JP6428997B2 - Nucleic acid amplification method - Google Patents

Description

The present invention relates to a nucleic acid amplification method by PCR. The present invention can be used not only for research but also for clinical diagnosis and environmental examination.

PCR (polymerase chain reaction) is (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primer to template single-stranded DNA, (3) DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating the cycle with three steps of extension of the primer using.
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 microbial testing in food and the environment. It has come to be used.

On the other hand, since PCR is a very sensitive detection method, false positives caused by carry-over of previously performed PCR amplification products become a problem. Therefore, PCR is performed using a substrate containing dUTP instead of dTTP, uracil base is incorporated into the amplification product, and uracil-N-glycosylase (UNG) treatment is performed during the next PCR, thereby allowing contamination (carrying). The method of decomposing the amplified product (dUTP / UDG contamination removal method) has been taken (Non-Patent Document 1).

However, in the dUTP / UDG contamination removal method, when PCR is carried out using dUTP instead of dTTP as a substrate, there has been a problem that the reaction efficiency is lowered and the amount of amplified product is reduced.

It is also known that PCR is easily affected by contaminants such as pigments, proteins, and sugars, and the contaminants inhibit the reaction (Non-patent Document 2). Usually, when a gene in a biological sample is amplified, purification of DNA is required. However, purification of DNA is complicated and requires time, and contamination occurs during the operation. There is a risk. In addition, when the target nucleic acid content in the sample is small, it may not be recovered. Therefore, there has been a demand for a method for amplifying a target nucleic acid by bringing a biological sample into a PCR reaction solution containing dUTP without going through a purification step.

Patent 4395377 Special table 2006-507012

Gene, Vol. 93 (1), 125-128 (1990) Journal of Clinical Microbiology, Vol. 39, no. 2, p485-493 (2001)

Therefore, an object of the present invention is to provide an efficient method for amplifying a target nucleic acid in PCR containing dUTP as a substrate. More specifically, the present invention provides a nucleic acid amplification method capable of performing PCR amplification even in the presence of a large amount of contaminants derived from biological samples in PCR containing dUTP as a substrate.
Furthermore, the other object of this invention is to provide the reagent kit suitable for said objective. In summary, an object of the present invention is to provide a PCR improvement method and a PCR reaction reagent suitable for amplification of genes present in animal and plant tissues and body fluids in the presence of dUTP.

The nucleic acid amplification method of the present invention for achieving the above object uses the DNA polymerase belonging to Family B to amplify DNA in a biological sample in the presence of dUTP without going through a purification step. This is a nucleic acid amplification method.

That is, the present inventor uses a DNA polymerase belonging to family B in a nucleic acid amplification method in which a biological sample itself and a gene amplification reaction solution are mixed and reacted, so that even if a large amount of biological contaminants exist, Found that PCR is possible even when a biological sample such as blood or oral mucosa is directly added to the PCR reaction solution, and the present invention has been achieved.

In addition, since a DNA polymerase belonging to the normal family B binds strongly to uracil, it could not be amplified by PCR containing dUTP as a substrate. For this reason, in PCR containing dUTP as a substrate, no DNA polymerase belonging to family B is used to amplify DNA in a biological sample without undergoing a purification step.

The representative invention of the present application is as follows.
[1]
A method of amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution, wherein the enzyme used for the amplification is a DNA polymerase belonging to Family B, and deoxyuridine (dUTP ) In the reaction solution.
[2]
The nucleic acid amplification method according to [1], wherein the biological sample is any of animal and plant tissues, body fluids, excreta, cells, bacteria, and viruses.
[3]
The nucleic acid amplification method according to [1] or [2], wherein the enzyme used for nucleic acid amplification is an archaeal DNA polymerase mutant having reduced base analog detection activity.
[4]
The nucleic acid amplification method according to any one of [1] to [3], wherein the enzyme used for nucleic acid amplification has a DNA synthesis rate of 30 bases / second or more.
[5]
The nucleic acid amplification method according to [3] or [4], wherein the archaeal DNA polymerase mutant is any one of the following (a) to (c):
(A) at least one amino acid among amino acids corresponding to the seventh, thirty-six, thirty-seventh, ninety-seventh to ninety-seventh and ninety-first to ninety-seventh amino acids of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 (corresponding to the Pfu wild-type sequence) It is an amino acid sequence having the following modification.
(B) A reduced base analog in which at least one amino acid is further modified in the amino acid sequence shown in (a), the amino acid sequence is 80% or more identical to the amino acid sequence shown in (a), and reduced An amino acid sequence encoding a DNA polymerase having detection activity.
(C) At least one amino acid is further modified in the amino acid sequence represented by (a), and one or several amino acids are deleted, substituted or added in the amino acid sequence represented by (a). And an amino acid sequence encoding a DNA polymerase having reduced base analog detection activity.
[6]
The mutant of archaebacterial DNA polymerase has at least one amino acid modification among amino acids corresponding to positions 36 and 93 in SEQ ID NO: 1 or SEQ ID NO: 2, according to any of [3] to [5] Nucleic acid amplification method.
[7]
The archaeal DNA polymerase variant is any one of SEQ ID NO: 1 or SEQ ID NO: 2, wherein Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36H / V93K, P36K, P36R or P36H The nucleic acid amplification method according to any one of [3] to [6], wherein
[8]
The nucleic acid amplification according to any one of [3] to [7], wherein the archaebacterial DNA polymerase mutant has any one of Y7A / V93K, Y7A / P36H, and P36H in SEQ ID NO: 1 or SEQ ID NO: 2. Law.
[9]
The nucleic acid amplification according to any one of [1] to [8], wherein the DNA polymerase belonging to Family B further has at least one amino acid modification in any of the amino acids constituting the 3′-5 ′ exonuclease active region Law.
[10]
Modification to the 3′-5 ′ exonuclease active region of DNA polymerase belonging to Family B is any one of the amino acids corresponding to D141, I142, E143, H147, N210 and Y311 in SEQ ID NO: 1 or SEQ ID NO: 2, The nucleic acid amplification method according to any one of [1] to [9], wherein the method has at least one amino acid modification.
[11]
The modification to the 3′-5 ′ exonuclease active region of DNA polymerase belonging to Family B is any one of D141A / E143A, I142R, H147E, H147D, N210D or Y311F in SEQ ID NO: 1 or SEQ ID NO: 2 [1 ] The nucleic acid amplification method according to any one of [10] to [10].
[12]
The nucleic acid amplification according to any of [3] to [11], wherein the archaebacterial DNA polymerase mutant has any one of the following modifications (1) to (4) in SEQ ID NO: 1 or SEQ ID NO: 2. Law.
(1) (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, (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36K, P36R, P36H, V93Q, V93K or V93R (3) (A) I142R and (B) Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36R, P36H, V93K, V93R or V93Q (4) (A) D141A / E143A and (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36R, P36H or One of the V93K [13]
The nucleic acid amplification method according to any one of [1] to [12], further comprising PCNA in the reaction solution.
[14]
The nucleic acid amplification method according to any one of [1] to [13], wherein PCNA is a mutant that is loaded alone into DNA.
[15]
PCNA is (a) N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109 of the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, and (b) amino acids corresponding to positions 139, 143 and 147 The nucleic acid amplification method according to any one of [1] to [14], which is a mutant having at least one modification in the C-terminal region.
[16]
PCNA modified amino acid corresponding to position 143 in SEQ ID NO: 21 or SEQ ID NO: 22 to basic amino acid, or modified from both amino acids corresponding to positions 82 and 143 to neutral amino acid, equivalent to position 147 The amino acid according to any one of [1] to [15], wherein the amino acid is a neutral amino acid, or the amino acids corresponding to the 109th and 143rd amino acids are both modified to neutral amino acids. Nucleic acid amplification method.
[17]
[1] A reagent for performing the nucleic acid amplification method according to any one of [16].
[18]
A kit comprising a reagent for performing the nucleic acid amplification method according to any one of [1] to [16].

The present invention eliminates the risk of loss and carryover during DNA purification, and further reduces time and cost. Also, in order to prevent contamination by the dUTP / UDG contamination removal method, not only in the research field, but also in the clinical field or forensic field such as genetic diagnosis in which the same sample is amplified many times, or in the microbiological examination in foods and the environment, etc. Can also be widely used

Evaluation of reduced base analog detection activity of DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of reduced base analog detection activity of DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of long-chain DNA amplification of DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of long-chain DNA amplification of DNA polymerase used in the nucleic acid amplification method of the present invention Comparison of DNA polymerase synthesis rates used in the nucleic acid amplification method of the present invention. Evaluation of amplification from blood using DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of PCR enhancement factor (PCNA) Comparison of synthesis rate of DNA polymerase used in nucleic acid amplification method of the present invention by addition of PCNA Evaluation of amplification from blood using DNA polymerase and PCNA used in the nucleic acid amplification method of the present invention Amplification evaluation from body tissues (nail, hair, oral mucosa) using DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of amplification from plant lysate using DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of amplification from the presence of feces using DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of amplification from blood using DNA polymerase and PCNA used in the nucleic acid amplification method of the present invention It is a figure which shows the amino acid area | region regarding the multimer formation of PCNA.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
In this specification, the base sequence, amino acid sequence, and individual components thereof may be simplified by alphabetical symbols, but all follow the practices in the fields of molecular biology and genetic engineering. Further, in this specification, in order to simply show the mutation of the amino acid sequence, for example, a notation such as “D143A” is used. “D143A” indicates that the 143rd aspartic acid was substituted with alanine, that is, the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. Further, the sequence numbers correspond to the sequence numbers 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 substituted with alanine and the 147th aspartic acid was substituted with alanine.
Further, in this specification, the term “mutant type” in the case of “mutant PCNA” means that it has an amino acid sequence different from the conventionally known PCNA, and it is due to artificial mutation or due to mutation in nature. There is no distinction.

(1) [Features of the present invention]
The nucleic acid amplification method of the present invention is a method of amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution, and the enzyme used for amplification is a polymerase belonging to family B And deoxyuridine (dUTP) is contained in the reaction solution.

(2) [Do not go through purification step]
In the nucleic acid amplification method of the present invention, nucleic acid in a biological sample that has not undergone a purification step is amplified without purification. Purification is a method for separating contaminants such as tissues and cell walls of biological samples from DNA in biological samples. Methods for separating DNA using phenol or phenol / chloroform, ion exchange resins, glass filters, etc. Alternatively, there is a method of separating DNA with a reagent having a protein aggregation action.

When nucleic acids to be amplified such as organs and cells are present in the tissue of the sample, an act of destroying the tissue to extract the nucleic acid (destruction by physical treatment, destruction using a surfactant, etc.) Does not correspond to the purification referred to in the present invention. Further, the act of diluting the sample obtained by the above method or the biological sample with a buffer solution does not correspond to the purification referred to in the present invention.

The nucleic acid amplification method in the present invention is a method in which a biological sample is added to a nucleic acid amplification reaction solution and amplified without taking these purification methods. In the present invention, the “biological sample that has not undergone the purification step” means that the biological sample itself, or a liquid biological sample diluted with a solvent such as water, or a solid biological sample is added to a solvent such as water and heated. However, it is not limited to these as long as it has not undergone a purification process.

In the present specification, the term “blood resistance” is used, which means that even when blood is present in a nucleic acid amplification reaction solution, it resists inhibition. The higher the blood concentration that can be amplified, the higher the blood resistance.

(3) [Biological sample]
The biological sample applied to the nucleic acid amplification method of the present invention is not particularly limited as long as it is a sample collected from a living body. For example, it refers to animal and plant tissues, body fluids, excreta, cells, bacteria, viruses and the like. Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.

(4) [Deoxyuridine (dUTP)]
In the nucleic acid amplification method of the present invention, the reaction solution contains dUTP. The route for obtaining dUTP is not particularly limited, but commercially available products can be used.
The concentration of dUTP in the reaction solution is not particularly limited, but the preferable lower limit is 0.5 μM or more, more preferably 50 μM or more, and more preferably 0.1 mM or more from the viewpoint of the removal efficiency of contamination. From the viewpoint of PCR efficiency, dUTP may be contained at a high concentration. A preferable upper limit is 1 mM or less, more preferably 0.6 mM or less.
Moreover, dTTP and dUTP may be mixed from the viewpoint of PCR efficiency. The ratio of dTTP and dUTP is preferably 100: 1 to 1: 100. More preferably, it is 10: 1 to 1:10, and further preferably 1: 1, but is not limited thereto.

The nucleic acid applied to the nucleic acid amplification method of the present invention is not limited by the difference in length, sequence, GC content, etc., but since dUTP is incorporated during amplification, the nucleic acid must contain T. It is preferable that 10 or more T is contained.

(5) [DNA polymerase belonging to family B]
The DNA polymerase used in the nucleic acid amplification method of the present invention is a DNA polymerase belonging to family B. The DNA polymerase belonging to the family B is preferably an archaea-derived DNA polymerase.

(6) [Analogous DNA polymerase]
Examples of DNA polymerases derived from archaea belonging to family B include DNA polymerases isolated from bacteria of the genera Pyrococcus and Thermococcus.
Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. Including, but not limited to, DNA polymerases isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii.
Examples of DNA polymerases derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including but not limited to DNA polymerase isolated from KS-1, Thermococcus celler, or Thermococcus sicili.
PCR enzymes using these DNA polymerases are commercially available, Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England) , Tgo (Roche), Pwo (Roche).

Among these, from the viewpoint of PCR efficiency, KOD DNA polymerase excellent in extensibility and thermal stability is preferable.

(7) [DNA polymerase mutant having reduced base analog detection activity]
The DNA polymerase belonging to family B used in the nucleic acid amplification method of the present invention may be a mutant having reduced base analog detection activity. Base analogs refer to bases other than adenine, cytosine, guanine, and thymine, and include uracil and inosine. Usually, a DNA polymerase belonging to Family B binds strongly when a base analog such as uracil or inosine is detected, and inhibits the polymerase function. The base analog detection activity refers to an activity that strongly binds to a base analog and inhibits the polymerase function. A DNA polymerase mutant belonging to family B having reduced base analog detection activity is a DNA polymerase mutant belonging to family B characterized by low binding ability to uracil and inosine.

As such a mutant having a decreased base analog detection activity of a DNA polymerase belonging to Family B, an archaeal DNA polymerase mutant can be exemplified.
Specifically, the amino acid sequence (uracil-binding pocket) relating to the binding of uracil formed by amino acids 1 to 40 and amino acids 78 to 130 is modified, and compared to wild-type DNA polymerase, uracil or inosine An archaeal DNA polymerase mutant characterized by low binding ability. Archaeal DNA polymerase mutants with low ability to bind to uracil and inosine do not show much functional degradation of DNA polymerases derived from archaea belonging to family B even in PCR in the presence of dUTP, and DNA polymerase elongation reaction by dUTP The impact on is reduced.

(8) [DNA synthesis rate of DNA polymerase]
The DNA polymerase mutant having reduced base analog detection activity used in the nucleic acid amplification method of the present invention preferably has a higher DNA synthesis rate than the wild type (WT).

The wild-type polymerase belonging to family B has high accuracy. This is to amplify while confirming whether the incorporated base is correct. Examples of bases that cause errors include uracil that is generated by thermal decomposition of cytosine and paired with alanine. Polymerases belonging to Family B have a pocket that binds strongly to uracil, and amplify the nucleic acid to be amplified while checking for the presence of this uracil one by one.

On the other hand, this pocket interacts strongly with uracil and slows (or stops) the reaction. This interaction is thought to extend to bases such as cytosine that are somewhat similar in structure, and thus the DNA synthesis rate may be suppressed. When the DNA polymerase mutant having reduced base analog detection activity used in the nucleic acid amplification method of the present invention is modified to the uracil-binding pocket, it will not interact with not only uracil but also cytosine, etc. Conceivable.

The extent to which the synthesis rate is increased by modifying the pocket that strongly binds to uracil is higher when the mutant that modified the exo region is further modified than when the wild type is modified.

The DNA polymerase belonging to the family B or the DNA polymerase derived from archaea desirably has a DNA synthesis rate of 30 bases / second or more by the following measurement method.
In the presence of dUTP, PCR is inhibited by strong binding between dUTP and polymerase. A polymerase having a higher DNA synthesis rate is less likely to cause dUTP binding and is less susceptible to dUTP in the presence of dUTP.

<Method for measuring DNA polymerase synthesis rate>
In the present invention, the synthesis rate of the DNA polymerase used in the nucleic acid amplification method is measured as follows. If the enzyme activity is strong, the sample is stored in a 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) Mix 2.5 μl of the following A liquid, 2.5 μl of B liquid, 1.5 μl of C liquid, 4.8 μl of D liquid, and 4 μl of E liquid, 10 minutes at 95 ° C., 10 minutes at 37 ° C. Place and create substrate.
(2) Add 5 μl of a 0.64 ng / μl enzyme solution to the substrate incubated at 75 ° C. for 30 seconds, and react at 75 ° C. for 30 seconds, 60 seconds, and 120 seconds. Then ice-cool, add 35 μl of solution F, and stir well.
(3) This solution is applied to 10 μl, 1% alkaline agarose gel, and electrophoresed using Biotinylated 2-Log DNA Ladder (0.1-10 kb) (manufactured by NEB) as a marker.
(4) Blotting to Hybond N + and detecting biotin according to NEB's Phototope-Star Chemiluminescent Detection Kit manual. The length of the chain extended per second is defined as the synthesis rate.
A: 10 × PCR Buffer for KOD-Plus-Ver. 2 (TOYOBO)
B: 2 mM dNTPs (manufactured by TOYOBO)
C: 25 mM MgSO ₄
D: 200 ng / μl M13 ssDNA (manufactured by TaKaRa)
E: 100 μM Biotinylated P7 primer (Sequence: Biotin-CGCCAGGGTTTCCCAGTCACGAC)
F: 59 mM NaOH, 59 mM EDTA, 0.1% BPB 30% Glycerol

The DNA synthesis rate can be further increased by adding a PCR enhancing factor (for example, PCNA) described later. A DNA polymerase having a DNA synthesis rate of 50 bases / second or more is preferable, but not limited thereto.

(9) [Arcobacterium DNA polymerase mutant]
The “DNA polymerase mutant having reduced base analog detection activity” will be described more specifically.
The amino acid sequence for uracil binding is highly conserved in DNA polymerases from Pyrococcus and DNA polymerases from Thermococcus. In DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis, it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus furiosus (SEQ ID NO: 2), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus gorgonarius (SEQ ID NO: 3), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus litoralis (SEQ ID NO: 4), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus sp. GB-D (SEQ ID NO: 5), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp. JDF-3 (sequence number 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 to 40 and amino acids 78 to 130. In Thermococcus sp KS-1 (SEQ ID NO: 8), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus cellar (SEQ ID NO: 9), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus cyclis (SEQ ID NO: 10), it is formed by amino acids 1 to 40 and amino acids 78 to 130.

More preferred as a DNA polymerase mutant for use in the nucleic acid amplification method of the present invention is the seventh, thirty-six, thirty-seven, thirty-nine, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-seventh, ninety-nineth, thirty-seventh, ninety-nineth, thirty-seventh, ninety-nineth, thirty-seventh, ninety-seventh, thirty-seventh, ninety-nine Archaebacterial DNA polymerase mutant in which at least one of amino acids is modified, that is, (a) 7, 36, 37, 90 to 97 and 112 to 119 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 Is an archaeal DNA polymerase variant represented by an amino acid sequence having at least one amino acid modification.

The archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (b).
(B) In the amino acid sequence represented by (a), at least one amino acid is further modified, and the amino acid sequence is 80% or more identical (preferably 85% or more identical to the amino acid sequence represented by (a), More preferably 90% or more, more preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.) And a reduced base analog An amino acid sequence encoding a DNA polymerase having detection activity.

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

The archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (c).
(C) At least one amino acid is further modified in the amino acid sequence represented by (a), and one or several amino acids are deleted, substituted or added in the amino acid sequence represented by (a). An amino acid sequence encoding a DNA polymerase having a reduced base analog detection activity.

Here, “several” is not limited as long as “decreased 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. Preferably it is 2-5.

The “amino acids corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 1” are amino acid sequences that are not completely identical to the amino acid sequence shown in SEQ ID NO: 1. In the DNA polymerase having the amino acid sequence, the 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 present specification, in a amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1, a position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (alignment) , A position corresponding to the position of SEQ ID NO: 1.
Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In the present specification, by using the default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja) of DNA Database of Japan (DDBJ), Compare the primary structure of the sequences.

The archaeal DNA polymerase mutant having reduced base analog detection activity used in the nucleic acid amplification method of the present invention is more preferably selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. Having at least one amino acid modification.
Here, for example, Y7 means a tyrosine (Y) residue that 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 has tyrosine (Y) substituted with a nonpolar amino acid, specifically selected from the group consisting of Y7A, Y7G, Y7V, Y7L, Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C. Amino acid substitution.

In another preferred example, the P36 amino acid is a proline (P) substituted with a positively charged polar amino acid, specifically a P36H, P36K, or P36R amino acid substitution. In another preferred example, the V93 amino acid is a valine (V) having a positive charge and substituted with a polar amine acid, specifically an amino acid substitution of V93H, V93K, or V93R. More preferably, it is V93K.

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

The archaeal DNA polymerase mutant having reduced base analog detection activity in the present invention modifies two or more amino acids selected from amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. What you did is fine. Specific examples include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferred examples include Y7A / P36H or Y7A / V93K. It is not limited to.

In Patent Document 1 or 2, any of the amino acids at positions 7, 36, 37, 90 to 97, and 112 to 119, which are assumed to be directly related to the interaction with uracil, was modified. Several archaeal DNA polymerase mutants are exemplified, but not all variants have good properties to meet the challenges of the present application, some of which have lost activity .

(10) [Method for evaluating 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 at a final concentration of 0.5 μM to 200 μM to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and a DNA polymerase to be evaluated, and thermal cycling is performed. . After the reaction, the presence or absence of a PCR product can be confirmed by ethidium bromide-stained agarose electrophoresis, and the detection activity of uracil can be evaluated by the allowable dUTP concentration. A DNA polymerase having a high uracil detection activity inhibits the extension reaction when a little dUTP is added, and the PCR product cannot be confirmed. In addition, DNA polymerase with low uracil detection activity can confirm gene amplification by PCR without problems even when a high concentration of dUTP is added.

An archaeal DNA polymerase mutant having reduced base analog detection activity is a result of optimal thermal cycling using any primer and DNA as a template in an enzyme optimal reaction buffer. Compared with the wild-type without DNA, it means a DNA polymerase in which the extension reaction is not inhibited 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 archaeal DNA polymerase mutant that can amplify PCR even when dUTP is added at a concentration of 0.5 μM is reduced compared to the wild type. It is presumed to have the activity of detecting a base analog.

The evaluation of the base analog detection activity in the present invention follows the following method.
KOD-Plus-Ver. 2 (Toyobo) attached 10 × PCR Buffer or Pfu DNA Polymerase (Agilent) attached 10 × PCR Buffer, 1 × PCR Buffer, 1.5 mM MgSO ₄ , 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), 15 pmol of the primer according to SEQ ID NOS: 13 and 14, which amplifies about 1.3 kb, 10 ng of human genomic DNA (Roche), and 1 U of each enzyme in a 50 μl reaction solution containing dUTP ( 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 on a schedule of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds repeated 30 cycles. After completion of the reaction, 5 μl of the reaction solution is subjected to agarose electrophoresis, ethidium bromide staining, and an amplified DNA fragment of about 1.3 kb is confirmed under ultraviolet irradiation to evaluate whether the base analog detection activity is reduced. .

The higher the dUTP concentration at which PCR can be amplified, the lower the base analog detection activity. In this specification, this is also expressed as “dUTP resistance is higher” as the dUTP concentration at which PCR can be amplified is higher.

(11) [Method for measuring DNA polymerase activity]
The DNA polymerase used in the nucleic acid amplification method of the present invention measures the activity as follows. If the enzyme activity is strong, the sample is stored in a 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) 25 μl of the following solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterilized water, and 5 μl of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes.
(2) Then, ice-cool, add 50 μl of E solution and 100 μl of D solution, and stir on ice for another 10 minutes after stirring.
(3) This solution 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 (manufactured by Packard), and the incorporation of nucleotides into the template DNA is measured. One unit of enzyme activity is defined as the amount of enzyme that takes 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction (that is, the fraction insolubilized when solution D is added) under these conditions.
A: 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15
mM dithiothreitol 100 μg / mL BSA (bovine serum albumin)
B: 1.5 μg / μl activated calf thymus DNA
C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
E: 1 mg / mL calf thymus DNA

(12) [Modification of 3′-5 ′ exonuclease region]
The modified DNA polymerase used in the nucleic acid amplification method of the present invention may further contain at least one amino acid modification in any of the amino acids constituting the 3′-5 ′ exonuclease active region.
3′-5 ′ exoase activity refers to the ability to remove incorporated nucleotides from the 3 ′ end of a DNA polymer, and the above 3′-5 ′ exonuclease region is a DNA polymerase belonging to family B and highly DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis, DNA polymerase (SEQ ID NO: 2) derived from Pyrococcus furiosus, DNA derived from Thermococcus gorgonarius Polymerase (SEQ ID NO: 3), DNA polymerase (SEQ ID NO: 4) derived from Thermococcus litoralis, DNA polymerase (SEQ ID NO: 5) derived from Pyrococcus sp. GB-D, Thermococcus sp. JDF-3 DNA polymerase (SEQ ID NO: 6), Thermoco DNA polymerase derived from Cass SP 9 ° N-7 (SEQ ID NO: 7), DNA polymerase derived from Thermococcus sp KS-1 (SEQ ID NO: 8), DNA polymerase derived from Thermococcus cellar (SEQ ID NO: 9) In the DNA polymerase (SEQ ID NO: 10) derived from Thermococcus cyclis, the amino acids 137 to 147, 206 to 222, and 308 to 318 are used. The present invention is also applicable to DNA polymerases other than the DNA polymerase specifically presenting the sequence. Further, in the archaeal DNA polymerase belonging to Family B other than the DNA polymerases shown in SEQ ID NOs: 1 to 10, 3′- consisting of amino acids 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1 The region corresponding to the 5 ′ exonuclease region is shown.

The “amino acids corresponding to the 137th to 147th, 206th to 222nd, and 308th to 318th amino acids shown in SEQ ID NO: 1” are DNA polymerases having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1. 3 is an expression including amino acid sequences corresponding to positions 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 1.

The modification to the 3'-5 'exonuclease region may consist of substitution, deletion, or addition. Modifications to 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, a DNA polymerase in which at least one of amino acids corresponding to positions 141, 142, 143, 210, 311 in SEQ ID NO: 1 or SEQ ID NO: 2 is modified is preferable. . These modified DNA polymerases are deficient in 3′-5 ′ exonuclease activity.
More preferably, it is a DNA polymerase deficient in 3′-5 ′ exonuclease activity, wherein the amino acid modification is any one selected from D141A, E143A, D141A / E143A, I142R, N210D, or Y311F.
In addition, 3'-5 'exonuclease activity-deficient (exo (-)) DNA polymerase includes a complete lack of activity, for example, 0.03%, 0.05% compared to the parent enzyme , 0.1%, 1%, 5%, 10%, 20%, or a maximum of 50% or less exonuclease activity.

As a DNA polymerase in which the 3'-5 'exonuclease active region is modified, another preferred form is one in which the amino acid corresponding to position 147 in SEQ ID NO: 1 or SEQ ID NO: 2 is modified. More preferably, it is any one selected from H147E and H147D. These modified DNA polymerases have improved PCR efficiency while maintaining exonuclease activity.

Based on the modification of the DNA polymerase exemplified in the above (9) and (12), various mutants can be considered as the modified DNA polymerase used in the nucleic acid amplification method of the present invention. Examples of such mutants include, but are not limited to, archaeal DNA polymerase mutants having any of the following modifications (1) to (4).
(1) (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, (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36K, P36R, P36H, V93Q, V93K or V93R (3) (A) I142R and (B) Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36R, P36H, V93K, V93R or V93Q (4) (A) D141A / E143A and (B) Y7A / V93K, Y7A / P36H, Y7A / P36R, P36R, P36H or One of the V93K

(13) Method for introducing amino acid modification In addition, a method for generating a DNA polymerase in which a 3′-5 ′ exonuclease active region is modified and a method for analyzing 3′-5 ′ exonuclease activity are known. This is disclosed in Japanese Patent No. 6946273. 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 the amount of the PCR product is increased as compared with the parent enzyme is described in Japanese Patent No. 3891330.

A method for modifying the DNA polymerase used in the nucleic acid amplification method of the present invention has already been established in the art. Therefore, it can modify | change according to a well-known method, The aspect in particular is not restrict | limited.

As one embodiment of a method for introducing an amino acid modification, a site-directed mutagenesis method based on the inverse PCR method can be used. For example, KOD-Plus-Mutageness Kit (manufactured by Toyobo) (1) denatures a plasmid into which a target gene has been inserted, anneals the mutation primer to the plasmid, and then performs an extension reaction using KOD DNA polymerase. (2) Repeat the cycle of (1) 15 times, (3) Selectively cleave only the plasmid using the restriction enzyme DpnI, (4) Phosphorylation of newly synthesized gene, Ligation (5) A kit that can transform a cyclized gene into Escherichia coli and obtain a transformant having a plasmid introduced with the target mutation.

The modified DNA polymerase gene is transferred to an expression vector as necessary, and, for example, E. coli 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 colony is inoculated in a nutrient medium such as LB medium or 2 × YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted. A vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, 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 host-derived polymerase, and the DNA polymerase activity is measured.

Any method may be used as a method for obtaining purified DNA polymerase from the strain selected by the above method, for example, the following method. After collecting the cells obtained by culturing in a nutrient medium, the crude enzyme solution is obtained by crushing and extraction by enzymatic or physical crushing methods. 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, it can be separated and purified by heparin sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to such an extent that it shows an almost single band.

(14) Nucleic Acid Amplification Method The nucleic acid amplification method of the present invention is a method of adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution to amplify a target nucleic acid in the biological sample. It is not particularly limited except that it is a DNA polymerase belonging to family B and contains deoxyuridine (dUTP) in the reaction solution.

A typical example of a method that can be amplified by a DNA polymerase is PCR. In the present invention, not only PCR but also DNA is used as a template, and one primer, dNTP (deoxyribonucleotide triphosphate) is reacted. It is also used in a method of synthesizing a DNA primer extension product by extending a primer by the above method. Specifically, a primer extension method, a sequencing method, a conventional method that does not perform temperature cycling, and a cycle sequence method are included.

In the method of the present invention, typical conditions in the case of PCR are shown below, but are not limited thereto.
Amplification target DNA obtained from an unpurified biological sample,
(A) an archaeal DNA polymerase variant having reduced base analog detection activity (b) DNA synthesis comprising a pair of primers (c) dUTP in which one primer is complementary to the DNA extension product of the other primer A substrate (deoxynucleotide triphosphate (dNTP)) and
(D) mixing a buffer solution containing magnesium ions, ammonium ions and / or potassium ions,
By raising or lowering the temperature of the reaction solution using a thermal cycler or the like, a specific DNA fragment is amplified by repeating thermal cycles of (1) DNA denaturation, (2) primer annealing, and (3) primer extension.
In the PCR method, a PCR enhancing factor (described later), BSA, a nonionic surfactant, and the like may be further used as necessary.

In the PCR method, if necessary, an antibody having the activity of suppressing the polymerase activity and / or 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 nonspecific amplification.

(15) [PCR enhancing factor]
Furthermore, the nucleic acid amplification reaction in the present invention may contain a PCR enhancing factor. The enhancement factor refers to a complex or protein having polynucleotide polymerase enhancing activity such as PEF, dUTPase, ssb, PCNA, RFC, helicase, etc. (Hogrefe et al., 1997, Strategies 10: 93-96; and US Pat. 6,183,997 etc.). PCR enhancing factors also include non-protein factors such as DMSO and betaine. The PCR enhancing factor used in the nucleic acid amplification method of the present invention is not limited to these, but PCNA is particularly preferred.

The PCNA used in the nucleic acid amplification method of the present invention is desirably heat-resistant to withstand the thermal cycle of PCR, and preferably remains active after PCR. More preferably, it is soluble even after heat treatment at 80 ° C. for 30 minutes, and the activity remains at 50% or more, more preferably 70% or more, more preferably 90% or more.

The PCNA used in the nucleic acid amplification method of the present invention more preferably includes PCNA isolated from bacteria of the genus Pyrococcus and Thermococcus. As PCNA derived from the genus Pyrococcus, Pyrococcus furiosus, Pyrococcus sp. Including, but not limited to, PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii. PCNAs derived from the genus Thermococcus include Thermococcus kodakaaraensis (SEQ ID NO: 21), Thermococcus gonorarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, PCNA isolated from KS-1, Thermococcus celler, or Thermococcus sicili.

PCNA used in the nucleic acid amplification method of the present invention may be a modified methionine corresponding to position 73 of SEQ ID NO: 21 or SEQ ID NO: 22 in order to increase the expression level. More preferably, it is modified to M73L, but is not limited thereto.

Furthermore, the PCNA used in the nucleic acid amplification method of the present invention may be a mutant that is loaded alone into DNA. PCNA usually forms a multimer and has a ring-like structure. Loading to DNA indicates that the DNA is allowed to pass inside the ring structure of the PCNA multimer, and PCNA can be loaded into DNA only in combination with a factor usually called RFC. Mutants that load DNA alone are those that modify the sites involved in PCNA multimer formation and destabilize multimer formation, making it easier to pass DNA into PCNA multimers without RFC. Show.

The site where PCNA is related to multimer formation is PCNA (SEQ ID NO: 21) derived from Thermococcus kodakaraensis, and PCNA of Pyrococcus furiosus (SEQ ID NO: 22) is an N consisting of amino acids 82, 84 and 109. Examples thereof include a terminal region and a C-terminal region consisting of amino acids 139, 143 and 147. The N-terminal region is positively charged, the C-terminal region is negatively charged, and multimers are formed by interaction.

What has been described above and below using SEQ ID NO: 21 or SEQ ID NO: 22 as an example also applies to PCNAs other than the PCNAs whose sequences are specifically presented herein. For example, in PCNA other than the PCNA shown in SEQ ID NOs: 21 and 22 as shown in FIG. 14, a region relating to multimer formation consisting of amino acids 82, 84, 109, 139, 143, and 147 of SEQ ID NO: 21 Indicates the corresponding area.

More preferably, the PCNA variant that is loaded into DNA alone is more preferably the amino acid sequence represented by SEQ ID NO: 21 or SEQ ID NO: 22, which is involved in PCNA multimer formation.
(A) N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109, or (b) C-terminal region consisting of amino acids corresponding to positions 139, 143 and 147, having at least one modification and no RFC Both include mutants that load into DNA and promote the elongation reaction of DNA polymerase.

For example, the amino acid corresponding to the 143rd position of SEQ ID NO: 21 is changed to a basic amino acid, the 82nd and 143rd positions are both changed to neutral amino acids, the 147th position is changed to a neutral amino acid, or 109 And the like, in which both the 145th and 143rd are modified to neutral amino acids.
Examples of the neutral amino acid of the present invention include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine. Preferably, alanine has the least influence on the three-dimensional structure of the peripheral site of the substitution site. Examples of basic amino acids include arginine, histidine and lysine as long as they are natural. Arginine is preferable.

More preferably, in addition to the PCNA variant described in WO2007 / 004654, a sequence in which the 147th amino acid residue is replaced with alanine (D147A), the 82nd and 143rd amino acid residues are alanine. (SEQ ID NO: 22: R82A / D143A or SEQ ID NO: 21: R82A / E143A), 109th and 143rd amino acid residues changed to alanine (SEQ ID NO: 22) R109A / D143A, or R109A / E143A in the case of SEQ ID NO: 21), and the like, but is not limited thereto.

Whether a PCNA variant can be loaded into DNA alone (whether it has amplification enhancing activity) can be evaluated by PCR. For example, to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, PCNA to be evaluated is added, PCNA is not added, and wild-type PCNA is added By comparing the amount of amplification with that of DNA, it can be confirmed whether it can be loaded into DNA alone (whether it has amplification enhancing activity). Even if wild-type PCNA or other PCNA that cannot be loaded into DNA alone is added, the PCR amplification amount does not change, but rather the amplification amount tends to decrease. On the other hand, a mutant that can be loaded into DNA alone can obtain an amplification amount superior to that without PCNA addition or with wild-type PCNA addition.

In the present invention, the evaluation of “whether the PCNA mutant can be loaded into DNA alone (whether it has amplification enhancing activity)” is based on an archaeal DNA polymerase having reduced base analog detection activity in a reaction system containing dUTP. Evaluate using mutants.
Specifically, the following “method for evaluating amplification enhancing activity” is followed.

"Evaluation method of amplification enhancing activity"
In this specification,
(I) PCR
KOD-Plus-Ver. 2 (Toyobo) attached 10 × PCR Buffer attached, 1 × PCR Buffer, and 1.5 mM MgSO ₄ ,
0.2 mM dNTPs (dATP, dUTP, dCTP, dGTP) containing dUTP instead of dTTP,
15 pmol primer (SEQ ID NO: 13 and 14 for 1.3 kb amplification),
10 ng of human genomic DNA (Roche),
250 ng of PCNA to be evaluated was added to 50 μl of a reaction solution containing 1 U KOD DNA polymerase V93K mutant (archebacterium DNA polymerase mutant having reduced base analog detection activity) mixed with 0.8 μg of KOD antibody,
After the pre-reaction at 94 ° C. for 30 seconds, PCR is performed on a schedule of 98 ° C., 10 seconds → 60 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds repeated 35 cycles.
(Ii) After completion of the PCR product analysis reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and under ultraviolet irradiation, the amplified DNA fragment was compared with the one not added with PCNA alone. Whether it can be loaded into DNA (whether it has amplification enhancing activity) can be evaluated. PCNA that can be loaded into DNA alone (high amplification enhancing activity) increases the amount of amplification upon addition.

(16) Reagent for carrying out nucleic acid amplification method, kit Reagent for carrying out nucleic acid amplification method of the present invention, kit comprises DNA polymerase belonging to family B and deoxyuridine (dUTP) in the reaction solution Other configurations are not particularly limited. The applied nucleic acid amplification method is not particularly limited.

When PCR is performed as a nucleic acid amplification method, examples of the reagent and kit of the present invention include the following configurations (a) to (d), but are not limited thereto.
(A) an archaeal DNA polymerase variant having reduced base analog detection activity (b) DNA synthesis comprising a pair of primers (c) dUTP in which one primer is complementary to the DNA extension product of the other primer A substrate (deoxynucleotide triphosphate (dNTP)) and
(D) a buffer solution containing magnesium ions, ammonium ions and / or potassium ions

The reagent and kit may further use other reagents, for example, a PCR enhancing factor, BSA, a nonionic surfactant and the like, if necessary.

Hereinafter, the present invention will be specifically described by way of examples. The description of the following examples does not limit the invention in any way.
(Example 1)
Production of KOD mutant A plasmid containing a modified thermostable DNA polymerase gene derived from Thermococcus kodakaraensis KOD1 strain was produced.
As a DNA template used for mutagenesis, a modified thermostable DNA polymerase gene (SEQ ID NO: 11) (pKOD) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBluescript was used. The mutation was introduced using KOD-Plus-Mutageness 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 Pfu mutant A plasmid containing a modified thermostable DNA polymerase gene derived from Pyrococcus furiosus was prepared.
As a DNA template used for mutagenesis, a modified thermostable DNA polymerase gene (SEQ ID NO: 12) (pPfu) derived from Pyrococcus furiosus cloned in pBluescript was used. The mutation was introduced using KOD-Plus-Mutageness 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.

The plasmids prepared in Example 1 and Example 2 are shown in Tables 1 and 2.

Example 3
Example 3-1
Production of modified thermostable DNA polymerase The cells obtained in Example 1 and Example 2 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin in advance in this medium. Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution 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 subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and heparin sepharose chromatography were performed. Finally, storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% nonidet P40, 50% glycerin) to obtain a modified thermostable DNA polymerase.

Example 3-2
Evaluation of the reduced base analog detection activity of the modified thermostable DNA polymerase The uracil detection activity was carried out according to the “Method for evaluating base analog detection activity” described in (10) above.

  As a result, when the wild-type DNA polymerase of Thermococcus kodakaraensis (KOD) is inhibited by the addition of 0.5 μM dUTP, PCR products cannot be confirmed. Y7A, P36H, P36K, P36R, V93Q, V93K, V93R In the mutant to the uracil-binding pocket, the PCR product could be confirmed even when some dUTP was added. In addition, when comparing P36K and P36K / Y7A, P36R and P36R / Y7A, etc., those with double mutations were more tolerant to high concentrations of dUTP and more amplified (Figure). 1, FIG. 2).

FIG. 1 shows a reaction system using a total of 8 types of wild type (KOD) and 7 types of KOD variants of Y7A, P36K, P36R, Y7A / P36K, Y7A / P36R, P36H, V93Q, and Y7A / P36H. This is a result of performing PCR reaction by adding water or dUTP (manufactured by Roche) prepared to a final concentration of 0.5, 5, 50, 100, 200 μM, and electrophoresis of the resulting product.
In the wild type, when dUTP was added, PCR products could not be confirmed (lanes 2-6), and when dUTP was not added (lane 1), amplification products were confirmed. On the other hand, in the 7 mutant types, amplification products were confirmed even when dUTP having a final concentration of 0.5 μM was added. Among them, amplification products were confirmed in the entire dUTP concentration range of 0.5 to 200 μM for 6 types of P36K, P36R, Y7A / P36K, Y7A / P36R, P36H, V93Q, and Y7A / P36H.

FIG. 2 shows that wild type (KOD), 3′-5 ′ exonuclease was deleted by mutation of N210D, KOD (KOD N210D) and I142R were deleted by mutation of N210D, and 3′-5 ′ exonuclease was deleted. Three types of KOD (KOD I142R) were prepared, and each of KOD and KOD N210D was added with any of the mutations of V93K, V93R, V93Q, and Y7A (8 types in total), and KOD I142R of V93K and Y7A Those with any mutation were prepared (two types). In addition, two types (2 types) were prepared by further adding any mutation of V93K and Y7A to KOD from which 3′-5 ′ exonuclease was deleted by double mutation of D141A / E143A. Using these 15 species in total, water or dUTP (Roche) prepared to a final concentration of 0.5, 5, 50, 100, and 200 μM was added to the reaction system, and PCR reaction was performed. It is the result of electrophoresis of the product.

In the wild type, when dUTP was added, PCR products could not be confirmed (lanes 2-6), and when dUTP was not added (lane 1), amplification products were confirmed. In contrast, in the mutant form in which any of the mutations of V93K, V93R, V93Q, and Y7A was added to KOD, an amplification product was confirmed when dUTP having a final concentration of 0.5 μM was added. In particular, amplification products were confirmed even when the concentration of dUTP was increased to 200 μM for V93K and V93R and to 100 μM for V93Q.

The effect of increasing dUTP resistance by adding any of the mutations V93K, V93R, V93Q, and Y7A to KOD is more deficient in 3′-5 ′ exonuclease (N210D, I142R or D141A / D141) than in adding mutation to wild-type KOD. E143A) It was shown that it is higher to add mutations to KOD. From these facts, it is understood that the resistance to dUTP is improved by adding a mutation that gives a deficiency to the 3′-5 ′ exonuclease in addition to the mutation to the uracil-binding pocket.

Similar experiments were performed with other KOD mutants, and the concentrations of dUTP in which amplification was observed in the column of dUTP resistance in Table 3 were summarized. 0 indicates that PCR was not possible with 0.5 μM dUTP. Similarly, 0.5, 5 and 100 showed amplification with 0.5, 5 and 100 μM dUTP, respectively, but one step higher. No amplification was seen with 5, 100, 200 μM dUTP. > 200 indicates that amplification was observed even when 200 μM was added.
As a result, mutations in Y7, P36 or V93 to the uracil-binding pocket tend to increase dUTP resistance levels, and further to mutants carrying 3′-5 ′ exonucleases such as H147E (exo (+)). Compared with the case where mutation is added, the dUTP resistance concentration is higher when mutation is further added to a mutant lacking 3′-5 ′ exonuclease such as N210D, I142R or D141A / E143A (exo (−)). A trend was confirmed.

In Pfu, a similar result was obtained in which the single mutants of Y7A, P36H, and V93K were compared with the multiple mutants of Y7A / P36H and Y7A / V93K, and the amount of amplification of the multiple mutants was larger.

Example 3-3
Evaluation of long-chain DNA amplification using a modified thermostable DNA polymerase Using the KOD mutant shown in Table 2, long-chain DNA exceeding 1.3 kB was obtained even under the condition of dUTP alone without dTTP. It was investigated whether it amplified. Table 2 shows both amino acid mutations in the exo region and amino acid mutations related to uracil bonds. Regarding amino acid mutations in the exo region, exo (+) is a mutation that retains 3'-5 'exonuclease including wild type, and exo (-) is missing 3'-5' exonuclease. It shows that it is a lost mutation.

In PCR with dUTP, 1.3 kbp and 2.8 kbp amplification of Human β-globin were compared. At this time, each enzyme used was a KOD mutant mixed with 1 μg of KOD antibody per 1U.

For PCR, KOD-Plus-Ver. 2 (manufactured by Toyobo), 1 × PCR Buffer, and 1.5 mM MgSO ₄ , dNTPs in which 2 mM dTTP is replaced with dUTP (dATP, dUTP, dCTP, dGTP), 15 pmol primer (1.3 kbp) For amplification, SEQ ID NO: 13 and 14, for 2.8 kbp amplification, SEQ ID NO: 15 and 16, for 3.6 kbp amplification, SEQ ID NO: 17 and 18), 10 ng of human genomic DNA (Roche), 1 U each mixed with antibody 50 μl of the reaction solution containing the enzyme was used. 94 ° C, 2 minutes before reaction, 98 ° C, 10 seconds → 65 ° C, 30 seconds → 68 ° C, 1 minute per 1kbp (1 minute 30 seconds for 1.3kbp amplification, 3 minutes for 2.8kbp amplification) PCR was performed with PCR system GeneAmp9700 (Applied Biosystem) on a schedule of 35 cycles of 3 minutes for amplification of 3.6 kbp and 4 minutes for amplification.

As a control, amplification with Taq DNA polymerase was also performed. Taq DNA polymerase was manufactured by Toyobo, and mixed with Anti-Taq High (manufactured by Toyobo). Reaction is 1 × BlendTaq with attached Buffer, 2 mM dTTP with dUTP (dATP, dUTP, dCTP, dGTP), 10 pmol primer (same as above), 10 ng human genomic DNA (Roche), mixed with antibody 50 μl of the reaction solution containing 2.5 U of the enzyme was added at 94 ° C. for 2 minutes, followed by 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., about 1 minute per 1 kbp (1.3 kbp PCR was performed with PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 35 cycles of 1 minute 30 seconds for amplification, 3 minutes for amplification of 2.8 kbp and 4 minutes for amplification of 3.6 kbp).
After completion of each reaction, 5 μl of the reaction mixture was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of about amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 3 shows that the wild type (KOD), V93K, Y7A / V93K, P36R, Y7A / P36R, P36H, Y7A / P36H, P36R / V93K, Y7A / P36R / V93K, P36H / V93K, Y7A / P36H / V93K Using a total of 11 species with various KOD mutants, PCR reaction was performed on human β-globin DNAs of different lengths in a reaction system containing 0.2 mM final concentration of dUTP (Roche) instead of dTTP. This is a result of electrophoresis of the obtained product. In FIG. 3, 1.3 kbp is Lane 1, 2.8 kbp is Lane 2, and 3.6 kbp is Lane 3. Further, as a control, the same examination was performed with Taq. In addition, the results of PCR using normal dTTP using wild-type KOD are also shown.

FIG. 4 shows that any of the mutations H147E, N210D, I142R, D141A / E143A was first added to the wild type (KOD) exo region, and V93K, Y7A / V93K, P36R, Y7A / P36R, P36H, Y7A were added to each. / P36H to which any of the mutations (total 24 types) were prepared, and these mutants were used to lengthen the reaction system containing 0.2 mM final concentration of dUTP (Roche) instead of dTTP. It is the result of having electrophoresed the product obtained by performing PCR reaction with respect to different human β-globin DNAs. In FIG. 4, 1.3 kbp is Lane 1, 2.8 kbp is Lane 2, and 3.6 kbp is Lane 3.

In FIG. 3, as a result of comparing the amplification amounts of V93K, P36H, and P36R, the mutant (P36R, P36H) in which mutation was added to the P36 position has a larger amplification amount than V93K, and can be amplified to a long target. Was confirmed.
Next, as a result of comparing the amplified amounts of V93K, P36H, and P36R, and the double mutant in which the mutation of Y7A was further added to each, the one in which the double mutation was added to the uracil-binding pocket rather than the single mutation, Amplification increased. These mutants were able to amplify long chain lengths that could not be amplified with Taq.
On the other hand, when the various multiple mutants combined for V93K, P36H, and P36R that were effective as described above were further examined, a synergistic effect was observed with P36H / V93K, but P36R, Y7A / P36R / V93K, Y7A / No amplification was observed with P36H / V93K.

3 and 4, wild-type DNA polymerases with mutations such as V93K and P36H and exo (−) mutants N210D, I142R, D141A / E143A mutants such as V93K and P36H As a result of comparing the mutated ones, the amount of amplification was larger when the exo (-) DNA polymerase was mutated.
Furthermore, in FIG. 3 and FIG. 4, mutations such as V93K and P36H are performed on wild-type DNA polymerase, and mutations such as V93K and P36H are performed on H147E which is an exo (+) mutant that improves PCR efficiency. As a result of comparison with the applied one, the exo (+) H147E mutant resulted in a larger amount of amplification.

The evaluation results of the KOD mutant are summarized in Table 3. In Table 3, the 11-step evaluation for dUTP resistance indicates that the closer to 0, the stronger the base analog detection activity, and the closer to 10, the lower the base analog detection activity. In Table 3, ◯ indicates that the signal is sufficiently amplified, Δ indicates that the signal is amplified to some extent, and × indicates that the signal is not amplified.

In Pfu, a single mutant of Y7A, P36H, and V93K was compared with a multiple mutant of Y7A / P36H and Y7A / V93K, and similar results were obtained in that the multiple mutant had a larger amount of amplification.

Example 3-4
Comparison of synthesis rate The synthesis rate was determined according to <DNA polymerase synthesis rate measurement method> described in (8) above.
FIG. 5 shows a total of 8 types of wild type (WT) and 7 types of KOD variants including Y7A / V93K, P36H, Y7A / P36H, Y7A / V93K / N210D, P36H / N210D, Y7A / P36H / N210D, and N210D. Is a result of electrophoresis of products obtained by performing PCR reaction for 30 seconds, 60 seconds and 120 seconds using Biotinylated P7 primer. In FIG. 5, “30” indicates that the PCR reaction was performed for 30 seconds. The same applies to 60 and 120. FIG. 5 shows that the longer the amplification product is detected at the top of the electrophoresis photograph, the longer the amplification product is taken and the faster the synthesis rate. On the left side of the photograph, 7000 bp and 3000 bp amplification products are detected at the respective arrows. The numbers shown at the bottom of the photograph are the synthesis rates of each DNA polymerase calculated based on the results.

In FIG. 5, the Y7A / V93K, P36H, and Y7A / P36H mutants showed faster synthesis rates than the wild type (WT).
In addition, the N210D mutant in which the exo region is modified has a faster synthesis rate than the wild type, and the mutant in which the N210D mutant is modified with Y7A / V93K, P36H, Y7A / P36H or the like has a higher synthesis rate. It was confirmed to be faster.

Example 3-5
Amplification from blood in PCR in the presence of dUTP Blood resistance was evaluated by changing the amount of blood added to the reaction solution. For comparison, Taq DNA polymerase (Taq DNA polymerase (Toyobo) mixed with KOD (wild type), KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant, and antibody mixed with 0.8 μg of KOD antibody per 1U. ) And Anti-Taq High (Toyobo) were mixed in equal amounts), and 482 bp PCR of HBg was performed to compare the differences in amplification.

As above, KOD PCR was performed using KOD-Plus-Ver. 2 (Toyobo Co., Ltd.) attached Buffer, using MgSO _4, 1 × PCR Buffer, and 1.5 mM MgSO _4, 15 pmol of primer (SEQ ID NO: 19 and 20 in the amplification of 482 bp), each enzyme 1U mixed with antibody Of normal dNTPs (dATP, dTTP, dCTP, dGTP) added at 0.2 mM, and dNTPs (dATP, dUTP, dCTP, dGTP) substituted with dUTP at 0.2 mM Each of these was added to be used, and blood itself and blood diluted with water were used as templates. PCR system GeneAmp 9700 (Applied) with a schedule that repeats 35 cycles of 94 ° C., 2 minutes pre-reaction, 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute per 1 bp (1 minute for 482 bp amplification) PCR was performed using Biosystem).

Taq DNA polymerase PCR was performed by adding ordinary dNTPs (in a 50 μl reaction solution containing Buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol primer (same as above), 2.5 U enzyme mixed with antibody. (dATP, dTTP, dCTP, dGTP) with 0.2 mM added and dNTPs with dTTP substituted with dUTP (dATP, dUTP, dCTP, dGTP) added to 0.2 mM, respectively. Blood itself and blood diluted with water were used. PCR system GeneAmp9700 (Applied Biosystem) with a schedule of 94 cycles at 94 ° C for 2 minutes followed by 35 cycles of 94 ° C, 30 seconds → 65 ° C, 30 seconds → 68 ° C, 1 minute per 1 bp (same as above) PCR was performed using
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 6 shows various DNA polymerases prepared by using blood as a sample and adjusting the reaction solution so that the proportion of blood in the reaction solution is 10, 5, 2, 0.2, 0.02, 0.002%. The result of having performed PCR reaction by and electrophoresis of the obtained product is shown. The DNA polymerases used are 4 types in total: Taq, KOD (wild type), and 2 types of KOD variants (Y7A / V93K, Y7A / P36H / N210D). The left side of each photograph uses dTTP, and the right side uses dUTP. One lane is the case of 10% of the blood in the reaction solution, and the following 2-6 lanes are the cases of 5%, 2%, 0.2%, 0.02%, and 0.002%, respectively.
As a result, amplification of Taq DNA polymerase and wild type KOD DNA polymerase was not confirmed in the presence of dUTP, but firm bands were confirmed in the KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants even in the presence of dUTP.
The KOD Y7A / P36H / N210D mutant, which has a faster synthesis rate than the KOD Y7A / V93K mutant, has higher blood resistance, and amplification was confirmed even when 10% blood was added.

Other variants of KOD (P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93R / H147E, V93R / H147E / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, Y7A / D36R, Y7A / P36R / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A / P36H / I14 R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E143A, Y7A / P36H / D141A / E143A, Y7A / P36R / 14) Under the reaction conditions, amplification was confirmed from the addition of 5% blood, and a firm band could be confirmed even in the presence of dUTP.

In the Pfu DNA polymerase mutant, amplification was confirmed from the addition of 5% blood under the same reaction conditions, and a firm band was confirmed even in the presence of dUTP in the mutants of P36H, V93R, Y7A / P36H, and Y7A / V93K. .

(Example 4)
Example 4-1
Preparation of KOD-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Thermococcus kodakaraensis KOD1 strain was prepared.
The DNA template used for mutagenesis was PCNA (SEQ ID NO: 23) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 strain cloned into pBluescript. The mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) 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 enzyme preparation.

Example 4-2
Preparation of Pfu-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Pyrococcus furiosus was prepared.
As a DNA template used for mutagenesis, PCNA (SEQ ID NO: 24) (pPfuPCNA) derived from Pyrococcus furiosus cloned in pBluescript was used. The mutation was introduced using KOD-Plus-Mutageness Kit (manufactured by Toyobo) 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 enzyme preparation.

Table 4 shows the plasmids prepared in Example 4-1 and Example 4-2.

Example 4-3
Production of modified heat-resistant PCNA The cells obtained in Example 4-1 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin in advance in this medium. E. coli DH5α (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution 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 subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid treatment using polyethyleneimine, ammonium sulfate salting out, and Q sepharose chromatography were performed. Finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 .1% Tween 20, 0.1% nonidet P40, 50% glycerin) to obtain modified heat-resistant PCNA.

Example 4-4
Evaluation of PCR enhancing factor (PCNA) To confirm the effect of PCNA, KOD-PCNA mutants (M73L, M73L / E143R, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, M73L / E143F, M73L / The difference in the amount of amplification by PCR in the presence of dUTP was compared by amplifying 1.3 kb of Human β-globin in accordance with “Method for measuring amplification enhancement activity” using E143A).

FIG. 7 shows the results of electrophoresis of the products obtained by adding 250 ng of various PCNA mutants and performing PCR reaction. The PCNA mutants used were 7 types in total: M73L, M73L / E143R, M73L / E143A, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, and M73L / E143F.
In the case where no PCNA was added (lane 8) or when the KOD-PCNA M73L mutant was used as PCNA (lane 1), only a small band was found, but other KOD PCNA mutants were not detected. When added, the amount of amplification increased and a clearer band was confirmed.

PCNA forms a multimer and promotes a nucleic acid synthesis reaction. Usually, however, the reaction cannot proceed without loading into DNA without the action of RFC. M73L / E143R, M73L / E143A, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, and M73L / E143F are modifications to sites involved in multimer formation, and multimer formation is moderately weakened. Therefore, it is conceivable that PCNA could be loaded onto DNA and the amount of PCR amplification was improved (FIG. 7).
PCNA has also been reported to increase the synthesis rate and shorten the elongation time. However, the improvement in the synthesis rate shortens the time for the interaction between uracil and the binding pocket, and weakens the detection activity of uracil. It is possible.

The same experiment was conducted with Pfu-PCNA mutants (M73L, M73L / D143R, M73L / R109A / D143A, M73L / D147A, M73L / R82A / D143A, M73L / D143A), and most of the Pfu-PCNA M73L mutants had bands. Although it could not be confirmed, a solid band was confirmed by addition of other Pfu-PCNA mutants (M73L / D143R, M73L / R109A / D143A, M73L / D147A, M73L / R82A / D143A, M73L / D143A).

Example 4-5
Comparison of synthesis rate by adding PCNA Various PCNA variants were added to the KOD V93K variant, and the synthesis rate was measured. KOD V93K was diluted with a 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). In addition, 250 ng of PCNA mutant was added to each reaction system, and the synthesis rate was measured.
Reagents and methods were performed according to <DNA polymerase synthesis rate measurement method> described in (8) above.

FIG. 8 shows the results of electrophoresis of products obtained by adding various PCNA mutants to the V93K mutant, performing PCR reaction for 30 seconds, 60 seconds, and 120 seconds. As the PCNA mutant, a PCNA mutant derived from KOD (D147A, E143R, R109A / E143A) was used. In FIG. 8, “30” indicates that the PCR reaction was performed for 30 seconds. The same applies to 60 and 120. FIG. 8 shows that the longer the amplified product is detected at the top of the electrophoretic photograph, the longer the amplified product is taken and the faster the synthesis rate. On the left side of the photograph, 7000 bp and 3000 bp amplification products are detected at the respective arrows. The numbers shown at the bottom of the photograph are the synthesis rates of each DNA polymerase calculated based on the results.

As a result, it was confirmed that the addition of PCNA significantly increased the synthesis rate (FIG. 8). This is thought to be due to the fact that PCNA performed the function of clamping, and DNA and polymerase were bound firmly.

Example 4-6
Effect of amplification from blood and addition of PCNA in PCR in the presence of dUTP PCNA was added to the evaluation of blood resistance to confirm the effect. For comparison, Taq DNA polymerase (Taq DNA polymerase (Toyobo) mixed with KOD (wild type), KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant, and antibody mixed with 0.8 μg of KOD antibody per 1U. PCNA D147A mutant was added in an amount of 250 ng, and 482 bp PCR of HBg was performed to compare the difference in amplification using a mixture of equal amounts of Anti-Taq High (produced by Toyobo).
As above, KOD PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using the attached Buffer, MgSO ₄ , 50 μl containing 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 19 and 20), 1 U of each enzyme mixed with the antibody In the reaction solution, 0.2 mM of normal dNTPs (dATP, dTTP, dCTP, dGTP) and dNTPs (dATP, dUTP, dCTP, dGTP) in which dTTP is replaced with dUTP are added to 0.2 mM. 250 ng of KOD-PCNA M73L / D147A mutant was added to each reaction solution. As the mold, blood itself and blood diluted with water were used. PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 2 minutes and 98 cycles of 10 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute.
Taq DNA polymerase PCR was performed by adding ordinary dNTPs (in a 50 μl reaction solution containing Buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol primer (same as above), 2.5 U enzyme mixed with antibody. (dATP, dTTP, dCTP, dGTP) with 0.2 mM added and dNTPs with dTTP substituted with dUTP (dATP, dUTP, dCTP, dGTP) added to 0.2 mM, respectively. Blood itself and blood diluted with water were used. PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute after 35 minutes at 94 ° C.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 9 shows various DNA polymerases prepared by using blood as a sample and adjusting the reaction solution so that the proportion of blood in the reaction solution is 10, 5, 2, 0.2, 0.02, 0.002%. 2 shows the result of electrophoresis of the product obtained by performing the PCR reaction in the presence of KOD-derived PCNA mutant (D147A). The DNA polymerases used are 4 types in total: Taq, KOD (wild type), and 2 types of KOD variants (Y7A / V93K, Y7A / P36H / N210D). The left side of each photograph uses dTTP, and the right side uses dUTP. One lane is the case of 10% of the blood in the reaction solution, and the following 2-6 lanes are the cases of 5%, 2%, 0.2%, 0.02%, and 0.002%, respectively.
As a result, it is known that PCNA binds to a site called PIP motif of polymerase belonging to family B. Since polymerases belonging to family A such as Taq have no PIP motif, no effect was seen even when PCNA was added (FIG. 9).
On the other hand, in the KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants having decreased base analog detection activity, it was found that when PCNA was added, the reaction occurred to a thicker blood.
In addition, the amount of amplification of KOD Y7A / P36H / N210D to which PCNA was added and KOD Y7A / V93K to which PCNA was added increased in descending order of the synthesis rate, which is also considered important for PCR in the presence of dUTP.

Other variants of KOD (P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93R / H147E, V93R / H147E / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, Y7A / D36R, Y7A / P36R / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A / P36H / I14 R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E143A, Y7A / P36H / D141A / E143A, Y7A / P36R / 14) Under the above reaction conditions, amplification was confirmed from the addition of 10% blood, and a firm band could be confirmed even in the presence of dUTP.

PCNA is also a variant of KOD-PCNA M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / D147A, M73L / R82A / D143A, M73L / R109A / D143A. The same reaction was carried out, amplification was confirmed from the addition of 10% blood, and a solid band could be confirmed even in the presence of dUTP.

In the Pfu DNA polymerase mutant, amplification was confirmed from the addition of 10% blood under the same reaction conditions, and a firm band was confirmed in the P36H, V93R, Y7A / P36H, Y7A / V93K mutant even in the presence of dUTP. .

Example 4-7
PCR from body tissues (nail, hair, oral mucosa) in the presence of dUTP
We examined whether PCR can be performed in the presence of dUTP using nails, hair, and oral mucosa as templates. A piece of nail cut with a nail clipper and one piece of hair were added to 180 μl of 50 mM NaOH, crushed by heat treatment at 95 ° C. for 10 minutes, and then neutralized by adding 20 μl of 1M Tris-HCl (pH 8.0). The supernatant was used as a template. The oral mucosa was obtained by suspending mucosa collected with a cotton swab in 200 μl of water as a template.
The enzyme includes Taq DNA polymerase (Taq DNA polymerase (Toyobo), which is a mixture of KOD (wild type), KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant, and antibody mixed with 0.8 μg of KOD antibody per 1 U. HBg 482 bp PCR was carried out using a mixture of equal amounts of (manufactured) and Anti-Taq High (manufactured by Toyobo)) to compare the differences in amplification.
As above, KOD PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using the attached Buffer, MgSO ₄ , 50 μl containing 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 19 and 20), 1 U of each enzyme mixed with the antibody In the reaction solution, 0.2 mM of normal dNTPs (dATP, dTTP, dCTP, dGTP) and dNTPs (dATP, dUTP, dCTP, dGTP) in which dTTP is replaced with dUTP are added to 0.2 mM. 1 μl of the above sample was used as a template. Moreover, what added 250 ng of KOD-PCNA E143R mutant which is a PCR enhancement factor to each reaction liquid was also implemented. PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 2 minutes and 98 cycles of 10 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute.
Taq DNA polymerase PCR was performed by adding ordinary dNTPs (in a 50 μl reaction solution containing Buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol primer (same as above), 2.5 U enzyme mixed with antibody. (dATP, dTTP, dCTP, dGTP) with 0.2 mM added and dNTPs with dTTP substituted with dUTP (dATP, dUTP, dCTP, dGTP) added to 0.2 mM, respectively. 1 μl of the above sample was used. Moreover, what added 250 ng of KOD-PCNA M73L / E143R mutant which is a PCR enhancement factor to each reaction liquid was also implemented. PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute after 35 minutes at 94 ° C.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 10 shows the results of electrophoresis of products obtained by performing PCR reaction with various DNA polymerases in the presence of KOD-derived PCNA mutant (E143R) using nail, hair and oral mucus as samples. . The DNA polymerases used are 4 types in total: Taq, KOD (wild type), and 2 types of KOD variants (Y7A / V93K, Y7A / P36H / N210D). The left side of each photograph uses dTTP, and the right side uses dUTP. When 1 lane is a nail sample, 2 lane is hair, 3 lane is oral mucosa. The respective “+” lanes are in the presence of KOD-derived PCNA mutant (E143R) and “−” indicates no PCNA.
As a result, Taq DNA polymerase and wild-type KOD DNA polymerase showed almost no amplification in the presence of dUTP, but KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants confirmed a firm band even in the presence of dUTP ( FIG. 10). In addition, the reaction solution to which PCNA was added resulted in a larger amount of amplification.
Other variants of KOD (P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93R / H147E, V93R / H147E / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, Y7A / D36R, Y7A / P36R / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A / P36H / I14 R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E143A, Y7A / P36H / D141A / E143A, Y7A / P36R / 14) Under the above reaction conditions, amplification from body tissues (nail, hair, oral mucosa) was confirmed, and a firm band could be confirmed even in the presence of dUTP. In addition, when KOD-PCNA mutants (M73L / D147A, M73L / R109A / E143A, M73L / E143R) and Pfu-PCNA mutants (M73L / D143R) are added to these, the amplification amount can be confirmed as described above. It was.

Example 4-8
Amplification from plant lysate Amplification from plant lysate was performed in a PCR reaction system containing dUTP.
For comparison, KOD Y7A / P36H / N210D mutant and Taq polymerase were used, and the difference in amplification amount of rbcL 1.3 kb was compared. The KOD Y7A / P36H / N210D mutant was also added with KOD-PCNA M73L / D147A added as a PCR enhancing factor.

As a template, rice leaf 3 mm square was added to 100 μl of Buffer A (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) and heat-treated at 95 ° C. for 10 minutes, and used as a lysate.
The PCR of KOD Y7A / P36H / N210D was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo) attached buffer, MgSO ₄ , 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NOs: 25 and 26), dNTPs in which 2 mM dTTP is replaced with dUTP (dATP, dUTP , DCTP, dGTP), a plant lysate was added to 50 μl of a reaction solution containing 1 U of enzyme mixed with KOD antibody so as to be 2% of the reaction solution. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 98 cycles of 98 ° C., 10 seconds → 65 ° C. 30 seconds → 68 ° C., 1.5 minutes for 35 cycles. 250 ng of KOD-PCNA M73L / D147A was added to the above reaction system and compared with that without PCNA.
Taq DNA polymerase was manufactured by Toyobo, and mixed with Anti-Taq High (manufactured by Toyobo). The reaction consists of Buffer attached to 1 × BlendTaq, 10 pmol primer (same as above), dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP is replaced by dUTP, and 50 μl of 2.5 U enzyme mixed with antibody. Plant lysate was added to the reaction solution so as to be 2% of the reaction solution, and after 94 ° C. for 2 minutes, 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C. The PCR was performed using a PCR system GeneAmp9700 (Applied Biosystem) on a schedule of repeating 35 cycles of minutes.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 11 shows that plant lysate was used as a sample, and the reaction solution was prepared so that the ratio of lysate in the reaction solution was 2%, and KOD Y7A / P36H / N210D mutant, KOD Y7A / P36H / N210D mutant were subjected to KOD. -Shows the result of electrophoresis of the product obtained by carrying out PCR reaction with a total of 3 types of Taq polymerase added with PCNA M73L / D147A.
In each photograph, 1 shows the result of Taq polymerase, 2 shows the result of KOD Y7A / P36H / N210D, and 3 shows the result of adding KOD-PCNA M73L / D147A to KOD Y7A / P36H / N210D.
As a result, amplification was confirmed even from unpurified plant lysate in KOD Y7A / P36H / N210D. Furthermore, a solid band with a larger amount of amplification was confirmed when the PCNA mutant was added than when the PCNA mutant was not added.
On the other hand, Taq polymerase could not directly amplify from plant lysate (FIG. 11).

Other variants of KOD (Y7A / V93K, P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93K / R147E / H147E, Y7A / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R7 / N210D, Y7A / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A P36H / I142R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E143A, Y7A / P36H / D141A / E143A, E143A, E14A / E143A However, amplification was confirmed from the plant lysate under the same reaction conditions, and a firm band could be confirmed even in the presence of dUTP.

PCNA is also a variant of KOD-PCNA M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / D147A, M73L / R82A / D143A, M73L / R109A / D143A. The same reaction was carried out, and it was confirmed that the amplification amount was improved as compared with the case where PCNA was not added.

In the Pfu DNA polymerase mutant, amplification was confirmed from the plant lysate under the same reaction conditions. P36H, V93R, Y7A / P36H, and Y7A / V93K mutants showed a firm band even in the presence of dUTP.

Example 4-9
It was evaluated whether amplified dUTP from stool and gene amplification can be performed in the presence of stool.
As the enzyme, KOD Y7A / P36H / N210D mutant and Taq polymerase were used, and the difference in amplification of about 700 bp of Salmonella invA gene was compared by real-time PCR using SYBR GREEN I and melting curves. The KOD Y7A / P36H / N210D mutant was also added with KOD-PCNA M73L / D147A added as a PCR enhancing factor.

As the inhibitor, a 10% fecal suspension was heat-treated at 95 ° C. for 10 minutes.
For the PCR of KOD Y7A / P36H / N210D, a buffer attached to KOD Dash (manufactured by Toyobo) was used. In 20 μl of a reaction solution containing 0.4 U of enzyme mixed with substituted dNTPs (dATP, dUTP, dCTP, dGTP), 1/30000 SYBR GREEN I, and KOD antibody, feces were added to the reaction solution at 0, 0, 0. 1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5%, 95 ° C., 30 seconds pre-reaction, 98 ° C., 10 seconds → 60 ℃ 10 seconds → 68 ℃, 30 seconds is repeated using LightCycler 2.0 (Roche) on a schedule of 50 cycles . 100 ng of KOD-PCNA M73L / D147A was added to the above reaction system and compared with that without PCNA.
Taq DNA polymerase was manufactured by Toyobo, and mixed with Anti-Taq High (manufactured by Toyobo). The reaction is as follows: Buffer attached to 1 × Taq (Mg attachment type), 50 copies of Salmonella genome, 4 pmol primer (same as above), 2 mM dTTP substituted with dUTP (dATP, dUTP, dCTP, dGTP), 4 mM In 20 μl of a reaction solution containing 1 U of enzyme mixed with MgSO ₄ , 1/30000 SYBR GREEN I, antibody, feces were added to the reaction solution at 0, 0.1, 0.25, 0.5, 1.0. , 1.5, 2.0, 2.5%, 95 ° C., 30 seconds pre-reaction, 98 ° C., 10 seconds → 60 ° C. 10 seconds → 68 ° C., 30 seconds repeated 50 cycles The schedule was performed using LightCycler 2.0 (Roche).
After completion of the reaction, a target peak appearing in the latter half of 80 ° C. was confirmed by melting curve analysis.

Table 5 shows the Cq value of Example 4-9.

Table 5 shows Cq values (default settings of LightCycler 2.0) of real-time PCR performed in the presence of dUTP and feces. N. D. Indicates that no amplification was observed and no Cq value was obtained.
As a result, amplification was not observed when 0.5% stool was added to Taq polymerase, but amplification was confirmed even when 2.5% stool was added to KOD Y7A / P36H / N210D. Further, comparing the presence or absence of PCNA, it was found that the one with PCNA added had a smaller Cq value and showed excellent PCR efficiency.

FIG. 12 shows the results of melting curve analysis of the obtained amplification product using PCR in the presence of dUTP and feces. The polymerases used were KOD Y7A / P36H / N210D mutant, KOD Y7A / P36H / N210D mutant with KOD-PCNA M73L / D147A added, and Taq polymerase in total.
1 shows the result of KOD Y7A / P36H / N210D, 2 shows the result of adding KOD-PCNA M73L / D147A to KOD Y7A / P36H / N210D, and 3 shows the result of Taq polymerase.
As a result, in KOD Y7A / P36H / N210D, amplification was confirmed even in the presence of dUTP and stool, and the peak decreased with the addition of stool, but the target peak could be confirmed even when 2.5% was added. . Similarly to the case of adding PCNA, the target peak could be confirmed even with the addition of 2.5%. However, with Taq polymerase, the target peak disappeared when 0.25% was added, suggesting that it was inhibited by the influence of feces (FIG. 12).

Other variants of KOD (Y7A / V93K, P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93K / R147E / H147E, Y7A / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R7 / N210D, Y7A / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A P36H / I142R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E143A, Y7A / P36H / D141A / E143A, E143A, E14A / E143A However, similarly, amplification was confirmed in a reaction system containing 2.5% feces, and a firm band could be confirmed even in the presence of dUTP.

PCNA is also a variant of KOD-PCNA M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / D147A, M73L / R82A / D143A, M73L / R109A / D143A. The same reaction was performed, and it was confirmed that the PCR efficiency was improved as compared with the case where PCNA was not added.

Similarly, amplification was confirmed in the Pfu DNA polymerase mutant in a reaction system containing 2.5% stool, and a solid band was confirmed in the P36H, V93R, Y7A / P36H, Y7A / V93K mutant even in the presence of dUTP. did it.

(Example 4-10)
Amplification from blood by addition of PCNA using reaction solution containing dUTP Similarly to FIG. 9, it was evaluated in the PCR reaction system containing dUTP whether crude (blood) resistance was improved by addition of PCNA.
As the enzyme, KOD Y7A / P36H / N210D and Taq polymerase were used, and the difference in the amount of amplification of 1.3 kb of HBg was compared. The KOD mutant was also added with Pfu-PCNA M73L / D147A.

PCR of KOD mutants was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer, MgSO ₄ attached, 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 13 and 14), dNTPs (dATP, dUTP in which 2 mM dTTP was replaced with dUTP) , DCTP, dGTP), and 50 μl of the reaction solution containing 1 U of each enzyme mixed with the KOD antibody, blood was added to 2% of the reaction solution, and 250 ng of Pfu-PCNA M73L / D147A without PCNA was added. The additions were compared.
Taq DNA polymerase PCR was mixed with 1 × BlendTaq attached Buffer (Toyobo product), 10 pmol primer (same as above), dNTPs (dATP, dUTP, dCTP, dGTP) with 2 mM dTTP replaced with dUTP, and antibody. A 50 μl reaction solution containing 2.5 U of enzyme was used by adding 2% of blood to the reaction solution.
The reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1.5 minutes. It was.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 13 shows a reaction solution containing 2% blood prepared by adding Pfu-PCNA M73L / D147A to KOD Y7A / P36H / N210D mutant and KOD Y7A / P36H / N210D mutant with Taq polymerase. The result of carrying out PCR reaction and electrophoresis of the obtained product is shown. In each photograph,-indicates no PCNA, and + indicates that PCNA was added.
As a result, using the polymerase of the present invention, it was shown that even a relatively long amplification of 1.3 kb can be amplified from crude conditions including blood components. In addition, a solid band was confirmed in the case where the PCNA mutant was added compared to the case where the PCNA mutant was not added (FIG. 13).

Other variants of KOD (P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, Y7A / P36R / N210D, Y7A / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A / P36H / I142R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E / D141A / E143A, Y7A / P36R / D141A / E143A) under the same reaction conditions, the amplification was confirmed and a firm band even in the presence of dUTP It was confirmed.

PCNA is also a variant of KOD-PCNA M73L / D147A, M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / R82A / D143A, M73L / R109A / D143A. The same reaction was carried out, and it was confirmed that the amplification amount was improved as compared with the case where PCNA was not added.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

The present invention eliminates the risk of loss and carryover during DNA purification, and further reduces time and cost. Also, in order to prevent contamination by the dUTP / UDG contamination removal method, not only in the research field, but also in the clinical field or forensic field such as genetic diagnosis in which the same sample is amplified many times, or in the microbiological examination in foods and the environment, etc. Can also be widely used

Claims (9)

A method of amplifying a target nucleic acid in a biological sample by adding a biological sample that has not undergone a purification step to a nucleic acid amplification reaction solution, wherein the enzyme used for the amplification belongs to the genus Pyrococcus and Thermococcus A DNA polymerase derived from a bacterium, wherein the modification is any one selected from the group consisting of Y7A / V93K, Y7A / V93R, Y7A / P36H, Y7A / P36R and P36H / V93K in SEQ ID NO: 1 or SEQ ID NO: 2. A nucleic acid amplification method, which is carried out under the condition that the DNA polymerase mutant has deoxyuridine (dUTP) in the reaction solution. The nucleic acid amplification method according to claim 1, wherein the biological sample is any one selected from the group consisting of animal and plant tissues, body fluids, excreta, cells, bacteria, and viruses. The nucleic acid amplification method according to claim 1 or 2, wherein the mutant of DNA polymerase has a modification of either Y7A / V93K or Y7A / P36H in SEQ ID NO: 1 or SEQ ID NO: 2. The DNA polymerase variant further comprises any modification selected from the group consisting of D141A / E143A, I142R, H147E, H147D, N210D and Y311F in SEQ ID NO: 1 or SEQ ID NO: 2. The nucleic acid amplification method described. The nucleic acid amplification method according to any one of claims 1 to 4, wherein the mutant of DNA polymerase has the following modification (1) or (2) in SEQ ID NO: 1 or SEQ ID NO: 2.
(1) (A) H147E and (B) any one selected from the group consisting of Y7A / V93K, Y7A / V93R, Y7A / P36H, Y7A / P36R and P36H / V93K (2) (A) N210D , (B) any one selected from the group consisting of Y7A / V93K, Y7A / P36H and Y7A / P36R The nucleic acid amplification method according to any one of claims 1 to 5, further comprising PCNA in the reaction solution. The nucleic acid amplification method according to claim 6, wherein PCNA is a mutant that is loaded alone into DNA. PCNA is (a) the N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109 of the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, and (b) amino acids corresponding to positions 139, 143 and 147 The nucleic acid amplification method according to claim 6 or 7, which is a mutant having at least one modification in the C-terminal region comprising: PCNA has any mutation selected from the group consisting of the following (a) to (d) with respect to the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22. The nucleic acid amplification method according to any one of the above.
(A) a modified amino acid corresponding to the 143rd amino acid to a basic amino acid;
(B) Amino acids corresponding to the 82nd and 143rd amino acids are both modified to neutral amino acids,
(C) a modified amino acid corresponding to the 147th amino acid, and
(D) Amino acids corresponding to the 109th and 143rd amino acids are both modified to neutral amino acids