙａｒｃｈａｅｏｔａ）及びクレンアーキオータ門（Ｃｒｅｎａｒｃｈａｅｏｔａ）に存在することが判っている。ユリアーキオータ門に属するＴＫ０５６６オルソログは、ＴＫ０５６６と比較すると、ＢＬＡＳＴ（登録商標）解析で、７７％のカバー率と４３％の最低値（ｉｄｅｎｔｉｔｙ）を示すことが判っている。また、クレンアーキオータ門に属するＴＫ０５６６オルソログは、ＴＫ０５６６と比較すると、ＢＬＡＳＴ（登録商標）解析で、４０％のカバー率と２７％の最低値（ｉｄｅｎｔｉｔｙ）を示すことが判っている。したがって、本発明では、ユリアーキオータ門及びクレンアーキオータ門に属する好熱菌由来のＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼを使用することができる。 TK0566 orthologs have been found to be present in particular at the gates of Euryarchaota and Clenarchaeota. It is known that the TK0566 ortholog belonging to the Yuriakiota gate shows 77% coverage and 43% minimum (identity) by BLAST (registered trademark) analysis as compared with TK0566. Further, it has been found that the TK0566 ortholog belonging to the Clenarchiota Gate shows a cover rate of 40% and a minimum value (identity) of 27% by BLAST (registered trademark) analysis as compared with TK0566. Therefore, in the present invention, a DEAD-box type RNA helicase derived from a thermophilic bacterium belonging to the urea chiota gate and the clenarchiota gate can be used.
　ユリアーキオータ門に属する好熱菌としては、特に限定されないが、例えば、サーモコッカス（Ｔｈｅｒｍｏｃｏｃｃｕｓ）属、パイロコッカス（Ｐｙｒｏｃｏｃｃｕｓ）属、メタノサーマス（Ｍｅｔｈａｎｏｔｈｅｒｍｕｓ）属、メタノサーモバクター（Ｍｅｔｈａｎｏｔｈｅｒｍｏｂａｃｔｅｒ）属、メタノコッカス（Ｍｅｔｈａｎｏｃｏｃｃｕｓ）属及びメタノカルドコッカス（Ｍｅｔｈａｎｏｃａｌｄｃｏｃｃｕｓ）属等に属するものが挙げられる。 The thermophile belonging to the Yuriakiota gate is not particularly limited. Examples include those belonging to the genus Methanococcus, the genus Methanococcus, and the like.
　サーモコッカス属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、上記のサーモコッカス・コダカレンシス由来（配列番号１）等が挙げられる。 Specific examples of DEAD-box RNA helicase derived from thermophilic bacteria belonging to the genus Thermococcus include, but are not limited to, those derived from Thermococcus kodakarensis (SEQ ID NO: 1) and the like.
　パイロコッカス属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、パイロコッカス・フリオサス（Ｐｙｒｏｃｏｃｃｕｓ　ｆｕｒｉｏｓｕｓ）由来（配列番号２）等が挙げられる。 Specific examples of thermophilic DEAD-box RNA helicase belonging to the genus Pyrococcus include, but are not limited to, those derived from Pyrococcus furiosus (SEQ ID NO: 2) and the like.
　メタノサーマス属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、メタノサーマス・フェルヴィドス（Ｍｅｔｈａｎｏｔｈｅｒｍｕｓ　ｆｅｒｖｉｄｕｓ）由来（配列番号３）等が挙げられる。 Specific examples of thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanothermus include, but are not limited to, those derived from Methanothermus fervidus (SEQ ID NO: 3) and the like.
　メタノサーモバクター属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、メタノサーモバクター・マルバージェンス（Ｍｅｔｈａｎｏｔｈｅｒｍｍｏｂａｃｔｅｒ　ｍａｒｂｕｒｇｅｎｓ）由来（配列番号４）等が挙げられる。 Specific examples of thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanothermobacter include, but are not particularly limited to, those derived from Methanothermobacter marburgens (SEQ ID NO: 4).
　メタノコッカス属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、メタノコッカス・ヴォルタエ（Ｍｅｔｈａｎｏｃｏｃｃｕｓ　ｖｏｌｔａｅ）由来（配列番号５）等が挙げられる。 Specific examples of thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanococcus include, but are not particularly limited to, those derived from Methanococcus voltae (SEQ ID NO: 5).
　メタノカルドコッカス属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、メタノカルドコッカス・ジャナシ（Ｍｅｔｈａｎｏｃａｌｄｏｃｏｃｃｕｓ　ｊａｎｎａｓｃｈｉｉ）由来（配列番号６）等が挙げられる。 Specific examples of thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Methanocadococcus include, but are not particularly limited to, those derived from Methanocardococcus jannaschii (SEQ ID NO: 6).
　クレンアーキオータ門に属する好熱菌としては、特に限定されないが、例えば、パイロバキュラム（Ｐｙｒｏｂａｃｕｌｕｍ）属及びスルフォロバス（Ｓｕｌｆｏｌｏｂｕｓ）属等に属するものが挙げられる。 The thermophilic bacterium belonging to the Clenarchiota is not particularly limited, and examples thereof include those belonging to the genus Pyrobaculum and the genus Sulfolobus.
　パイロバキュラム属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、パイロバキュラム・カリディフォンティス（Ｐｙｒｏｂａｃｕｌｕｍ　ｃａｌｉｄｉｆｏｎｔｉｓ）由来（配列番号７）等が挙げられる。 Specific examples of thermophilic bacterium-derived DEAD-box RNA helicase belonging to the genus Pyrobaculum include, but are not limited to, those derived from Pyrobaculum califontis (SEQ ID NO: 7) and the like. .
　スルフォロバス属に属する好熱菌由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの具体例としては、特に限定されないが、スルフォロバス・トコダイ（Ｓｕｌｆｏｌｏｂｕｓ　ｔｏｋｏｄａｉｉ）由来（配列番号８）等が挙げられる。 Specific examples of DEAD-box RNA helicases derived from thermophiles belonging to the genus Sulfolobus include, but are not limited to, those derived from Sulfolobus tokodaii (SEQ ID NO: 8) and the like.
　２．好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの改変体
　本発明においては、前記好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの改変体であって、耐熱性及びヘリカーゼ活性を有するものも同様に使用できる。 2. Thermophilic archaic-derived DEAD-box RNA helicase variant In the present invention, the thermophilic archaic-derived DEAD-box RNA helicase variant, which has heat resistance and helicase activity, is also used. it can.
　上記改変体は、特に限定されないが、好ましくは９０℃においてヘリカーゼ活性を示すものである。 The above-mentioned variant is not particularly limited, but preferably exhibits helicase activity at 90 ° C.
　また、上記改変体は、特に限定されないが、好ましくは５０℃において二本鎖核酸の巻き戻し活性を示し、かつ９０℃において二本鎖核酸の存在下でＡＴＰａｓｅ活性を示すものである。 Further, the above-mentioned variant is not particularly limited, but preferably exhibits the unwinding activity of the double-stranded nucleic acid at 50 ° C. and the ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C.
　なお、本発明において、二本鎖核酸の巻き戻し活性は以下のようにして測定する。 In the present invention, the unwinding activity of the double-stranded nucleic acid is measured as follows.
　巻き戻し活性測定には放射性標識した基質を用いる（表１）。５０ｐｍｏｌの６３ｍｅｒＲＮＡ（ｓｓＲＮＡ６３）を、[γ-３２Ｐ] ＡＴＰ （６０００Ｃｉ／ｍｍｏｌ） （ＰａｒｋｉｎＥｌｍｅｒ, Ｂｏｓｔｏｎ, ＵＳＡ） 及びＴ４ ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ ｋｉｎａｓｅ （Ｔａｋａｒａ ｂｉｏ） を用いて３７℃で３０分反応させ、３２Ｐ放射性標識する。Ｍｉｃｒｏｓｐｉｎ Ｇ－２５ （ＧＥ Ｈｅａｌｔｈｃａｒｅ） を用いて未反応のγ－ＡＴＰを除去する。ラベルしたｓｓＲＮＡ６３ １０ｐｍｏｌにそれぞれ２０ｐｍｏｌのｓｓＲＮＡ３３、ｓｓＲＮＡ４８－５’、ｓｓＲＮＡ４８－３’を加えて、２ｍＭ ＭｇＣｌ２及び５０ｍＭ ＨＥＰＥＳ （ｐＨ７．６） を含むバッファー中で９５℃、５分処理し、室温で２時間放置しアニールさせることで、両端突出型ｄｓＲＮＡ、５’突出型ｄｓＲＮＡ、３’突出型ｄｓＲＮＡを作製する。これをＭｉｃｒｏｓｐｉｎ Ｓ－４００ＨＲに供し、アニールしなかったＲＮＡを除去する。得られたｄｓＲＮＡ及びアニールさせていないｓｓＲＮＡを７％非変性アクリルアミドゲルを用いて確認する。このｓｓＲＮＡと作製したｄｓＲＮＡを基質として巻き戻し活性測定を行う。 A radiolabeled substrate is used for measuring the unwinding activity (Table 1). 50 pmol of 63merRNA (ssRNA63) was reacted with [γ- 32 P] ATP (6000 Ci / mmol) (ParkinElmer, Boston, USA) and T4 polynucleotide kinase (Takara bio) at 37 ° C. for 30 minutes and labeled for 32 minutes. To do. Unreacted γ-ATP is removed using Microspin G-25 (GE Healthcare). 20 pmol of ssRNA33, ssRNA48-5 ′, and ssRNA48-3 ′ were added to 10 pmol of labeled ssRNA63, respectively, and treated at 95 ° C. for 5 minutes in a buffer containing 2 mM MgCl 2 and 50 mM HEPES (pH 7.6). By allowing to stand for a period of time and annealing, both-end protruding dsRNA, 5 ′ protruding dsRNA, and 3 ′ protruding dsRNA are prepared. This is subjected to Microspin S-400HR to remove RNA that has not been annealed. The obtained dsRNA and unannealed ssRNA are confirmed using a 7% non-denaturing acrylamide gel. The unwinding activity is measured using this ssRNA and the prepared dsRNA as a substrate.
　巻き戻し活性測定は以下のようにして行う。３２Ｐ標識核酸基質、０．２μＭの精製タンパク質、１ｍＭ ＡＴＰ、２ｍＭ ＭｇＣｌ２、２ｍＭ ＤＴＴ、４ｕｎｉｔのＲＮａｓｅ ｉｎｈｉｂｉｔｏｒ、５０ｍＭ ＨＥＰＥＳ （ｐＨ７．６） を含む反応液１０μＬを５０℃で３０分反応させ、反応後直ちに氷上へ移すことにより反応を停止する。反応産物を７％アクリルアミドゲル （ＮＡＴＩＶＥ－ＰＡＧＥ） （表２）に供し、ゲルを乾燥後、反応産物の放射活性をＢＡＳ－２５００ （ＦＵＪＩＦＩＬＭ） により検出し、Ｘ線フィルムに感光させる。 The rewinding activity is measured as follows. 10 μL of a reaction solution containing 32 P-labeled nucleic acid substrate, 0.2 μM purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of RNase inhibitor, 50 mM HEPES (pH 7.6) is reacted at 50 ° C. for 30 minutes. Immediately afterwards, stop the reaction by transferring to ice. The reaction product is applied to a 7% acrylamide gel (NATIVE-PAGE) (Table 2). After drying the gel, the radioactivity of the reaction product is detected by BAS-2500 (FUJIFILM) and exposed to X-ray film.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　また、本発明において、二本鎖核酸の存在下におけるＡＴＰａｓｅ活性は以下のようにして測定する。 In the present invention, ATPase activity in the presence of double-stranded nucleic acid is measured as follows.
　ＡＴＰａｓｅ活性はＡＴＰがＡＤＰに変換される際に放出される遊離リン酸量を測ることにより測定する。まず核酸依存性について調べるため、基質としてｓｓＲＮＡである６３ｍｅｒＲＮＡ（ｓｓＲＮＡ６３（配列番号９））を用いる。反応液の総量は１０μＬとし、５ｎＭの核酸基質、０．２μＭの精製タンパク質、１ｍＭＡＴＰ、２ｍＭＭｇＣｌ２、２ｍＭＤＴＴ、４ｕｎｉｔのＲｉｂｏｎｕｃｌｅａｓｅｉｎｈｉｂｉｔｏｒ（Ｈｕｍａｎｐｌａｃｅｎｔａ）（Ｔａｋａｒａｂｉｏ）、５０ｍＭＨＥＰＥＳ（ｐＨ７．６）を含む。これを９０℃で３０分反応させ、反応停止のために直ちに氷上へ移す。反応液の遊離リン酸量をＢＩＯＭＯＬＧＲＥＥＮＴＭ（ＢＩＯＭＯＬ）を用いて測定し、Ａｂ５３０をＭｕｌｔｉｓｋａｎＳｐｅｃｔｒｕｍ（ＴｈｅｒｍｏＬａｂｓｙｓｔｅｍｓ）を用いて測定する。 ATPase activity is measured by measuring the amount of free phosphate released when ATP is converted to ADP. First, in order to examine nucleic acid dependency, 63merRNA (ssRNA63 (SEQ ID NO: 9)) which is ssRNA is used as a substrate. The total amount of the reaction solution is 10 μL, and contains 5 nM nucleic acid substrate, 0.2 μM purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of Ribonuclease inhibitor (Humanplacenta) (Takarabio), 50 mM HEPES (pH 7.6). This is reacted at 90 ° C. for 30 minutes and immediately transferred to ice to stop the reaction. The amount of free phosphoric acid in the reaction solution is measured using BIOMOLGREEN ™ (BIOMOL), and Ab 530 is measured using Multiskan Spectrum (ThermoLabsystems).
　前記改変体は、特に限定されないが、配列番号１～８のいずれかで表されるアミノ酸配列からなるＴＫ０５６６又はＴＫ０５６６オルソログにおいて１～１００個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなるものであってもよい。前記改変体は、配列番号１～８のいずれかで表されるアミノ酸配列からなるＴＫ０５６６又はＴＫ０５６６オルソログにおいて、好ましくは１～５０個、より好ましくは１～２０個、さらに好ましくは１～１０個、最も好ましくは１～数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる。 The variant is not particularly limited, and consists of an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, or added in TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8. It may be a thing. The variant is preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, in TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8, Most preferably, it consists of an amino acid sequence in which one to several amino acids are deleted, substituted or added.
　前記改変体は、あるいは、配列番号１～８のいずれかで表されるアミノ酸配列からなるＴＫ０５６６又はＴＫ０５６６オルソログと相同性が８０％以上のアミノ酸配列からなるものであってもよい。前記改変体は、配列番号１～８のいずれかで表されるアミノ酸配列からなるＴＫ０５６６又はＴＫ０５６６オルソログと、より好ましくは相同性が９０％以上のアミノ酸配列からなるものであり、さらに好ましくは相同性が９５％以上のアミノ酸配列からなるものである。 The modified body may alternatively be composed of an amino acid sequence having a homology of 80% or more with TK0566 or TK0566 ortholog comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 8. Said variant is a TK0566 or TK0566 ortholog consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, more preferably an amino acid sequence having a homology of 90% or more, more preferably a homology Consists of 95% or more amino acid sequence.
　なお、ＴＫ０５６６オルソログは、ＳＦ２ヘリカーゼであり、その構造解析が既になされている。大きく分けてＮ末端側から、Ｎ末端領域、ヘリカーゼコア及びＣ末端領域に分けることができ、さらにヘリカーゼコアはドメイン１及びドメイン２がＮ末端側からこの順で連結された構造を有する。ＤＥＡＤモチーフはドメイン１内に存在する。また、その高次構造解析もなされている。これらの情報を参考にして、前記改変体を作成することができる。具体的には、耐熱性及びヘリカーゼ活性の維持のために重要な領域を保存ないし改変の程度を最小限に留めつつ、必要に応じて他の領域においてアミノ酸改変を導入することにより、前記改変体を作成すればよい。 In addition, TK0566 ortholog is SF2 helicase, and its structural analysis has already been done. It can be roughly divided into an N-terminal region, a helicase core and a C-terminal region from the N-terminal side, and the helicase core has a structure in which domain 1 and domain 2 are connected in this order from the N-terminal side. The DEAD motif is present in domain 1. In addition, higher-order structural analysis has also been performed. With reference to these information, the modified body can be prepared. Specifically, the above-mentioned variant is introduced by introducing amino acid alterations in other regions as necessary while preserving the regions important for maintaining heat resistance and helicase activity or minimizing the degree of modification. Should be created.
　上記において、相同性とは、最適に整列させた２つの配列間を比較することにより決定される。配列を整列させて比較する方法としては、例えばＢａｓｉｃ Ｌｏｃａｌ Ａｌｉｇｎｍｅｎｔ Ｓｅａｒｃｈ Ｔｏｏｌ（ＢＬＡＳＴ）のアルゴリズムを用いて行うことができる。この場合、本発明における相同性は、ＢＬＡＳＴにおける「同一性」のことを指す。また、上記アルゴリズムを使用した場合と比べて結果に差が生じない限り、別のアルゴリズムを用いて相同性を算出してもよい。 In the above, homology is determined by comparing two optimally aligned sequences. As a method for aligning and comparing the sequences, for example, an algorithm of Basic Local Alignment Search Tool (BLAST) can be used. In this case, homology in the present invention refers to “identity” in BLAST. Moreover, as long as there is no difference in the results compared to the case where the above algorithm is used, the homology may be calculated using another algorithm.
　３．ＰＣＲによるＤＮＡ増幅方法
　本発明のＰＣＲによるＤＮＡ増幅方法は、
好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ、又は
耐熱性及びヘリカーゼ活性を有するその改変体
の存在下でＰＣＲを行うことを特徴とする方法である。 3. DNA amplification method by PCR The DNA amplification method by PCR of the present invention comprises:
It is a method characterized in that PCR is carried out in the presence of a thermophilic archaic-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
　好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体の作用により、非特異的な増幅を抑制することができる。かかる作用効果は、好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの機能及び特性のうち、耐熱性及びヘリカーゼ活性に由来するものである。 Nonspecific amplification can be suppressed by the action of a thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof. Such effects are derived from heat resistance and helicase activity among the functions and properties of thermophilic archaea-derived DEAD-box RNA helicase.
　好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体は、その耐熱性のため、ＰＣＲにおいてそのヘリカーゼ活性を失うことがない。また、好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体は、そのヘリカーゼ活性により、図１に模式的に示したように、非特異的にプライマーが核酸にアニールすることによりできた二本鎖の核酸部分をアンワインドさせる。このはたらきにより、非特異的なＤＮＡ増殖が抑制される。 A thermophilic archaia-derived DEAD-box RNA helicase or a variant thereof does not lose its helicase activity in PCR due to its heat resistance. In addition, the thermophilic archaia-derived DEAD-box RNA helicase or a modified product thereof was obtained by non-specifically annealing the primer to the nucleic acid due to its helicase activity, as schematically shown in FIG. Unwind the nucleic acid portion of the strand. This function suppresses nonspecific DNA growth.
　ＰＣＲは、特に限定されず、幅広い条件で行うことができる。 PCR is not particularly limited and can be performed under a wide range of conditions.
　特に限定されないが、２０～１５０ｎＭの好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体の存在下でＰＣＲを行うことが好ましい。２０ｎＭ以上の好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体の存在下でＰＣＲを行うと、非特異的な増幅を効果的に抑制することができる。また、１５０ｎＭ以下の好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ又はその改変体の存在下でＰＣＲを行うと、特異的に増幅されるＤＮＡの量が良好となる。 Although not particularly limited, it is preferable to perform PCR in the presence of 20 to 150 nM thermophilic archaea-derived DEAD-box type RNA helicase or a modified form thereof. When PCR is performed in the presence of 20 nM or more thermophilic archaic DEAD-box type RNA helicase or a variant thereof, non-specific amplification can be effectively suppressed. In addition, when PCR is performed in the presence of a thermophilic archaic-derived DEAD-box type RNA helicase of 150 nM or less or a modified form thereof, the amount of specifically amplified DNA is improved.
　特に限定されないが、本発明の効果の面で、マグネシウムイオンの存在下でポリメラーゼ連鎖反応を行うことが好ましい。この場合、特に限定されないが、０．５ｍＭ～３ｍＭのマグネシウムイオンの存在下でポリメラーゼ連鎖反応を行うことがより好ましい。上記濃度のマグネシウムイオンの存在下でポリメラーゼ連鎖反応を行うことにより、特異的に増幅されるＤＮＡの量を十分な程度に維持しつつ、非特異的な増幅を効果的に抑制することができる。 Although not particularly limited, it is preferable to perform the polymerase chain reaction in the presence of magnesium ions in terms of the effects of the present invention. In this case, the polymerase chain reaction is more preferably carried out in the presence of 0.5 mM to 3 mM magnesium ions, although not particularly limited. By performing the polymerase chain reaction in the presence of the magnesium ion at the above concentration, it is possible to effectively suppress nonspecific amplification while maintaining a sufficient amount of specifically amplified DNA.
　上記において、マグネシウムイオンは反応系に、特に限定されないが、例えば、硫酸マグネシウムとして添加されていてもよい。 In the above, magnesium ions are not particularly limited to the reaction system, but for example, magnesium ions may be added as magnesium sulfate.
　４．ＰＣＲによるＤＮＡ増幅方法のために用いられる組成物
　本発明の組成物は、ＰＣＲによるＤＮＡ増幅方法のために用いられるものであり、
好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ、又は
耐熱性及びヘリカーゼ活性を有するその改変体
を含有する。 4). Composition used for DNA amplification method by PCR The composition of the present invention is used for DNA amplification method by PCR,
It contains a thermophilic archaea-derived DEAD-box type RNA helicase, or a variant thereof having heat resistance and helicase activity.
　本発明の組成物は、上記に加えてさらに、例えば、Ｔｒｉｓ－ＨＣｌ等の緩衝液、ＫＣｌ等の塩、ジチオトレイトール等の酸化還元剤、エチレンジアミン四酢酸（ＥＤＴＡ）等のキレート剤、グリセロール並びにＴｗｅｅｎ－２０及びＮＰ－４０等の界面活性剤からなる群より選択される少なくとも一種を含んでいてもよい。 In addition to the above, the composition of the present invention further includes, for example, a buffer such as Tris-HCl, a salt such as KCl, a redox agent such as dithiothreitol, a chelating agent such as ethylenediaminetetraacetic acid (EDTA), glycerol, It may contain at least one selected from the group consisting of surfactants such as Tween-20 and NP-40.
　以下に実施例及び試験例を掲げて本発明をさらに詳しく説明するが、本発明はこれら実施例のみに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.
　１．　TK0566の発現及び精製
T. kodakarensis の総DNAを鋳型にし、プライマー（TK0566EX-F（配列番号１０）、TK0566EX-R（配列番号１１））を用いてTK0566遺伝子を増幅した。得られたTK0566遺伝子を含むDNA断片はNdeI 及び EcoRIを用いて切断しpET28a に挿入した。そして構築された pET-TK0566プラスミドを用いて、E. coli BL21-Codon-Plus(DE3)-RIL を形質転換した。5 mLのLB培地（1% tryptone, 0.5% yeast extract, 1% NaCl; adjusted to pH 7.3 with NaOH）で37 ℃、6時間前々培養を行い、20 mLのLB培地で前培養 (1%植菌、37 ℃、10時間) を行った。さらにジャーファーメンターを用いて、37℃で1L培養を行い、OD660が0.4付近でβ-D-1-thiogalactopyranoside (IPTG) (終濃度1 mM) を添加し、誘導した。誘導4時間後に集菌し、SDS-PAGEによりTK0566の発現を確認した。LB培地には20μg/mLカナマイシン及び30μg/mLクロラムフェニコールが含まれている。培養菌体を回収し、10mLのバッファーD：20 mM Tris-HCl、500 mM NaCl、 0.1% Triton X-100、 pH 7.9で懸濁後、超音波破砕した。破砕液を8000g 10分間遠心し、上清を80 ℃で15 分間熱処理した。そして再び8000g、10分間遠心して上清を回収した。発現したTK0566のN末端側にはHis6 tagが付加されているため、Niカラムを使用して精製した。バッファーDでカラムを平衡化し、そこに上清をアプライした。そしてバッファーE：20 mM Tris-HCl、500 mM NaCl、20 mM imidazole、0.1% Triton X-100、pH 7.9で洗浄した後、バッファーF：20 mM Tris-HCl、500 mM NaCl、250 mM imidazole、0.1% Triton X-100、pH 7.9で溶出させた。溶出画分はバッファーDで透析した。 1. Expression and purification of TK0566
Using the total DNA of T. kodakarensis as a template, the TK0566 gene was amplified using primers (TK0566EX-F (SEQ ID NO: 10), TK0566EX-R (SEQ ID NO: 11)). The obtained DNA fragment containing the TK0566 gene was cleaved with NdeI and EcoRI and inserted into pET28a. The constructed pET-TK0566 plasmid was used to transform E. coli BL21-Codon-Plus (DE3) -RIL. Pre-culture for 6 hours at 37 ° C in 5 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl; adjusted to pH 7.3 with NaOH) and pre-culture in 20 mL of LB medium (1% Bacteria, 37 ° C., 10 hours). Furthermore, 1 L culture was performed at 37 ° C. using a jar fermenter, and β-D-1-thiogalactopyranoside (IPTG) (final concentration 1 mM) was added and induced when OD 660 was around 0.4. Bacteria were collected 4 hours after induction, and the expression of TK0566 was confirmed by SDS-PAGE. The LB medium contains 20 μg / mL kanamycin and 30 μg / mL chloramphenicol. The cultured cells were collected, suspended in 10 mL of buffer D: 20 mM Tris-HCl, 500 mM NaCl, 0.1% Triton X-100, pH 7.9, and sonicated. The disrupted solution was centrifuged at 8000 g for 10 minutes, and the supernatant was heat-treated at 80 ° C. for 15 minutes. Then, the supernatant was collected again by centrifugation at 8000 g for 10 minutes. Since His6 tag was added to the N-terminal side of expressed TK0566, it was purified using a Ni column. The column was equilibrated with buffer D, and the supernatant was applied thereto. Then, after washing with buffer E: 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 0.1% Triton X-100, pH 7.9, buffer F: 20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.1 Elute with% Triton X-100, pH 7.9. The eluted fraction was dialyzed against buffer D.
　２．　ATPase 活性測定
　ATPase活性はATPがADPに変換される際に放出される遊離リン酸量を測ることにより測定した。まず核酸依存性について調べるため、基質としてssRNAである63mer RNA (ssRNA63)を用いた。反応液の総量は10μLとし、5nMの核酸基質、0.2μMの精製タンパク質、1mM ATP、2mM MgCl2、2mM DTT、4unitのRibonuclease inhibitor (Human placenta) (Takara bio)、50mM HEPES (pH7.6) を含む。これを50℃から110℃で30分反応させ、反応停止のために直ちに氷上へ移した。反応液の遊離リン酸量をBIOMOL GREENTM(BIOMOL) を用いて測定し、Ab530をMultiskan Spectrum (Thermo Labsystems) を用いて測定した。図２に測定結果を示す。 2. Measurement of ATPase activity ATPase activity was measured by measuring the amount of free phosphate released when ATP was converted to ADP. First, 63mer RNA (ssRNA63) which is ssRNA was used as a substrate in order to investigate nucleic acid dependency. The total volume of the reaction solution is 10 μL, and 5 nM nucleic acid substrate, 0.2 μM purified protein, 1 mM ATP, 2 mM MgCl 2 , 2 mM DTT, 4 units of Ribonuclease inhibitor (Human placenta) (Takara bio), 50 mM HEPES (pH 7.6) Including. This was reacted at 50 ° C. to 110 ° C. for 30 minutes and immediately transferred onto ice to stop the reaction. The amount of free phosphoric acid in the reaction solution was measured using BIOMOL GREEN ™ (BIOMOL), and Ab 530 was measured using Multiskan Spectrum (Thermo Labsystems). FIG. 2 shows the measurement results.
　３．　SF2 ヘリカーゼ添加によるPCR増幅への影響の検証
３．１　鋳型DNAの合成（T. kodakarensis 由来16S rDNA、Pseudomonas aeruginosa由来 toxA）
　高次構造を形成するT. kodakarensis由来の16S rDNA 及び高GC含量であるP. aeruginosaのエキソトキシンA（タンパク質合成に重要な役割を果たす伸長因子（EF‐2）のADP‐リボシル化による阻害）をコードする毒素遺伝子toxA を対象にし、鋳型DNAの合成を行った。T. kodakarensis の総DNAゲノムを鋳型とし、プライマー（TKr05-Fw（配列番号１２） 、TKr05-Rv（配列番号１３））を用いて16S rDNAをPCRにより増幅した。また、P. aeruginosa の総DNAゲノムを鋳型とし、プライマー（PA1148-Fw（配列番号１４）、PA1148-Rv（配列番号１５））を用いてtoxA 遺伝子を増幅した。得られた増幅断片をゲル抽出し、Nucleo Spin Gel and PCR Clean-up (Takara)を用いて精製した。PAO1株ゲノムDNAの分譲元はNBRC, NITE, Kisarazu, Japan (NBRC Number：106052G)である。
３．２　TK0566添加によるPCR増幅への影響（鋳型：合成 16S rDNA）
　精製したSF2ヘリカーゼがPCR増幅へ影響を及ぼすかを検証するため以下のPCR反応組成（表３）及びPCR条件（表４）でPCRを行った結果を図３に示す。図３に示すように、TK0566を添加したときPCRの誤増幅の低減効果が見られた。 3. Verification of PCR amplification effect by adding SF2 helicase 3.1 Synthesis of template DNA (16S rDNA from T. kodakarensis, toxA from Pseudomonas aeruginosa)
16S rDNA from T. kodakarensis forming higher-order structure and P. aeruginosa exotoxin A with high GC content (inhibition by ADP-ribosylation of elongation factor (EF-2) that plays an important role in protein synthesis) The template DNA was synthesized using the toxin gene toxA encoding. Using the total DNA genome of T. kodakarensis as a template, 16S rDNA was amplified by PCR using primers (TKr05-Fw (SEQ ID NO: 12) and TKr05-Rv (SEQ ID NO: 13)). Further, the toxA gene was amplified using the total DNA genome of P. aeruginosa as a template and using primers (PA1148-Fw (SEQ ID NO: 14), PA1148-Rv (SEQ ID NO: 15)). The obtained amplified fragment was gel-extracted and purified using Nucleo Spin Gel and PCR Clean-up (Takara). The distributor of PAO1 strain genomic DNA is NBRC, NITE, Kisarazu, Japan (NBRC Number: 106052G).
3.2 Effect on PCR amplification by adding TK0566 (template: synthetic 16S rDNA)
FIG. 3 shows the results of PCR under the following PCR reaction composition (Table 3) and PCR conditions (Table 4) in order to verify whether the purified SF2 helicase affects PCR amplification. As shown in FIG. 3, when TK0566 was added, the effect of reducing PCR misamplification was observed.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　４．　MgSO 4 添加によるPCR効率の検証（鋳型：合成16S rDNA）
　通常のPCRでは1mM MgSO4を添加しているが、TK0566によってMg2+が奪われ、KODポリメラーゼの機能効率が低下している可能性が考えられた。よってこの可能性を検証するためMgSO4の添加量を増やし、以下の組成及び条件でPCRを行った。その結果を図４に示す。 4). Verification of PCR efficiency by adding MgSO 4 (template: synthetic 16S rDNA)
In normal PCR, 1 mM MgSO 4 was added, but it was considered that Mg 2+ was deprived by TK0566 and the functional efficiency of KOD polymerase was reduced. Therefore, in order to verify this possibility, the amount of MgSO 4 added was increased, and PCR was performed under the following composition and conditions. The result is shown in FIG.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　図４に示すように、100nMのヘリカーゼを添加時にMgSO4の濃度を上昇させたところ、1mM MgSO4よりも2mM MgSO4の方が増幅効率が高かった。このことから、Mg2+がTK0566によって奪われたため、KODポリメラーゼの機能が低下したと考えられる。そのため、2mM MgSO4を用いてPCRを行うことが好ましいことが判った。 As shown in FIG. 4, when the concentration of MgSO 4 was increased when 100 nM helicase was added, amplification efficiency of 2 mM MgSO 4 was higher than that of 1 mM MgSO 4 . From this, it is considered that Mg 2+ was deprived by TK0566 and the function of KOD polymerase was lowered. Therefore, it was found preferable to perform PCR using 2 mM MgSO4.
　５．　サイクル数増加によるPCR 増幅の特異性検証　（鋳型：16S rDNA）
　非特異のバンドが消失した理由として、目的のバンドと非特異のバンド両方の強度が低くなった可能性が懸念される。このことから、PCR反応のサイクル数を45に増やし、TK0566を加えたときに非特異のバンドが検出されるかを検証した。PCR反応組成、条件は以下に示した。結果は図５に示す。 5. Specificity verification of PCR amplification by increasing the number of cycles (Template: 16S rDNA)
As a reason for the disappearance of the non-specific band, there is a concern that the intensity of both the target band and the non-specific band may be reduced. From this, the number of PCR reaction cycles was increased to 45, and it was verified whether a non-specific band was detected when TK0566 was added. The PCR reaction composition and conditions are shown below. The results are shown in FIG.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　図５に示すように、PCR反応サイクルを45にした際、TK0566を添加すると非特異的なバンドは見られなかったことから、TK0566はプライマーのミスアニールを抑制することが出来ると考えられる。 As shown in FIG. 5, when the PCR reaction cycle was set to 45, when TK0566 was added, no non-specific band was observed, so it is considered that TK0566 can suppress primer misannealing.
　６．　TK0566添加によるPCR増幅特異性の検証　（鋳型：T. kodakarensisの総DNA）
　T. kodakarensis の総DNAを鋳型にし、TK0566添加によるPCR増幅の特異性を検証した。
TK0566の上流1000bpを増幅するプライマー（TK0566-up1000-Fw（配列番号１６）、TK0566-up1000-Rv（配列番号１７））を用いた。PCR反応組成及び条件を以下に示し、結果を図６に示す。 6). Verification of PCR amplification specificity by adding TK0566 (Template: Total DNA of T. kodakarensis)
Using T. kodakarensis total DNA as a template, the specificity of PCR amplification by adding TK0566 was verified.
Primers (TK0566-up1000-Fw (SEQ ID NO: 16), TK0566-up1000-Rv (SEQ ID NO: 17)) that amplify 1000 bp upstream of TK0566 were used. The PCR reaction composition and conditions are shown below, and the results are shown in FIG.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　図６に示すように、完全に非特異のバンドは消失しなかったが、総DNAを鋳型にした場合においてもTK0566添加によりミスアニールの割合が減少した。 As shown in FIG. 6, a completely non-specific band did not disappear, but even when total DNA was used as a template, the rate of misannealing decreased with the addition of TK0566.
　７．　TK0566添加によるPCR増幅特異性の検証（鋳型：Gluconacetobacter europaeusの総DNA）
　G. europaeus の総DNAを用いてPCRを行った。増幅領域は多数の非特異バンドが検出されるilvI 遺伝子の上流領域である。TK0566添加によるPCR増幅特異性を検証した結果を図７に示す。PCRの組成及び条件は以下に示す。 7). Verification of PCR amplification specificity by adding TK0566 (template: total DNA of Gluconacetobacter europaeus)
PCR was performed using the total DNA of G. europaeus. The amplified region is the upstream region of the ilvI gene where many non-specific bands are detected. The result of verifying the PCR amplification specificity by adding TK0566 is shown in FIG. The composition and conditions of PCR are shown below.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　図７に示すように、完全に非特異のバンドは消失しなかったが、T. kodakarensisと同様にG. europaeusの総DNAを鋳型にした場合においてもTK0566添加によりミスアニールの割合が減少した。 As shown in FIG. 7, the completely non-specific band did not disappear, but the rate of misannealing decreased with the addition of TK0566 even when the total DNA of G. opapaeus was used as a template, as in T. kodakarensis.
　８．　高GC含量鋳型に対するTK0566添加による PCR増幅特異性の検証（鋳型：合成DNA GC含量69%のPa-toxA）
　Pa-toxA はGC含量が高いため、アニーリング温度を上昇させても非特異のバンドを消失させることが出来ない。GC含量が高い鋳型DNAを用いた時のPCR増幅の特異性を検証した。
以下に示す組成及び条件でPCRを行った結果を図８に示す。 8). Verification of PCR amplification specificity by adding TK0566 to high GC content template (Template: Pa-toxA with 69% synthetic DNA GC content)
Since Pa-toxA has a high GC content, a non-specific band cannot be lost even if the annealing temperature is increased. The specificity of PCR amplification when template DNA with high GC content was used was verified.
The results of PCR performed under the following composition and conditions are shown in FIG.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　図８に示すように、TK0566を添加することでミスアニールの割合が減少したことが判る。 As shown in FIG. 8, it can be seen that the rate of mis-annealing was reduced by adding TK0566.
　９．　Tth ポリメラーゼ、Taq ポリメラーゼを用いた時のTK0566添加によるPCR増幅特異性への影響
　TK0566がKOD ポリメラーゼと特異的に相互作用することでPCR増幅の特異性が増強している可能性が考えられた。この可能性を検証するため、KODポリメラーゼ以外の酵素（Thermus aquaticus、 Thermus thermophilus由来のポリメラーゼ）に対する効果を検証した。以下にPCR組成及び条件を示し結果を図９に示す。 9. Effect of TK0566 addition on the PCR amplification specificity when Tth polymerase or Taq polymerase is used TK0566 may have increased the specificity of PCR amplification by specifically interacting with KOD polymerase. In order to verify this possibility, the effect on enzymes other than KOD polymerase (thermus aquaticus, polymerase derived from Thermus thermophilus) was verified. The PCR composition and conditions are shown below and the results are shown in FIG.
　10×standard buf : Taq DNA Polymerase with Standard Taq Buffer (New England Bio Labs)
　10×thermo buf：Taq DNA Polymerase with ThermoPol Buffer (New England Bio Labs)
　Taq pol：Taq DNA Polymerase (New England Bio Labs) 10 × standard buf: Taq DNA Polymerase with Standard Taq Buffer (New England Bio Labs)
10 x thermo buf: Taq DNA Polymerase with ThermoPol Buffer (New England Bio Labs)
Taq pol: Taq DNA Polymerase (New England Bio Labs)
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　図９に示すように、TK0566添加による誤増幅の低減効果はKOD plus ポリメラーゼだけではなくTthポリメラーゼ及びTaqポリメラーゼにも見られた。 As shown in FIG. 9, the effect of reducing misamplification by adding TK0566 was observed not only in KOD plus polymerase but also in Tth polymerase and Taq polymerase.
　１０．　ARMS法を用いたALDH2遺伝子型の検出
　髪の毛からDNAを抽出し、総DNAからallele 特異的合成プライマー（ASP）を使ったPCR（ARMS法）を用いてアルデヒドでヒドロゲナーゼ2 (ALDH2) 遺伝子型の検出を行った。ALDH2はエタノールが代謝されてできるアルデヒドを酸化する酵素であり、その活性の欠損はALDH2遺伝子の点突然変異E487Kによるものである。対立遺伝子の組み合わせから正常型ホモ接合体（NN型）、ヘテロ接合体（NM型）、変異型ホモ接合体（MM型）の3種類の遺伝子が存在する。このうちのどの遺伝子型を持っているかを調べることでアルコールに対する感受性を判断することが出来る。以下にPCR反応組成及び条件を示し、結果を図１０に示す。 10. Detection of ALDH2 genotype using ARMS method Extraction of DNA from hair and detection of hydrogenase 2 (ALDH2) genotype with aldehyde using PCR (ARMS method) using allele specific synthetic primer (ASP) from total DNA Went. ALDH2 is an enzyme that oxidizes aldehydes produced by the metabolism of ethanol, and the lack of activity is due to the point mutation E487K of the ALDH2 gene. There are three types of genes from combinations of alleles: normal homozygotes (NN type), heterozygotes (NM type), and mutant homozygotes (MM type). Sensitivity to alcohol can be determined by examining which of these genotypes are present. The PCR reaction composition and conditions are shown below, and the results are shown in FIG.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　図１０に示すように、SNP解析において2回エタノール沈澱した被験者Aのゲノムは非特異的な増幅が見られた。そこに25mMのTK0566を添加することでミスアニールが低減され、検出感度が高くなったといえる。これにより被験者AのALDH2遺伝子型は[NM]と判断できる。しかしながら被験者Bのゲノムに関しては全くバンドが検出されなかった。理由としてはゲノムが微量であったことが考えられる。以上のことよりPCR反応時のTK0566添加はSNP解析等の遺伝子診断の検出感度を向上させると考えられる。 As shown in FIG. 10, nonspecific amplification was observed in the genome of subject A that had been ethanol precipitated twice in the SNP analysis. It can be said that the addition of 25 mM TK0566 reduced misannealing and increased the detection sensitivity. Thus, the ALDH2 genotype of subject A can be determined as [NM]. However, no band was detected for subject B's genome. The reason may be that the genome was very small. From the above, it is considered that the addition of TK0566 during PCR reaction improves the detection sensitivity of gene diagnosis such as SNP analysis.

Claims (9)

  1. ポリメラーゼ連鎖反応によるＤＮＡ増幅方法であって、
    好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ、又は
    耐熱性及びヘリカーゼ活性を有するその改変体
    の存在下でポリメラーゼ連鎖反応を行う工程を含む方法。     A DNA amplification method by polymerase chain reaction,
    A method comprising a step of performing a polymerase chain reaction in the presence of a thermophilic archaea-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
  2. 前記好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼが、配列番号１～８のいずれかで表されるアミノ酸配列からなる、請求項１に記載の方法。 The method according to claim 1, wherein the thermophilic archaea-derived DEAD-box RNA helicase consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.
  3. 前記改変体が、配列番号１～８のいずれかで表されるアミノ酸配列と相同性が８０％以上のアミノ酸配列からなる、請求項２に記載の方法。 The method according to claim 2, wherein the variant comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.
  4. 前記改変体が、９０℃においてヘリカーゼ活性を示す、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the variant exhibits helicase activity at 90 ° C.
  5. 前記改変体が、５０℃において二本鎖核酸の巻き戻し活性を示し、かつ９０℃において二本鎖核酸の存在下でＡＴＰａｓｅ活性を示す、請求項１～４のいずれか一項に記載の方法。 The method according to any one of claims 1 to 4, wherein the variant exhibits an unwinding activity of a double-stranded nucleic acid at 50 ° C and an ATPase activity in the presence of the double-stranded nucleic acid at 90 ° C. .
  6. 前記工程が、２０～１５０ｎＭの前記好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼの存在下でポリメラーゼ連鎖反応を行う工程である、請求項１～５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein the step is a step of performing a polymerase chain reaction in the presence of 20 to 150 nM of the thermophilic archaea-derived DEAD-box type RNA helicase.
  7. 前記工程が、マグネシウムイオンの存在下でポリメラーゼ連鎖反応を行う工程である、請求項１～６のいずれか一項に記載の方法。 The method according to any one of claims 1 to 6, wherein the step is a step of performing a polymerase chain reaction in the presence of magnesium ions.
  8. 前記工程が、０．５ｍＭ～３ｍＭのマグネシウムイオンの存在下でポリメラーゼ連鎖反応を行う工程である、請求項７に記載の方法。 The method according to claim 7, wherein the step is a step of performing a polymerase chain reaction in the presence of 0.5 mM to 3 mM magnesium ions.
  9. ポリメラーゼ連鎖反応によるＤＮＡ増幅方法のために用いられる、
    好熱性アーキア由来ＤＥＡＤ－ｂｏｘ型ＲＮＡへリカーゼ、又は
    耐熱性及びヘリカーゼ活性を有するその改変体
    を含有する、組成物。     Used for DNA amplification method by polymerase chain reaction,
    A composition comprising a thermophilic archaea-derived DEAD-box RNA helicase or a variant thereof having heat resistance and helicase activity.
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