JP7014256B2 - Nucleic acid amplification reagent - Google Patents

Description

The present invention relates to a nucleic acid amplification reagent used for PCR (polymerase chain reaction) having excellent reaction specificity and amplification efficiency.

PCR is (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) the primer using a DNA polymerase. This is a method of amplifying the target nucleic acid in the sample by repeating the three steps of extension of the DNA as one cycle. The target nucleic acid can be amplified hundreds of thousands of times even from a small amount of sample such as several copies, and it is widely used not only in the research field but also in the forensic field such as genetic diagnosis and clinical diagnosis, or in the microbiological examination in food and environment. It is becoming used.

In PCR, reaction specificity and amplification efficiency are important. Here, the reaction specificity is the ability to specifically amplify only the target DNA of interest, and if the reaction specificity is low, non-specific amplification or primer dimer occurs, and the amplification amount of the target product decreases. It leads to. Further, the amplification efficiency indicates the efficiency of amplifying the target DNA, and in an ideal PCR with an amplification efficiency of 100%, the target DNA is amplified twice in one cycle. If the amplification efficiency is low, the amount of amplification of the target DNA in one cycle is small, which leads to a small amount of product obtained and the inability to detect the amplified target DNA.

Various PCR compositions have been studied in order to enhance reaction specificity and amplification efficiency. For example, salts such as KCl, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ , and tetramethylammonium hydroxide have a function of regulating the annealing force of the primer, and the optimum concentration of these has a function of adjusting the reaction specificity and amplification efficiency. It becomes important to enhance it. In addition, additives such as betaine, glycerol, glycols, and sugars are said to prevent primer misannealing and also to stabilize proteins, making it possible to increase reaction specificity and amplification efficiency by adding them. Become.

As an additive for PCR, the addition of a protein factor that works in collaboration with DNA polymerase as a replication factor is also being investigated. Single-stranded binding proteins (SSBs) have been shown to specifically bind to single strands and destabilize the double helix structure of nucleic acids, thus preventing primer misannealing. It has also been shown that the addition of PCNA, which acts as a DNA clamp, prevents dissociation of the polymerase from synthetic nucleic acids and enhances amplification efficiency (Patent Document 1).

DNA polymerase that carries out PCR is also very important for improving reaction specificity and amplification efficiency. In the commonly used Taq DNA polymerase and Tth DNA polymerase, various mutations have been investigated in order to improve PCR efficiency and reaction specificity. There is also a DNA polymerase fused with SSB in order to strengthen the binding force with nucleic acid. Recently, it has also been shown that DNA polymerases belonging to Family B have higher amplification efficiency than DNA polymerases belonging to Family A (general-purpose Taq DNA polymerases, Tth DNA polymerases, etc.). ..

Further, Patent Document 2 shows a composition containing betaine in nucleic acid amplification using a DNA polymerase belonging to Family B, and suggests that various additives are used in order to enhance PCR performance. .. However, even the invention described in Patent Document 2 cannot be said to satisfy sufficient required characteristics from the viewpoint of its versatility.

In spite of such various studies, it is sometimes found that PCR does not sufficiently amplify. In particular, DNA amplification of a long-chain target or a target having a region with a high GC ratio is unlikely to cause amplification, and the reaction is inhibited even under conditions containing impurities, and DNA amplification may not occur. There is a demand for a PCR composition having a high success rate (which means high reaction specificity and amplification efficiency; the same applies hereinafter) that can be efficiently amplified even under such difficult conditions.

International Publication No. 2007/004654 Pamphlet Japanese Unexamined Patent Publication No. 2010-213689

Carol J. Butt et al., Science, Vol. 273, 1996, pp. 1058-1073.

It is an object of the present invention to provide a reaction composition having a high success rate in DNA replication. Still another object of the present invention is to provide a nucleic acid amplification reagent suitable for the above-mentioned purpose. In summary, an object of the present invention is to provide a PCR reaction reagent suitable for the amplification of genes in the presence of base analogs.

The nucleic acid amplification reagent of the present invention for achieving the above object is characterized by using a DNA polymerase belonging to Family B, PCNA, and betaine.

That is, as a result of diligent studies in view of the above circumstances, the present inventors have added betaine to a reaction system using DNA polymerase belonging to Family B and PCNA to obtain a long-chain target DNA or a region having a high GC rate. We have found that efficient PCR can be performed even in a reaction system containing an inhibitor and amplification of a target DNA having an inhibitory substance, and have completed the present invention. Typical inventions are as follows.

[Item 1]
A nucleic acid amplification reagent containing DNA polymerase, Proliferating Cell Nuclear Antigen (PCNA), and betaine belonging to Family B, wherein the PCNA comprises either of the following (1) or (2). A characteristic nucleic acid amplification reagent.
(1) In the amino acid sequence set forth in SEQ ID NO: 13, a polypeptide consisting of an amino acid sequence in which one to several amino acid residues are substituted, deleted, inserted or added, and has DNA polymerase amplification enhancing activity (1). 2) A polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 13 and having a DNA polymerase amplification enhancing activity [Item 2].
A nucleic acid amplification reagent containing a DNA polymerase, PCNA and betaine belonging to Family B, wherein the PCNA comprises any of the following (1) or (2).
(1) In the amino acid sequence set forth in SEQ ID NO: 14, a polypeptide consisting of an amino acid sequence in which one to several amino acid residues are substituted, deleted, inserted or added, and has DNA polymerase amplification enhancing activity (1). 2) A polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 14 and having a DNA polymerase amplification enhancing activity [Item 3].
A nucleic acid amplification reagent containing a DNA polymerase, PCNA and betaine belonging to Family B, wherein the PCNA comprises any of the following (1) or (2).
(1) In the amino acid sequence set forth in SEQ ID NO: 19, a polypeptide consisting of an amino acid sequence in which one to several amino acid residues are substituted, deleted, inserted or added, and has DNA polymerase amplification enhancing activity (1). 2) A polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 19 and having a DNA polymerase amplification enhancing activity [Item 4].
Item 6. The nucleic acid amplification reagent according to any one of Items 1 to 3, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from archaea.
[Item 5]
Item 6. The nucleic acid amplification reagent according to any one of Items 1 to 4, which is an archaeal DNA polymerase variant having a base analog detection activity in which DNA polymerase belonging to Family B is reduced.
[Item 6]
Item 6. The nucleic acid amplification reagent according to any one of Items 1 to 5, wherein the DNA polymerase belonging to Family B has a modification of at least one amino acid in any of the amino acids constituting the 3'-5'exonuclease active region.
[Item 7]
Item 6. The nucleic acid amplification reagent according to any one of Items 1 to 6, wherein the PCNA is a mutant PCNA having an amplification enhancing activity.

INDUSTRIAL APPLICABILITY The present invention provides a PCR composition having excellent reaction specificity and amplification efficiency in DNA amplification. It can be widely used not only in research fields but also in clinical fields such as genetic diagnosis, forensic fields, and microbiological tests in foods and the environment.

Amplification of long-chain target DNA Amplification of target DNA with regions with high GC content DNA amplification from plant lysate The figure which shows the amino acid region about the multimer formation of PCNA.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
In the present specification, for the base sequence, amino acid sequence and individual constituents thereof, simplified symbols in the one-letter alphabet may be used, but all of them follow the practices in the fields of molecular biology and genetic engineering. Further, in the present specification, for the sake of concisely indicating the variation in the amino acid sequence, for example, a notation such as "D143A" is used. "D143A" indicates that the 143rd aspartic acid was replaced with alanine, that is, the type of amino acid residue before the substitution, its location, and the type of the amino acid residue after the substitution. Further, the sequence number corresponds to the sequence number described in the sequence listing unless otherwise specified. In the case of multiple mutants, the above notation is connected by "/". For example, "D143A / D147A" indicates that the 143rd aspartic acid was replaced with alanine and the 147th aspartic acid was replaced with alanine. For multiple mutants of triple mutant or higher, information on the mutant site such as "P36H" is added after the "/" symbol. Further, in the present specification, the term "mutant" in the case of "mutant PCNA" or the like means that it has an amino acid sequence different from that of a conventionally known PCNA, and is due to an artificial mutation or a mutation in the natural world. It does not distinguish whether it is due to.

(1) Nucleic Acid Amplification Reagent One of the embodiments of the present invention is a reagent for amplifying nucleic acid.
(A) DNA polymerase belonging to family B, and (b) PCNA
(C) A nucleic acid amplification reagent characterized by carrying out a DNA synthesis reaction in the presence of betaine.

(1.1)
The nucleic acid amplification reagent in the present invention is not particularly limited as long as it can be amplified by DNA polymerase. PCR is a typical nucleic acid amplification method, but the nucleic acid amplification reagent of the present invention is not limited to PCR, but uses DNA as a template and reacts with one kind of primer, dNTP (deoxyribonucleotide triphosphate). It is also used as a method for synthesizing a DNA primer extension product by extending the DNA primer. Specifically, it includes a primer extension method, a sequence method, a conventional method without a temperature cycle, a cycle sequence method, and the like.

The nucleic acid applied to the nucleic acid amplification reagent of the present invention is not particularly limited as long as it can be amplified by DNA polymerase without being limited by differences in length, sequence, GC content and the like. The nucleic acid is typically a DNA composed of adenine (A), cytosine (C), guanine (G), and thymine (T), but in the nucleic acid amplification reagent of the present invention, it is "decreased" as a DNA polymerase. (In the present specification, bases other than adenine, cytosine, guanine, and thymine are referred to as base analogs). Since a variant having "detection activity" may be used, adenine, cytosine, guanine, and thymine may be used. It may contain a base other than, for example, uracil, inosin, or the like. Hereinafter, in the present specification, unless otherwise specified, the bases constituting the DNA may contain any of the above-mentioned A, C, G, T and base analogs.

In the nucleic acid amplification reagent of the present invention, typical compositions in the case of PCR are shown below, but the composition is not limited thereto. For example, in the DNA to be amplified,
(A) DNA polymerase belonging to family B (b) PCNA
(C) In addition to betaine
(D) A pair of primers in which one primer is complementary to the DNA extension product of the other primer (e) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)), and
(F) A buffer solution containing magnesium ion, ammonium ion and / or potassium ion is mixed and mixed.
By raising and lowering the temperature of the reaction solution in the cycle shown by the following (I) to (IV) using a thermal cycler or the like, (1) DNA denaturation, (2) primer annealing, and (3) primer extension The thermal cycle is repeated to amplify specific DNA fragments.
(I) The reaction solution is heated to about 94 ° C., and the temperature is maintained for 30 seconds to 1 minute to separate the double-stranded DNA into single strands.
(II) Rapidly cool to about 60 ° C. (slightly different depending on the primer), and anneal the single-stranded DNA and the primer.
(III) The primer is not separated and the DNA polymerase is heated again to the optimum temperature range for its activity. For experimental purposes, the temperature is set to about 60-72 ° C. This temperature is usually maintained for 1 to 2 minutes, depending on the time required for DNA to be synthesized and the length of amplification.
(IV) Up to this point is one cycle, and thereafter, by repeating the steps (I) to (III), a specific DNA fragment is amplified.

In the above PCR, BSA and a nonionic surfactant may be further used, if necessary. Further, an antibody having an activity of suppressing the polymerase activity and / or the 3'-5'exonuclease activity of the thermostable DNA polymerase may be used. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective for increasing the sensitivity of PCR and reducing non-specific amplification.

(1.2)
[DNA polymerase belonging to Family B]
The DNA polymerase used in the nucleic acid amplification reagent of the present invention is a DNA polymerase belonging to Family B. In the present invention, the DNA polymerase belonging to Family B refers to a DNA polymerase having 3'-5'exonuclease activity and not 5'-3'exonuclease activity. It is preferably a DNA polymerase derived from archaea (Archea). The DNA polymerase belonging to the family B is preferably a DNA polymerase derived from archaea.

[DNA polymerase derived from archaea]
Examples of the archaeal-derived DNA polymerase belonging to Family B include DNA polymerases isolated from bacteria of the genus Pyrococcus and Thermococcus. Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. DNA polymerases isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii, but not limited to these. Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gogonarius, Thermococcus litoralis, and Thermococcus sp. JDF-3, Thermococcus sp. 9degres North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Includes, but is not limited to, DNA polymerases isolated from KS-1, Thermococcus celer, or Thermococcus siculi. These DNA polymerases are commercially available, Pfu (Starage), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Deep Vent (New England Biolabs), DeepVent (New England Biolabs) and so on. Among them, KOD DNA polymerase having excellent extensibility and thermal stability is preferable from the viewpoint of PCR efficiency.

Further, the DNA polymerase may be a mutant in which the 3'-5'exonuclease region described later is modified and / or modified so as to have a reduced base analog detection activity.

(1.3)
[Modification of DNA polymerase (I) Modification of 3'-5'exonuclease region]
The modified DNA polymerase used in the nucleic acid amplification reagent of the present invention may further contain a modification of at least one amino acid in any of the amino acid sequences of the 3'-5'exonuclease active region.

The 3'-5'exonuclease activity refers to the ability to remove the incorporated nucleotide from the 3'end of the DNA polymer, and the 3'-5'exonuclease region described above is a DNA polymerase belonging to Family B. It is a highly conserved site and is derived from the DNA polymerase derived from Thermococcus kodakaraensis (SEQ ID NO: 1), the DNA polymerase derived from Pyrococcus friosus (SEQ ID NO: 2), and Thermococcus gorgonarius. DNA polymerase (SEQ ID NO: 3), DNA polymerase derived from Thermococcus litralis (SEQ ID NO: 4), DNA polymerase derived from Pyrococcus SP GB-D (SEQ ID NO: 5), derived from Thermococcus SP JDF-3 DNA polymerase (SEQ ID NO: 6), DNA polymerase derived from Thermococcus SP 9 ° N-7 (SEQ ID NO: 7), DNA polymerase derived from Thermococcus SP KS-1 (SEQ ID NO: 8), Thermococcus. In the DNA polymerase derived from cellar (SEQ ID NO: 9) or the DNA polymerase derived from Thermococcus shikuri (SEQ ID NO: 10), it is the amino acids 137 to 147, 206 to 222, and 308 to 318. In the present invention, it is also applied to a DNA polymerase other than the DNA polymerase specifically presented with a sequence. Further, in the archaeal-derived DNA polymerases belonging to Family B other than the DNA polymerases shown in SEQ ID NOs: 1 to 10, the 3'-consisting of the amino acids 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1 5'Indicates the region corresponding to the exonuclease region.

The "amino acid corresponding to positions 137 to 147, 206 to 222, and 308 to 318 shown in SEQ ID NO: 1" is a DNA polymerase having an amino acid sequence that is not completely the same as the amino acid sequence shown in SEQ ID NO: 1. , 137-147, 206-222, and 308-318 of SEQ ID NO: 1. In the present specification, the meaning of "corresponding" in the same form of notation as described above is the same as the above-mentioned example.

In the present invention, in the amino acid sequence that is not exactly the same as the amino acid sequence shown in SEQ ID NO: 1, a certain position (order) on SEQ ID NO: 1 and the position of the corresponding amino acid are compared (aligned) with the primary structure of the sequence. When, the position corresponds to the position of SEQ ID NO: 1. In the present specification, the meaning of "corresponding position" in the notation of the same form as described above is the same as that of the above example.

Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using a commercially available analysis tool available through a telecommunication line (Internet). In the present invention, by using the default (initial setting) parameter in ClustalW (http://clustalw.dbbj.nig.ac.jp/index.php/lang=ja) of DNA Databank of Japan (DDBJ), Compare the primary structure of the array.

The modification of the 3'-5'exonuclease region described above may consist of substitutions, deletions, or additions, but is not particularly limited. For example, the modifications to the amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 1 are shown.

As the DNA polymerase in which the 3'-5'exonuclease active region is modified, it is preferable that at least one of the amino acids corresponding to positions 141, 142, 143, 210 and 311 in SEQ ID NO: 1 or SEQ ID NO: 2 is modified. .. These modified DNA polymerases lack 3'-5'exonuclease activity. More preferably, it is a DNA polymerase lacking 3'-5'exonuclease activity, wherein the amino acid modification is any one selected from D141A, E143A, D141A / E143A, I142R, N210D, or Y311F. In addition, the (exo (-)) DNA polymerase lacking 3'-5'exonuclease activity includes a complete lack of activity, for example, 0.03% and 0.05% as compared with the parent enzyme. , 0.1%, 1%, 5%, 10%, 20%, or a modified DNA polymerase having an exonuclease activity of up to 50% or less.

Another preferred form of the DNA polymerase modified from the 3'-5'exonuclease active region is any one selected from H147E or H147D in SEQ ID NO: 1 or SEQ ID NO: 2. These modified DNA polymerases have improved PCR efficiency while maintaining exonuclease activity.

A method for producing a DNA polymerase obtained by modifying a 3'-5'exonuclease active region and a method for analyzing 3'-5'exonuclease activity are known, and are disclosed in, for example, US Pat. No. 6,946,273. There is. A DNA polymerase with improved PCR efficiency refers to a modified DNA polymerase in which the amount of PCR product is increased compared to the parent enzyme. A method for analyzing whether or not the amount of PCR product is increased as compared with the parent enzyme is described in Japanese Patent No. 3891330 and the like.

(1.4)
[Modification of DNA polymerase (II) Modification to prepare a DNA polymerase variant having reduced base analog detection activity]
The DNA polymerase belonging to Family B used in the nucleic acid amplification reagent of the present invention may be a mutant having reduced base analog detection activity. The base analog indicates a base other than adenine, cytosine, guanine, and thymine, and examples thereof include uracil and inosine. Normally, DNA polymerases belonging to Family B bind strongly when they detect base analogs such as uracil and inosine, and inhibit the polymerase function. The base analog detection activity indicates an activity that strongly binds to a base analog and inhibits the polymerase function. The DNA polymerase variant belonging to Family B having a reduced base analog detection activity is a DNA polymerase variant belonging to Family B characterized by a low binding ability to uracil and inosine.

Such variants can be made by modifying at least one of the amino acid sequences for uracil binding (uracil binding pockets). Specifically, uracil-binding pockets formed by positions 1-40 and 78-130 of the amino acid sequence (SEQ ID NO: 1) of a paleobacterial DNA polymerase belonging to Family B, for example, a DNA polymerase derived from the Thermococcus kodakaraensis KOD1 strain. An example is a DNA polymerase variant in which at least one of the DNA polymerases is modified to reduce the ability to bind to uracil and inosin as compared with the wild-type DNA polymerase. DNA polymerase variants with low binding ability to uracil and inosin do not show much decrease in DNA polymerase function even in PCR in the presence of dUTP, and the effect of dUTP on the elongation reaction of DNA polymerase is reduced.

The amino acid sequences for uracil binding are highly conserved in DNA polymerases from the genus Pyrococcus and those from the genus Thermococcus. In the DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis, it is formed by amino acids 1-40 and amino acids 78-130. In Pyrococcus furiosus (SEQ ID NO: 2), it is formed by amino acids 1-40 and 78-130. In Thermococcus gorgonarius (SEQ ID NO: 3), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus litralis (SEQ ID NO: 4), it is formed by amino acids 1-40 and amino acids 78-130. In Pyrococcus SP GB-D (SEQ ID NO: 5), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus SP JDF-3 (SEQ ID NO: 6), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus SP 9 ° N-7 (SEQ ID NO: 7), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus SP KS-1 (SEQ ID NO: 8), it is formed by amino acids 1-40 and amino acids 78-130. In the thermococcus cellar (SEQ ID NO: 9), it is formed by amino acids 1-40 and 78-130. In Thermococcus shikuri (SEQ ID NO: 10), it is formed by amino acids 1-40 and amino acids 78-130.

(1.5)
More preferred as the DNA polymerase variants with reduced base analog detection activity used in the nucleic acid amplification reagents of the present invention are 7, 36, 37, 90, which are assumed to be directly related to the interaction with uracil. An paleobacillary DNA polymerase variant in which at least one of the 97 and 112-119th amino acids has been modified, eg, (a) 7, 36, 37 of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. , 90-97 and 112-119, which is a paleobacterial DNA polymerase variant represented by an amino acid sequence having a modification of at least one amino acid.

(1.6)
The above-mentioned DNA polymerase variant may be the one represented by the amino acid sequence of (b) below.
(B) In the amino acid sequence shown in (a), at least one amino acid is further modified at sites other than positions 7, 36, 37, 90 to 97 and 112 to 119, and the amino acid sequence and SEQ ID NO: 1 are used. The identity or the identity of the amino acid sequence thereof and SEQ ID NO: 2 is 80% or more (preferably 85% or more, further preferably 90% or more, still more preferably 95% or more, still more preferably 98%. The above, more preferably 99% or more), and an amino acid sequence encoding a DNA polymerase having a reduced base analog detection activity.

Various methods are known as methods for calculating the identity of amino acid sequences. For example, it can be calculated using a commercially available analysis tool available through a telecommunication line (Internet). In the present invention, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: // www. ncbi. nlm. nih. The amino acid sequence identity is calculated by using the default (default) parameters in gov / BLAST /.

(1.7)
The above DNA polymerase variant may be the one represented by the amino acid sequence of (c) below.
(C) In the amino acid sequence shown in (a), one or several amino acids are deleted, substituted or added at sites other than positions 7, 36, 37, 90 to 97 and 112 to 119, and the amino acid sequence is further deleted. , An amino acid sequence encoding a DNA polymerase with reduced base analog detection activity.

Here, "several" is not limited as long as "reduced base analog detection activity" is maintained, but is, for example, a number corresponding to less than about 20% of all amino acids, preferably less than about 15%. It is a corresponding number, more preferably a number corresponding to less than about 10%, even more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 160, preferably 2 to 120, more preferably 2 to 80, still more preferably 2 to 40, and even more. The number is preferably 2 to 5.

The "amino acid corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 1" is an amino acid sequence that is not completely the same as the amino acid sequence shown in SEQ ID NO: 1. It is an expression including an amino acid sequence relating to the binding of uracil corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 of SEQ ID NO: 1 in the DNA polymerase having the above.

In the present invention, in an amino acid sequence that is not exactly the same as the amino acid sequence shown in SEQ ID NO: 1, a certain position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (aligned). The position corresponds to the position of SEQ ID NO: 1. In the present specification, the meaning of "corresponding" in the same form of notation as described above is the same as the above-mentioned example.

In the present invention, a certain position (order) on SEQ ID NO: 6 and a corresponding position in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 are when the primary structure of the sequence is compared (aligned). The position corresponds to the position of SEQ ID NO: 1. In the present specification, the meaning of "corresponding position" in the notation of the same form as described above is the same as that of the above example.

Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using a commercially available analysis tool available through a telecommunication line (Internet). In the present invention, by using the default (initial setting) parameter in ClustalW (http://clustalw.dbbj.nig.ac.jp/index.php/lang=ja) of DNA Databank of Japan (DDBJ), Compare the primary structure of the array.

(1.8)
The DNA polymerase variant having reduced base analog detection activity used in the nucleic acid amplification reagent of the present invention is more preferably selected from at least amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. Has one amino acid modification. Here, for example, Y7 means a tyrosine (Y) residue which is the seventh amino acid, and one letter of the alphabet represents an abbreviation of a commonly used amino acid. In a preferred example, the Y7 amino acid is selected from the group consisting of tyrosine (Y) substituted with a non-polar amino acid, specifically Y7A, Y7G, Y7V, Y7L, Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C. It is an amino acid substitution. In another preferred example, the P36 amino acid is a proline (P) substituted with a positively charged polar amino acid, specifically an amino acid substitution of P36H, P36K, or P36R. In another preferred example, the V93 amino acid is valine (V) substituted with a positively charged polar amine acid, specifically an amino acid substitution of V93H, V93K, or V93R.

More preferably, the modification is a modification of at least one amino acid selected from the group consisting of Y7A, P36H, P36K, P36R, V93Q, V93K, and V93R. More preferably, it is P36K, P36R or P36H. Particularly preferably, it is P36H.

The DNA polymerase variant having reduced base analog detection activity in the present invention is a modification of two or more amino acids selected from the amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. But it's okay. Specific examples thereof include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferably Y7A / P36H or Y7A / V93K. Not limited.

In Japanese Patent No. 4395377 or Japanese Patent Application Laid-Open No. 2006-507012, amino acids 7, 36, 37, 90 to 97, and 112 to 119, which are assumed to be directly related to the interaction with uracil. Some archaeal DNA polymerase variants modified in any of the above are exemplified. However, not all of the variants have good properties to the extent that the problems of the present invention can be solved, and some of them have lost their activity.

(1.9)
Based on the modification of the DNA polymerase exemplified above, various variants can be considered as the modified DNA polymerase used in the nucleic acid amplification reagent of the present invention. Examples of such mutants include, but are not limited to, variants of archaeal DNA polymerase having any of the following modifications (1)-(4).
(1) Either (A) H147E and (B) Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36H / V93K, P36K, P36R, P36H, V93R or V93Q (2). ) (A) N210D and (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36K, P36R, P36H, V93Q, V93K or V93R (3) (A) I142R and (B) Y7A / Either (4) (A) D141A / E143A and (B) Y7A / V93K, Y7A / Either P36H, Y7A / P36R, P36R, P36H or V93K

(1.10)
[Evaluation method for base analog detection activity]
The base analog detection activity in the present invention can be evaluated by PCR. The base analog is typically uracil. For example, a dUTP solution is added to a normal PCR reaction solution containing a template DNA, a buffer material, magnesium, dNTPs, a primer, and a DNA polymerase to be evaluated at a final concentration of 0.5 μM to 200 μM, and a thermal cycle is performed. After the reaction, the presence or absence of a PCR product can be confirmed by ethidium bromide-stained 1% agarose gel electrophoresis, and the detection activity of uracil can be evaluated by the acceptable dUTP concentration. For DNA polymerase having high uracil detection activity, the extension reaction is inhibited by the addition of a small amount of dUTP, and the PCR product cannot be confirmed. In addition, DNA polymerase with low uracil detection activity can be confirmed to have DNA amplification by PCR without any problem even if a high concentration of dUTP is added.

DNA polymerase variants with reduced base analog detection activity are free of mutations as a result of performing an optimal thermal cycle using arbitrary primers and template DNA in the enzyme-optimal reaction Buffer. Compared to the wild type, this is a DNA polymerase that does not inhibit the elongation reaction even when a high concentration of dUTP is added, and the PCR product can be confirmed. However, if it is difficult to compare with the wild type, the number of archaeal DNA polymerase mutants that can be amplified by PCR even if dUTP is added at a concentration of 0.5 μM is reduced compared to the wild type. It is presumed that it has the activity of detecting base analogs.

The evaluation of the base analog detection activity in the present invention follows the following method.
KOD-Plus-Ver. Using the 10 × PCR Buffer attached to 2 (manufactured by Toyo Spinning Co., Ltd.) or the 10 × PCR Buffer attached to Pfu DNA Polymerase (manufactured by Agent), 1 × PCR Buffer, and 1.5 mM ו ₄ , 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), the primer set forth in SEQ ID NOs: 11 and 12 of 15 pmol that amplifies about 1.3 kb, 10 ng of human genomic DNA (Roche Human Genometric DNA; model number 11691112001), in 50 μl of a reaction solution containing 1 U of each enzyme. Add dUTP (manufactured by Roche) to a final concentration of 0.5, 5, 50, 100, 200 μM. After the pre-reaction at 94 ° C. for 30 seconds, PCR is performed with PCR system GeneAmp9700 (Applied Biosystem) on a schedule of repeating 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds for 30 cycles. After completion of the reaction, 1% agarose gel electrophoresis was performed on 5 μl of the reaction solution, stained with ethidium bromide, and the amplified DNA fragment of about 1.3 kb was confirmed under ultraviolet irradiation to see if the base analog detection activity was reduced. Can be evaluated.

(1.11)
[Introduction method of amino acid modification]
A method for modifying the DNA polymerase used in the nucleic acid amplification reagent of the present invention has already been established in the art. Therefore, the modification can be performed according to a known method, and the mode thereof is not particularly limited.

As one aspect of the method for introducing the modification of amino acids, a site-specific mutagenesis method based on the Inverse PCR method can be used. For example, KOD-Plus-Mutagenesis Kit (manufactured by Toyo Spinning Co., Ltd.) (1) denatures a plasmid into which a gene of interest has been inserted, anneals a mutation primer to the plasmid, and then carries out an extension reaction using a KOD DNA polymerase. , (2) Repeat the cycle of (1) 15 times, (3) Selectively cleave only the plasmid used as a template using the restriction enzyme DpnI, (4) Phosphorize the newly synthesized gene and perform transformation. It is a kit that can be carried out and cyclized, (5) transforming the cyclized gene into Escherichia coli, and obtaining a transformant carrying a plasmid into which the desired mutation has been introduced.

The modified DNA polymerase gene is transferred to an expression vector as needed, and Escherichia coli, for example, as a host is transformed with the expression vector and then applied to an agar medium containing a drug such as ampicillin to form colonies. The colonies are inoculated into a nutrient medium such as LB medium or 2 × YT medium, cultured at 37 ° C. for 12 to 20 hours, and then the cells are disrupted to extract a crude enzyme solution. The vector is preferably derived from pBluescript. Any known method may be used for crushing the cells, and for example, sonication, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the polymerase derived from the host, and the DNA polymerase activity is measured.

Any method may be used for obtaining the purified DNA polymerase from the strain selected by the above method, but for example, the following method is preferable. That is, after collecting the bacterial cells obtained by culturing in a nutrient medium, they are crushed and extracted by an enzymatic or physical crushing method to obtain a crude enzyme solution. The obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the DNA polymerase fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, heparin sepharose column chromatography can be used for separation and purification to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to the extent that it exhibits a nearly single band.

(1.12)
[Method for measuring DNA polymerase activity]
In the present invention, the activity of the DNA polymerase used as the nucleic acid amplification reagent shall be measured as follows. If the enzyme activity is strong, sample with storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). Dilute and measure.
(1) Add 25 μl of the following solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterile water, and 5 μl of enzyme solution to a microtube and react at 75 ° C. for 10 minutes. (2) After that, the mixture is ice-cooled, 50 μl of E solution and 100 μl of D solution are added, and after stirring, ice-cooled for another 10 minutes. (3) This liquid is filtered through a glass filter (GF / C filter manufactured by Whatman) and thoroughly washed with 0.1N hydrochloric acid and ethanol. (4) The radioactivity of the filter is measured with a liquid scintillation counter (TriCarb 2810TR manufactured by PerkinElmer), and the uptake of nucleotides in the template DNA is measured. One unit of enzyme activity is the amount of enzyme that takes up 10 nmol nucleotides per 30 minutes into an acid-insoluble fraction (that is, a fraction that is insolubilized when solution D is added) under these conditions.
Solution A: 40 mM Tris-HCl buffer (pH 7.5), 16 mM magnesium chloride 15 mM dithiothreitol, 100 μg / mL BSA (bovine serum albumin)
Solution B: 1.5 μg / μl Activated calf thymus DNA
Solution C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
Liquid D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
Solution E: 1 mg / mL calf thymus DNA

(2) PCNA
(2.1)
PCNA (Proliferating Cell Nuclear Antigen) used in the nucleic acid amplification reagent of the present invention is a kind of PCR enhancing factor. The PCNA is not particularly limited, but a heat-resistant one that can withstand the heat cycle of PCR is desirable, and one that remains active even after PCR is preferably desired. It is more preferably soluble even after heat treatment at 80 ° C. for 30 minutes, and it is desirable that the activity remains 50% or more, more preferably 70% or more, and particularly preferably 90% or more.

Such PCNAs include, for example, PCNAs isolated from bacteria of the genus Pyrococcus and Thermococcus. PCNAs derived from the genus Pyrococcus include Pyrococcus furiosus (SEQ ID NO: 13) and Pyrococcus sp. PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi or Pyrococcus horikoshii, but not limited to these. PCNAs derived from the genus Thermococcus include Thermococcus kodakaransis (SEQ ID NO: 14), Thermococcus gogonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9degres North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. KS-1, Thermococcus celer, or Thermococcus siculi, Methanocaldococcus jannaschii (Mja) or Methanobacterium thermoautotropic (Mth) are not particularly limited to PCNA isolated from these, including but not limited to PCNA isolated from these.

The gene encoding the PCNA can be cloned from an organism carrying the PCNA. It can also be artificially synthesized based on amino acid sequence information and nucleic acid sequence information.

Furthermore, the PCNA used in the nucleic acid amplification reagent of the present invention may be a mutant (having a DNA polymerase amplification enhancing activity) that is loaded into DNA by itself. PCNAs usually form multimers and have a ring-like structure. Loading into DNA means passing DNA through the structure of the ring of PCNA multimers, and PCNA can only be loaded into DNA in collaboration with a factor, usually called RFC. A mutant that loads into DNA alone is a mutant that makes it easier for DNA to pass inside the PCNA multimer by modifying the site involved in PCNA multimer formation and destabilizing the multimer formation. show.

Specific examples of the PCNA used in the nucleic acid amplification reagent of the present invention include the following. In the amino acid sequence set forth in SEQ ID NO: 13, 14 or 19, a polypeptide consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added and has DNA polymerase amplification enhancing activity. Is. Further, it may be a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 13, 14 or 19, and having DNA polymerase amplification enhancing activity. More preferably, the homology with the amino acid sequence set forth in SEQ ID NO: 13, 14 or 19 is 85% or more, more preferably 88% or more, still more preferably 90% or more, still more preferably 93% or more, further. It is preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. A polypeptide consisting of an amino acid sequence having such an identity of a certain level or more can be prepared based on a known genetic engineering technique as described above.

(2.1.1) KOD-PCNA and Pfu-PCNA
The sites where PCNA is involved in multimeric formation are PCNA derived from Thermococcus kodakaraensis (hereinafter, also referred to as "KOD-PCNA") (SEQ ID NO: 14), PCNA of Pyrococcus furiosus (hereinafter, "Pfu-PCNA"). (Also described) (SEQ ID NO: 13) includes an N-terminal region consisting of the 82nd, 84th, and 109th amino acids and a C-terminal region consisting of the 139th, 143rd, and 147th amino acids. The N-terminal region is positively charged, the C-terminal region is negatively charged, and interacts to form a multimer.

The description using SEQ ID NO: 13 or SEQ ID NO: 14 as an example also applies to a PCNA other than the PCNA specifically presented with the sequence herein. For example, as shown in FIG. 4, in the PCNA other than the PCNA shown in SEQ ID NOs: 13 and 14, the region relating to the multimer formation consisting of the 82nd, 84th, 109th, 139th, 143th, and 147th amino acids of SEQ ID NO: 13 Indicates the corresponding area. Here, when there are two different amino acid sequences, the amino acid or region "corresponds" in one of the reference amino acid sequences means that the reference sequence is the same when the primary structure of the amino acid sequence is compared (aligned). The position corresponds to the position.

PCNA variants that load DNA alone are more preferably involved in PCNA multimer formation.
(A) has at least one modification in the N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109, or (b) the C-terminal region consisting of amino acids corresponding to positions 139, 143 and 147, and has no RFC. Examples thereof include mutants that load into DNA and promote the elongation reaction by DNA polymerase.

For example, the amino acid corresponding to the 143rd position of SEQ ID NO: 13 is modified to a basic amino acid, the 82nd and 143rd positions are both modified to a neutral amino acid, the 147th position is modified to a neutral amino acid, or 109. Examples thereof include those obtained by modifying both the 143rd and 143rd amino acids to neutral amino acids. Examples of the neutral amino acid of the present invention include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline, serine, threonine, cysteine, methionine, asparagine and glutamine as long as they are natural. Preferably, alanine has the least effect on the three-dimensional structure of the site surrounding the substitution site. Examples of the basic amino acid include arginine, histidine, lysine, and tryptophan if they are naturally occurring. It is preferably arginine or lysine.

More preferably, the PCNA variant described in Patent Document 1 is exemplified, and the sequence in which the 147th amino acid residue is replaced with alanine (D147A), and the 82nd and 143rd amino acid residues are alanine. (R82A / D143A or R82A / E143A), a sequence in which the amino acid residues at positions 109 and 143 are replaced with alanine (R109A / D143A or R109A / E143A), and the like can be mentioned. Not limited to.

Further, the PCNA used in the nucleic acid amplification reagent of the present invention may be a modified methionine corresponding to the 73rd position of SEQ ID NO: 13 or SEQ ID NO: 14 in order to increase the expression level. More preferably, it may be modified to M73L, but the present invention is not limited to this.

(2.1.2) Mja-PCNA
Further, the PCNA used in the nucleic acid amplification reagent of the present invention is preferably the PCNA monomer represented by any of the following (1) to (3).
[1] A polypeptide consisting of an amino acid sequence in which the 142nd amino acid residue of the amino acid sequence set forth in SEQ ID NO: 19 is replaced with a basic amino acid residue.
[2] In the PCNA monomer represented by [1], one or several amino acid residues other than the amino acid residue corresponding to the 142nd amino acid residue in the amino acid sequence set forth in SEQ ID NO: 19 are substituted or deleted. A polypeptide consisting of an amino acid sequence that has been lost, inserted and / or added (collectively referred to as "mutation") and having DNA polymerase amplification enhancing activity.
[3] A polypeptide consisting of an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 19 and having DNA polymerase amplification enhancing activity.

SEQ ID NO: 19 is an amino acid sequence of PCNA derived from Methanocaldococcus jannaschii (hereinafter, also simply referred to as Mja). This amino acid sequence has been elucidated in Non-Patent Document 1 and the like.

In SEQ ID NO: 19, the 142nd amino acid residue is one of the amino acid residues involved in PCNA multimer formation. Amino acid residues involved in PCNA multimer formation are present in the N-terminal region and the C-terminal region of each monomer. The PCNA multimer is formed by joining the N-terminal region of one monomer and the C-terminal region of the other monomer as an interface. In eukaryotic cells and archaea, PCNAs often form trimers. In the amino acid sequence shown by SEQ ID NO: 19, the N-terminal region corresponds to the position of the group shown in (a) below, and the C-terminal region corresponds to the position of the group shown in (b) below.
(A) Amino acid residue group at positions 80, 82 and 108 (b) Amino acid residue group at positions 138, 142 and 146

In the above-mentioned polypeptide (2), one or several amino acid residue is substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 19 as long as it retains the DNA polymerase amplification enhancing activity. (Hereinafter, these are collectively referred to as "mutation".) It is a polypeptide consisting of an amino acid sequence. One or several mutations can be treated with restriction enzymes, treatments with exonucleases, DNA ligases, etc., position-specific mutation introduction methods and random mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New). It can be carried out by introducing a mutation into the DNA encoding the PCNA monomer of the present invention described later by using a known method such as York). The variant PCNA monomer can also be obtained by other methods such as ultraviolet irradiation. Variant PCNA monomers also include naturally occurring variants (eg, single nucleotide polymorphisms), such as when based on individual differences in microorganisms carrying PCNA, differences in species or genera.

The polypeptide of [3] above is a polypeptide consisting of an amino acid sequence that retains the DNA polymerase amplification enhancing activity and has an identity of 80% or more as compared with the amino acid sequence shown in SEQ ID NO: 19. be. Preferably, the amino acid sequence of the PCNA monomer of the present invention has an identity of 85% or more, more preferably 88% or more, still more preferably 90% or more, still more. It is preferably 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. A polypeptide consisting of an amino acid sequence having such an identity of a certain level or more can be prepared based on a known genetic engineering technique as described above.

The PCNA as described in [2] or [3] above is more preferably one to which an affinity tag such as a His tag inserted at the N-terminal is added to facilitate the purification of PCNA. Not particularly limited.

In the above PCNA monomer, the 142nd amino acid residue in SEQ ID NO: 19 is replaced with a basic amino acid residue, but the type of basic amino acid to be replaced is not particularly limited. Examples of the basic amino acid include arginine, histidine, lysine, and tryptophan if they are naturally occurring. It is preferably arginine or lysine.

(2.2)
In the method for obtaining PCNA, the PCNA gene is transferred to an expression vector as needed, Escherichia coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin, and colonies are obtained. To form. The colonies are inoculated into a nutrient medium such as LB medium or 2 × YT medium, cultured at 37 ° C. for 12 to 20 hours, and then the cells are disrupted to extract a crude enzyme solution. The vector is preferably derived from pBluescript. Any known method may be used for crushing the cells, but for example, sonication, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. .. The expression of the target protein can be confirmed by heat-treating this crude enzyme solution at 80 ° C. for 30 minutes, centrifuging it to remove the protein derived from the host, and subjecting it to SDS-PAGE.

Any method may be used to obtain purified PCNA from the strain selected by the above method, and for example, the following methods are available. After collecting the cells obtained by culturing in a nutrient medium, they are crushed and extracted by an enzymatic or physical crushing method to obtain a crude enzyme solution. The obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the PCNA fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by Q Sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to the extent that it exhibits a nearly single band.

(2.3) DNA polymerase amplification-enhancing activity Whether or not the above PCNA variant can be loaded into DNA alone (whether it has DNA polymerase amplification-enhancing activity) can be evaluated by PCR. PCNA to be evaluated was added to the PCR reaction solution containing the template DNA, buffer material, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, and the one without PCNA addition and the one with wild PCNA addition and DNA. By comparing the amount of amplification, it is possible to confirm whether or not it can be loaded into DNA alone. Even if PCNA that cannot be loaded into DNA by itself, such as wild-type PCNA, is added, the amount of DNA amplification by PCR does not change, but rather tends to decrease the amount of DNA amplification. On the other hand, mutants that can be loaded into DNA alone can be compared with those without PCNA addition to evaluate the DNA polymerase amplification-enhancing activity.

In the present invention, the evaluation of "whether the PCNA mutant can be loaded into DNA alone" (evaluation of the DNA polymerase amplification enhancing activity) shall follow the following method.
Using the 10 × PCR Buffer (concentrated 10 times the concentration used for the reaction) attached to KOD Dash (Toyobo),
1 × PCR Buffer,
0.2 mM dNTPs,
Primers according to SEQ ID NOs: 15 and 16 of 15 pmol, which amplifies about 3.6 kb of DNA.
10 ng of human genomic DNA (Roche Human Genome DNA; model number 11691112001),
Prepare the reaction solution to contain 1U KOD-Plus-DNA polymerase.
250 ng of PCNA to be evaluated is added to 50 μl of the reaction solution, and PCR is performed on a schedule of repeating 98 ° C., 10 seconds → 68 ° C., 30 seconds for 30 cycles after the prereaction at 94 ° C. for 30 seconds. After completion of the reaction, 1% agarose gel electrophoresis was performed on 5 μl of the reaction solution, stained with ethidium bromide, and the amplified DNA fragment of about 3.6 kb under ultraviolet irradiation was compared with the one to which wild-type PCNA was added. It is possible to evaluate whether or not the PCNA can be loaded into. The amount of DNA amplification increases with the addition of PCNA, which can be loaded into DNA by itself.

In the present invention, as a method for quantitatively evaluating the increase in the amount of DNA amplification, the amount of DNA amplification is quantified by using Gel Pro Analyzer (Media Cybernetics), which is software for electrophoresis pattern analysis. .. When the amount of DNA amplification is compared by such a method, the amount of DNA amplification when PCNA is added is 1.0 times (preferably 1.2 times, more preferably 1.2 times) the amount of DNA amplification when PCNA is not added. Is more than 1.5 times, more preferably 2 times and 3 times). Alternatively, if the unamplified target DNA is amplified, it is determined that the PCNA "has DNA polymerase amplification enhancing activity".

The modification of PCNA can be performed in the same manner as the modification of DNA polymerase.

In the above-mentioned nucleic acid amplification reagent of the present invention, the DNA synthesis substrate is typically composed of four types of deoxynucleotide triphosphates, dATP, dCTP, dGTP, and dTTP, but other than dATP, dCTP, dGTP, and dTTP. Deoxynucleotide triphosphates such as dUTP and dITP may be contained.

The primer used in the above-mentioned nucleic acid amplification reagent of the present invention is typically composed of a nucleotide consisting of four bases of adenine, cytosine, guanine and thymine, whereas in the nucleic acid amplification reagent of the present invention, adenine, cytosine and guanine are used. , It may contain nucleotides other than thymine, such as uracil and inosin.

Hereinafter, the present invention will be specifically described with reference to Examples. The description of the following examples is not particularly limited to the present invention.

(Example 1-1)
Preparation of KOD DNA polymerase mutant
A plasmid containing the modified thermostable DNA polymerase (Y7A / V93K / H147E and Y7A / P36H / N210D mutant) genes derived from the Thermococcus-Kodakaraensis KOD1 strain was prepared for use in the examples described later. As the DNA template used for mutagenesis, a modified thermostable DNA polymerase gene (SEQ ID NO: 17) (pKOD) derived from Thermococcus Kodakaraensis KOD1 strain cloned into pBluescript was used. Mutagenesis was performed using KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.
(Example 1-2)
Preparation of Pfu DNA Polymerase Variant A plasmid containing the modified heat-resistant DNA polymerase (Y7A / P36H / N210D mutant) gene derived from the Pyrococcus furiosus strain was prepared for use in the examples described later. As the DNA template used for mutagenesis, a modified thermostable DNA polymerase gene (SEQ ID NO: 35) (pPfu) derived from the Pyrococcus furiosus strain cloned into pBluescript was used. Mutagenesis was performed using KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.

(Example 2)
Preparation of modified thermostable DNA polymerase
The culture of the bacterial cells obtained in Example 1 was carried out as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Eshericia cultivated in 3 mL of LB medium (1% bactotrypton, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin in advance at 37 ° C. for 16 hours. Kori JM109 (plasmid transformant) (using a test tube) was inoculated and aerated and cultured at 37 ° C. for 16 hours. The cells are collected from the culture medium by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells are treated by sonication. It was crushed to obtain a cell crushed solution. Next, the cell disruption solution was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin Sepharose chromatography were performed, and finally a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithioslateol, 0) was performed. .1% Tween20, 0.1% nonidet P40, 50% glycerin) was substituted to obtain a modified heat-resistant DNA polymerase. The DNA polymerase activity measurement in the above purification step was performed according to the above DNA polymerase activity measurement method. When the enzyme activity was high, the sample was diluted for measurement.

(Example 3)
Preparation of KOD-PCNA1 mutant
A plasmid containing a modified thermostable PCNA1 (M73L / D147A mutant) gene derived from Thermococcus kodakaraensis KOD1 strain was prepared. As the DNA template used for mutagenesis, PCNA (SEQ ID NO: 18) (pKODPCNA) derived from Thermococcus Kodakaraensis KOD1 strain cloned into pBlueScript was used. KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) was used for mutagenesis, and the method was performed according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5α was transformed with the obtained plasmid and used for the preparation of the enzyme.

(Example 4)
Preparation of KOD-PCNA1
The culture of the bacterial cells obtained in Example 3 was carried out as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Eshericia cultivated in 3 mL of LB medium (1% bactotrypton, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin in advance at 37 ° C. for 16 hours. Cory DH5α (plasmid transformant) (using a test tube) was inoculated and aerated and cultured at 37 ° C. for 16 hours. The cells are collected from the culture medium by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells are treated by sonication. It was crushed to obtain a cell crushed solution. Next, the cell disruption solution was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and Q-sepharose chromatography were performed, and finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0) was performed. .1% Tween 20, 0.1% nonidet P40, 50% glycerin) was substituted to obtain a modified heat-resistant PCNA.

(Example 5)
Preparation of plasmid for Mja-PCNA mutant
A plasmid containing a modified thermostable PCNA gene variant (E142K) derived from the Methanocaldococcus jannaschii strain was prepared. As the DNA template used for mutagenesis, PCNA (SEQ ID NO: 34) (pMjaPCNA) derived from the Methanocaldococcus jannaschii strain cloned into pET23b was used. KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) was used for mutagenesis, and the method was performed according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli BL21 (DE3) pLysS was transformed with the obtained plasmid and used for the preparation of the enzyme.

(Example 6)
Fabrication of Mja-PCNA
The culture of the bacterial cells obtained in Example 1 was carried out as follows. First, 80 mL of LB medium (1% bactotrypton, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. This medium was inoculated with Escherichia coli BL21 (DE3) pLysS (plasmid transformant) (using a test tube) cultured at 37 ° C. for 16 hours in 3 mL LB medium containing 100 μg / mL ampicillin in advance, and heated to 37 ° C. The cells were aerated and cultured until the OD600 became 0.3 to 0.6. Then, IPTG was added to a final concentration of 0.5 mM, and the cells were aerated and cultured for 4 hours. The cells are collected from the culture medium by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells are treated by sonication. It was crushed to obtain a cell crushed solution. Next, the cell disruption solution was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and Q-sepharose chromatography were performed, and finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0) was performed. .1% Tween20, 0.1% nonidet P40, 50% glycerin) was replaced to obtain an Mja-PCNA variant.

(Example 7)
Amplification of long-chain target DNA
Using KOD DNA polymerase mutant (Y7A / V93K / H147E) and KOD-PCNA1 M73L / D147A mutant, a system for amplifying 17.5 kb of DNA was used to compare DNA amplification with and without PCNA. rice field. In each of the above cases, DNA amplification was compared between the case with and without further betaine. For PCR, KOD-Plus-Ver. Using the 10 × PCR Buffer attached to 2 (Toyobo),
1 × PCR Buffer,
1.5 mM ו ₄ ,
0.2 mM dNTPs,
Primers according to SEQ ID NOs: 20 and 21 of 15 pmol, which amplifies about 8.5 kb of DNA.
Human genomic DNA equivalent to 300, 30, 3 copies (Roche Human Genome DNA; model number 11691112001), and
500 ng of KOD-PCNA1 mutant (M73L / D147A) was added (or not added) to 50 μl of the reaction solution containing the 1.25U KOD DNA polymerase variant (Y7A / V93K / H147E) mixed with 1 μg of KOD antibody. And 1M betaine was added (or not added) and the reaction systems were compared.

As a control, the one without adding PCNA was also carried out. The cycle is 94 ° C, 2 minutes pre-reaction, then 98 ° C, 10 seconds → 65 ° C, 10 seconds → 68 ° C, 4 minutes 30 seconds, repeating 40 cycles of PCR system GeneAmp® 9700 (Applied Biosystem). ) Was used for PCR. After completion of the reaction, 1% agarose gel electrophoresis was performed on 5 μl of the reaction solution, stained with ethidium bromide, and the amplified amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 1 amplifies approximately 8.5 kb of DNA from a low copy template with and without the KOD-PCNA1 mutant, and with and without betaine in each of the above cases, and compares the amplification. It will be the one that was done.

As a result, no DNA amplification could be confirmed without PCNA and betaine. In addition, with PCNA and without betaine, DNA amplification was confirmed, but the specificity was poor, and there were some lanes where DNA amplification was not observed. Although DNA amplification was confirmed without PCNA and with betaine, the amplification efficiency was poor, and the amount of DNA amplification was small when amplification from 3 copies. On the other hand, with PCNA and betaine, the specificity was high, and sufficient DNA amplification was confirmed even from a low copy such as 3 copies. Long-chain target DNA has a long elongation time and is prone to non-specific amplification. Although PCNA is excellent in enhancing the amplification efficiency, it also amplifies DNA from a misannealed primer, resulting in poor reaction specificity. On the other hand, betaine has the function of not only increasing the amplification efficiency but also improving the reaction specificity, and it is considered that the combination of these has the effect of obtaining excellent amplification efficiency while increasing the reaction specificity. Be.

(Example 8)
Amplification of target DNA with regions with high GC content
Using KOD DNA polymerase mutant (Y7A / V93K / H147E) and KOD-PCNA1 M73L / D147A mutant, 5 types of DNA amplification system of about 10 kb (these 5 types have a GC rate of more than 70% and a region of 500 bp or more). In), DNA amplification was compared with and without PCNA. In each of the above cases, DNA amplification was compared between the case with and without further betaine. For PCR, KOD-Plus-Ver. Using the 10 × PCR Buffer attached to 2 (Toyobo),
1 × PCR Buffer,
1.5 mM ו ₄ ,
0.2 mM dNTPs,
6 pmol primers that amplify about 10 kb (ATCB: SEQ ID NOs: 22 and 23, BRAF: SEQ ID NOs: 24 and 25, TGFb: SEQ ID NOs: 26 and 27, ACE: SEQ ID NOs: 28 and 29, CASP3: SEQ ID NOs: 30 and 31).
20 ng of human genomic DNA (Roche Human Genome DNA; model number 11691112001), and
0.6U KOD DNA polymerase variant (Y7A / V93K / H147E) mixed with 0.5 μg KOD antibody
The reaction systems were compared by adding (or not adding) betaine to 1 M in 20 μl of the reaction solution containing, and adding (or not adding) 400 ng of the PCNA mutant. As a control, PCNA and betaine were not added. The cycle was 94 ° C., followed by a pre-reaction for 2 minutes, followed by a schedule of repeating 30 cycles of 98 ° C., 10 seconds → 60 ° C., 10 seconds → 68 ° C., 2 minutes using PCR system GeneAmp® 9700 (Applied Biosystem). PCR was performed.

FIG. 2 shows the results of PCR and electrophoresis of the obtained product with and without the KOD-PCNA1 mutant, and with and without betaine in each of the above cases.

As a result, when neither betaine nor PCNA was present, DNA amplification was not observed in all five species. In the case where betaine was not added and the KOD-PCNA1 mutant was added, DNA amplification was confirmed only in CASP3 in lane 5, but smearing occurred and the band of the amplification product could not be confirmed, and no band was seen. The success rate was low. When betaine was added and PCNA was not added, it was confirmed that there was a thin band in ATCB in lane 1 and CASP3 in lane 5, but the amplification efficiency was poor. On the other hand, DNA amplification could be confirmed in all 5 types of those to which betaine and PCNA were added, although the ACE band in lane 4 was thin. The region containing GC is difficult to amplify, and excellent amplification efficiency is required. In addition, this target DNA is as long as 10 kb, and reaction specificity is also important. The combination of PCNA and betaine has enabled efficient DNA amplification while maintaining excellent reaction specificity.

(Example 9)
DNA amplification from plant lysate
Two types of DNA polymerases, KOD DNA polymerase mutant (Y7A / P36H / N210D) and Pfu polymerase mutant (Y7A / P36H / N210D), KOD-PCNA1 mutant (M73L / D147A) and Mja-PCNA mutant (M73L / D147A) as PCNA. Using two types of E142K), it was compared and examined whether PCR could be performed from plant lysate without purification. Tobacco leaves 3 mm square were added to 100 μl of Buffer A (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) as a mold, and heat-treated at 95 ° C. for 10 minutes was used as a lysate. For PCR, use the 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo).
1 × PCR Buffer,
0.2 mM dNTPs,
Primers according to SEQ ID NOs: 32 and 33 of 15 pmol, which amplifies about 1.3 kb.
1000 ng of PCNA to be evaluated was added (or not added) to 50 μl of the reaction solution containing the 1.25U DNA polymerase mutant (KOD and Pfu, respectively, Y7A / P36H / N210D mutant) mixed with 1 μg of KOD antibody. In addition, betaine was compared with those containing 1M and those without. As a control, PCNA and betaine were not added. The cycle is 94 ° C., followed by a prereaction of 2 minutes, then 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., with a schedule of repeating 1.5 minutes for 35 cycles. PCR was performed using.
After completion of the reaction, 1% agarose gel electrophoresis was performed on 5 μl of the reaction solution, stained with ethidium bromide, and the amplified amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

In FIG. 3, a PCR reaction was carried out by adding 1000 ng of KOD-PCNA1 mutant or Mja-PCNA mutant with and without betaine of KOD DNA polymerase mutant (Y7A / P36H / N210D), and plant lysate. The results of PCR performed without purification and electrophoresis are shown. For comparison, the one without PCNA added was also carried out.

Plant lysates are rich in PCR inhibitors and are known to cause inhibition when subjected to PCR. When neither betaine nor PCNA was added, addition of 2 μl of lysate to a reactivity of 50 μl caused inhibition. The one without betaine and using the Mja-PCNA mutant caused inhibition at 4 μl, and the one without betaine and using the KOD-PCNA1 mutant caused inhibition at 8 μl. In each of the above cases, by adding betaine to the betaine, inhibition did not occur even if double lysate was added. It was confirmed that the combination of betaine and PCNA makes it more resistant to inhibition. It was found that betaine and PCNA have independent functions, and that the combination of these functions exhibits excellent amplification performance.

Similar results were obtained with the Pfu DNA polymerase variant (Y7A / P36H / N210D), and when neither betaine nor PCNA was added, addition of 2 μl of lysate to a reactivity of 50 μl caused inhibition. The one without betaine and using the Mja-PCNA mutant caused inhibition at 4 μl, and the one without betaine and using the KOD-PCNA1 mutant caused inhibition at 8 μl. In each of the above cases, by adding betaine to the betaine, inhibition did not occur even if double lysate was added. It was found that the combination of betaine and PCNA does not depend on the type of polymerase.

INDUSTRIAL APPLICABILITY The present invention is useful in biotechnology-related industries, and is particularly useful in technologies related to DNA synthesis, regardless of research use or diagnostic use.

Claims (6)

A nucleic acid amplification reagent containing a DNA polymerase belonging to Family B, Proliferating Cell Nuclear Antigen (PCNA), and betaine, which is used for nucleic acid amplification of a long-chain target of 8.5 kb or more, wherein the PCNA is SEQ ID NO: 13. In the amino acid sequence described in 1, it has at least one modification of the amino acid residue corresponding to positions 82, 84, 109, 139, 143 and 147, and consists of any of the following (1) or (2). A nucleic acid amplification reagent characterized by being a thing.
(1) In the amino acid sequence set forth in SEQ ID NO: 13, the activity consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added, and the activity of loading into DNA even without RFC . Polypeptide (2) A polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 13 and having an activity of loading into DNA even in the absence of RFC. A nucleic acid amplification reagent containing a DNA polymerase, PCNA and betaine belonging to Family B and used for nucleic acid amplification of a long-chain target of 8.5 kb or more, wherein the PCNA is used in the amino acid sequence set forth in SEQ ID NO: 14. It is characterized by having at least one modification of the amino acid residue corresponding to positions 82, 84, 109, 139, 143 and 147, and consisting of any of the following (1) or (2). Nucleic acid amplification reagent.
(1) In the amino acid sequence set forth in SEQ ID NO: 14, the amino acid sequence consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added, and has an activity of loading into DNA even in the absence of RFC . Polypeptide (2) A polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 14 and having an activity of loading into DNA even in the absence of RFC. A nucleic acid amplification reagent containing a DNA polymerase, PCNA and betaine belonging to Family B and used for nucleic acid amplification of a long-chain target of 8.5 kb or more, wherein the PCNA is used in the amino acid sequence set forth in SEQ ID NO: 19. It is characterized by having at least one modification of the amino acid residue corresponding to positions 80, 82, 108, 138, 142 and 146, and consisting of any of the following (1) or (2). Nucleic acid amplification reagent.
(1) In the amino acid sequence set forth in SEQ ID NO: 19, the activity consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added, and the activity of loading into DNA even without RFC . Polypeptide (2) A polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 19 and having an activity of loading into DNA even in the absence of RFC. The nucleic acid amplification reagent according to any one of claims 1 to 3, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from archaea. The nucleic acid amplification reagent according to any one of claims 1 to 4, which is an archaeal DNA polymerase variant having a base analog detection activity in which DNA polymerase belonging to family B is reduced. The nucleic acid amplification reagent according to any one of claims 1 to 5, wherein the DNA polymerase belonging to Family B has a modification of at least one amino acid in any of the amino acids constituting the 3'-5'exonuclease active region. ..

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