JP6648749B2 - Nucleic acid amplification method - Google Patents

Description

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

PCR (polymerase chain reaction) means (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of primers to single-stranded DNA template, (3) DNA polymerase This method is a method of amplifying a target nucleic acid in a sample by repeating the three steps of elongation of the primer using the above as one cycle and repeating this cycle.
The target nucleic acid can be amplified hundreds of thousands times even from a small number of samples, such as several copies.It is widely used not only in research fields but also in forensic fields such as genetic diagnosis and clinical diagnosis, and microbiological testing in food and the environment. It is being used.

On the other hand, since PCR is a very sensitive detection method, there is a problem of false positives due to carryover of PCR amplification products performed previously. Therefore, PCR is performed using a substrate containing dUTP instead of dTTP, a uracil base is incorporated into the amplification product, and a Uracil-N-Glycosylase (UNG) treatment is performed at the time of the next PCR, whereby contamination (carrying) is performed. A technique (dUTP / UDG contamination removal method) for decomposing the over-product is used (Non-Patent Document 1).

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

In addition, PCR is easily affected by contaminants such as dyes, proteins, and saccharides, and it is also known that contaminants inhibit the reaction (Non-Patent Document 2). Usually, when a gene in a biological sample is amplified, DNA purification is required. However, in the purification of DNA, the purification operation is complicated and time-consuming, and contamination occurs during the operation. There is a risk. Further, when the content of the target nucleic acid in the sample is small, it may not be possible to collect the nucleic acid. For these reasons, 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 has been required.

Patent 4395377 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 in a PCR containing dUTP as a substrate even when a large amount of impurities derived from a biological sample are present.
Still another object of the present invention is to provide a reagent kit suitable for the above purpose. In summary, an object of the present invention is to provide an improved PCR method and a PCR reaction reagent suitable for amplifying 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 is characterized in that DNA in a biological sample is amplified in the presence of dUTP without using a purification step, using a DNA polymerase belonging to Family B. This is a nucleic acid amplification method.

That is, in the nucleic acid amplification method of mixing and reacting a biologically-derived sample itself and a gene amplification reaction solution, the present inventor uses a DNA polymerase belonging to Family B, so that even if a large amount of biologically-derived impurities are present, Has found that PCR can be performed even when a biological sample such as blood or oral mucosa is directly added to a PCR reaction solution, and has accomplished the present invention.

It has been considered that a normal DNA polymerase belonging to Family B strongly binds to uracil, and therefore cannot be amplified by PCR containing dUTP as a substrate. Therefore, there has been no report on a case where a DNA in a biological sample was amplified without going through a purification step using a DNA polymerase belonging to Family B in PCR containing dUTP as a substrate.

A typical invention of the present application is as follows.
[1]
A method for amplifying a target nucleic acid in a biological sample by adding a biological sample that has not been subjected to a purification step to a nucleic acid amplification reaction solution, wherein the enzyme used for amplification is a DNA polymerase belonging to Family B and deoxyuridine (dUTP ) In a reaction solution.
[2]
The nucleic acid amplification method according to [1], wherein the biological sample is any of animal and plant tissues, body fluids, excrement, 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 a 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 positions 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 (corresponding to the wild-type sequence of Pfu) Is an amino acid sequence having the following modification.
(B) 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 the reduced base analog is This is an amino acid sequence encoding a DNA polymerase having a detection activity.
(C) at least one amino acid is further modified in the amino acid sequence shown in (a), and one or several amino acids are deleted, substituted or added in the amino acid sequence shown in (a). And an amino acid sequence encoding a DNA polymerase having reduced base analog detection activity.
[6]
The mutant according to any of [3] to [5], wherein the mutant of the archaeal DNA polymerase has at least one amino acid modification among the amino acids corresponding to positions 36 and 93 in SEQ ID NO: 1 or SEQ ID NO: 2. Nucleic acid amplification method.
[7]
The mutant of archaeal DNA polymerase is any one of Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36H / V93K, P36K, P36R or P36H in SEQ ID NO: 1 or SEQ ID NO: 2. The method for amplifying nucleic acid according to any one of [3] to [6], which has the following modification.
[8]
The nucleic acid amplification according to any of [3] to [7], wherein the archaeal DNA polymerase mutant has any of Y7A / V93K, Y7A / P36H or 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]
The modification of the DNA polymerase belonging to Family B to the 3′-5 ′ exonuclease active region is performed by modifying any of 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 of the DNA polymerase belonging to Family B to the 3′-5 ′ exonuclease active region is any 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 one of [3] to [11], wherein the mutant of the archaeal DNA polymerase has any of the following (1) to (4) in SEQ ID NO: 1 or SEQ ID NO: 2. Law.
(1) Any one of (A) H147E and (B) any of Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36H / V93K, P36K, P36R, P36H, V93R or V93Q ) (A) N210D, (B) any of Y7A / V93K, Y7A / P36H, Y7A / P36R, P36K, P36R, P36H, V93Q, V93K or V93R, (3) (A) I142R, and (B) Y7A / Any of 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 including PCNA in the reaction solution.
[14]
The method for amplifying nucleic acid according to any one of [1] to [13], wherein PCNA is a mutant that is loaded alone into DNA.
[15]
PCNA is derived from the amino acid sequence represented by SEQ ID NO: 21 or SEQ ID NO: 22 from the (a) N-terminal region consisting of amino acids corresponding to positions 82, 84, and 109; and (b) the 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 among the C-terminal regions.
[16]
PCNA in which the amino acid corresponding to position 143 in SEQ ID NO: 21 or SEQ ID NO: 22 has been changed to a basic amino acid, or amino acid corresponding to positions 82 and 143 in which both amino acids have been changed to neutral amino acids, corresponding to position 147 Any one of [1] to [15], which is a mutant in which amino acids corresponding to amino acids 109 and 143 are both changed to neutral amino acids. Nucleic acid amplification method.
[17]
A reagent for performing the nucleic acid amplification method according to any one of [1] to [16].
[18]
A kit comprising a reagent for performing the nucleic acid amplification method according to any one of [1] to [16].

According to the present invention, the risk of loss and carryover during DNA purification can be eliminated, and the time and cost can be further reduced. In addition, to prevent contamination due to the dUTP / UDG contamination removal method, it can be used not only in the research field, but also in the clinical field or forensic field such as genetic diagnosis where the same sample is amplified many times, or in microbiological tests in food and the environment. Can also be widely used

Evaluation of reduced activity of detecting a base analog of DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of reduced activity of detecting a base analog of DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of long DNA amplification of DNA polymerase used in the nucleic acid amplification method of the present invention Evaluation of long DNA amplification of DNA polymerase used in the nucleic acid amplification method of the present invention Comparison of the synthesis rates of DNA polymerases 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 enhancer (PCNA) Comparison of synthesis rate of DNA polymerase used in the nucleic acid amplification method of the present invention by adding PCNA Evaluation of amplification from blood using DNA polymerase and PCNA used in the nucleic acid amplification method of the present invention Evaluation of amplification 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 nucleic acid amplification method of the present invention Amplification evaluation in 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 FIG. 4 is a view showing an amino acid region related to multimer formation of PCNA.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
In the present specification, the base sequence, the amino acid sequence, and their individual constituent factors may be represented by simplified symbols in alphabetical notation, but all follow the practices in the field of molecular biology and genetic engineering. Further, in the present specification, a notation such as “D143A” is used to briefly show mutations in the amino acid sequence. "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. Unless otherwise specified, the sequence numbers correspond to the sequence numbers described in the sequence listing. 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 the present specification, the “mutant type” in the case of “mutant PCNA” means having an amino acid sequence different from conventionally known PCNA, and is caused by an artificial mutation or a mutation in nature. It does not distinguish between them.

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

(2) [No purification step]
In the nucleic acid amplification method of the present invention, a nucleic acid in a biological sample that has not undergone a purification step is amplified without purification. Purification is a method for separating DNA in a biological sample from contaminants such as tissues and cell walls of the biological sample. Methods for separating DNA using phenol or phenol / chloroform, ion-exchange resins, glass filters Alternatively, there is a method of separating DNA using a reagent having a protein aggregation action.

When nucleic acid to be amplified, such as an organ or a cell, is present in the tissue of a 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 mentioned in the present invention. Further, the act of diluting a sample or a biological sample obtained by the above method with a buffer or the like 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 using these purification methods. In the present invention, the term "biological sample that has not been subjected to the purification step" refers to a biological sample itself or a liquid biological sample diluted with a solvent such as water, or a solid biological sample added to a solvent such as water to generate heat. Examples thereof include those crushed and crushed, but are not limited thereto unless they have undergone a purification step.

In this specification, the term "blood resistance" is used, which means that even if blood is present in the nucleic acid amplification reaction solution, it can withstand 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, excrement, cells, bacteria, viruses, and the like. Body fluids include 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 a commercially available route can be used.
The concentration of dUTP in the reaction solution is not particularly limited, but the lower limit is preferably 0.5 μM or more, more preferably 50 μM or more, and more preferably 0.1 mM or more, from the viewpoint of the efficiency of removing contamination. From the viewpoint of PCR efficiency, dUTP may be contained at a high concentration. A preferred upper limit is 1 mM or less, more preferably 0.6 mM or less.
Further, dTTP and dUTP may be mixed from the viewpoint of PCR efficiency. The ratio between dTTP and dUTP is preferably from 100: 1 to 1: 100. The ratio is more preferably from 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 restricted by its length, sequence, GC content, etc., but it incorporates dUTP during amplification, so it is necessary that the nucleic acid contains T, It is preferable that T is contained 10 or more.

(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 Family B is preferably a DNA polymerase derived from archaea (Archea).

(6) [A DNA polymerase derived from archaea]
Examples of DNA polymerases derived from archaea belonging to the family B include DNA polymerases isolated from bacteria belonging to the genus Pyrococcus and the genus Thermococcus.
Pyrococcus DNA polymerases 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, and 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 siculi.
PCR enzymes using these DNA polymerases are commercially available, and include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), and Deep Vent (New England). , Tgo (Roche), Pwo (Roche) and the like.

Among them, from the viewpoint of PCR efficiency, KOD DNA polymerase having excellent extensibility and heat stability is preferable.

(7) [DNA polymerase mutant having reduced base analog detection activity]
The DNA polymerase belonging to Family B used for the nucleic acid amplification method of the present invention may be a mutant having a reduced activity of detecting a base analog. The base analog refers to a base other than adenine, cytosine, guanine, and thymine, and includes uracil and inosine. Usually, a DNA polymerase belonging to Family B binds strongly when detecting a base analog, uracil or inosine, and inhibits the polymerase function. The term “base analog detection activity” refers to an activity of strongly binding to a base analog and inhibiting a polymerase function. A DNA polymerase mutant belonging to Family B having a reduced activity of detecting a base analog is a DNA polymerase mutant belonging to Family B, which has a low ability to bind to uracil or inosine.

Examples of such a mutant having a reduced activity of detecting a base analog of a DNA polymerase belonging to Family B include an archaeal DNA polymerase mutant.
Specifically, the amino acid sequence relating to the binding of uracil formed by amino acids 1 to 40 and amino acids 78 to 130 (uracil binding pocket) is modified, and compared with wild-type DNA polymerase, to uracil and inosine. An archaeal DNA polymerase mutant characterized by low binding ability. The archaeal DNA polymerase mutant having a low ability to bind to uracil or inosine showed little decrease in the function of archaea-derived DNA polymerase belonging to family B even in PCR in the presence of dUTP, and the DNA polymerase elongation reaction by dUTP was not observed. Impact has been reduced.

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

Wild-type, family B polymerases have high accuracy. This is because amplification is performed while confirming that the incorporated base is correct. Bases that cause errors include uracil, which results from the thermal degradation of cytosine and pairs with alanine. The polymerase belonging to Family B has a pocket that strongly binds to uracil, and amplifies the nucleic acid to be amplified while confirming the presence of uracil one by one.

On the other hand, these pockets interact strongly with uracil and slow (or stop) the reaction. This interaction is considered to extend to bases such as cytosine having a similar structure to some extent, which may have reduced the DNA synthesis rate. The DNA polymerase mutant having reduced base analog detection activity used in the nucleic acid amplification method of the present invention, by modifying the uracil binding pocket, does not interact with not only uracil but also cytosine and the like, and the synthesis rate increases. Conceivable.

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

It is desirable that the DNA polymerase belonging to the family B or the archaebacterial DNA polymerase has a DNA synthesis rate of 30 bases / second or more by the following measurement method.
In the presence of dUTP, a strong binding between dUTP and the polymerase causes inhibition of PCR. A polymerase having a higher DNA synthesis rate is less likely to bind to dUTP and is less susceptible to dUTP in the presence of dUTP.

<DNA polymerase synthesis rate measurement method>
In the present invention, the synthesis rate of 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). Measure after dilution. (1) 2.5 μl of solution A, 2.5 μl of solution B, 1.5 μl of solution C, 4.8 μl of solution D, and 4 μl of solution E are mixed, and the mixture is mixed at 95 ° C. for 10 minutes and at 37 ° C. for 10 minutes. Place and make substrate.
(2) Add 5 μl of 0.64 ng / μl enzyme solution to the incubated substrate at 75 ° C. for 30 seconds and react at 75 ° C. for 30 seconds, 60 seconds and 120 seconds. Then, the mixture is cooled on ice, 35 μl of solution F is added, and the mixture is stirred well.
(3) This solution is applied to 10 μl of a 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 in accordance with the instructions of the NEB's Phototope-Star Chemiluminescent Detection Kit. 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 (TaKaRa)
E: 100 μM Biotinylated P7 primer (sequence: Biotin-CGCCAGGGTTTTCCCAGTCACGAC)
F: 59 mM NaOH, 59 mM EDTA, 0.1% BPB 30% Glycerol

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

(9) [Analyte 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 Pyrococcus and Thermococcus DNA polymerases. In the DNA polymerase derived from Thermococcus kodakaraensis (SEQ ID NO: 1), 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 1-40 and amino acids 78-130. 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 (SEQ ID NO: 6), it is formed by amino acids 1 to 40 and amino acids 78 to 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 siculi (SEQ ID NO: 10), it is formed by amino acids 1 to 40 and amino acids 78 to 130.

More preferred as the DNA polymerase mutant used in the nucleic acid amplification method of the present invention are nucleotides 7, 36, 37, 90 to 97, and 112 to 119, which are assumed to be directly involved in the interaction with uracil. An archaeal DNA polymerase mutant in which at least one of the 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 mutant represented by an amino acid sequence having at least one amino acid modification among amino acids corresponding to

The archaeal DNA polymerase mutant described above may have the amino acid sequence of the following (b).
(B) at least one amino acid is further modified in the amino acid sequence shown in (a), and the amino acid sequence is 80% or more identical to the amino acid sequence shown in (a) (preferably 85% or more, More preferably 90% or more identical, more preferably 95% or more identical, more preferably 98% or more identical, more preferably 99% or more identical), and a reduced base analog Amino acid sequence encoding a DNA polymerase having detection activity.

Various methods are known for calculating amino acid sequence identity. 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) of the National Center for Biotechnology Information (NCBI) http: // www. ncbi. nlm. nih. The amino acid sequence identity is calculated by using the default (initial setting) parameters in gov / BLAST /.

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

Here, “several” is not limited as long as “reduced base analog detection activity” is maintained, but is, for example, a number corresponding to less than about 20% of all amino acids, and 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, and still more preferably 2 to 40, Preferably, the number is 2 to 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. Is an expression containing an amino acid sequence relating to binding of uracil corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 of SEQ ID NO: 1 in a DNA polymerase having SEQ ID NO: 1.

In the specification of the present application, in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1, a certain position (order) on SEQ ID NO: 1 corresponds to a position corresponding to the primary structure of the sequence (alignment). , Corresponding to the position of SEQ ID NO: 1.
Various methods are known for comparing the primary structure of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In this specification, the default (initial setting) is used in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja) of DNA Databank of Japan (DDBJ). Compare the primary structures of the sequences.

The archaeal DNA polymerase mutant having reduced activity of detecting a base analog 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 made.
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 non-polar amino acid, and is 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) substituted with a positively charged polar amino acid, specifically a V93H, V93K, or V93R amino acid substitution. 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 a reduced base analog detection activity according to the present invention has 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 May be done. Specific examples include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferably Y7A / P36H or Y7A / V93K. However, the present invention is not limited to this.

In addition, in patent document 1 or 2, modification was added to any of the amino acids 7, 36, 37, 90 to 97, and 112 to 119, which are assumed to be directly related to the interaction with uracil. Several archaeal DNA polymerase variants have been exemplified, but not all variants have good properties to meet the task of the present application, some of which have lost activity. .

(10) [Evaluation method for base analog detection activity]
The activity of detecting a base analog in the present invention can be evaluated by PCR. The base analog is typically uracil. For example, a dUTP solution is added to a normal PCR reaction solution containing a template DNA, a buffer, magnesium, dNTPs, a primer, and a DNA polymerase to be evaluated at a final concentration of 0.5 μM to 200 μM, and a thermal cycle is performed. . After the reaction, the presence or absence of the PCR product is confirmed by ethidium bromide-stained agarose electrophoresis, and the uracil detection activity can be evaluated based on the allowable dUTP concentration. For a DNA polymerase having a high uracil detection activity, the elongation reaction is inhibited by the addition of a little dUTP, and a PCR product cannot be confirmed. In addition, gene amplification by PCR can be confirmed without any problem even when a high concentration of dUTP is added to a DNA polymerase having a low uracil detection activity.

An archaeal DNA polymerase mutant having reduced base analog detection activity is defined as an enzyme-optimized reaction buffer, using an arbitrary primer and a DNA serving as a template, and performing optimal thermal cycling. Compared with a wild type without any, the addition of a high concentration of dUTP does not inhibit the extension reaction, and means a DNA polymerase in which a PCR product can be confirmed. However, when it is difficult to compare with the wild-type, the archaeal DNA polymerase mutant that can perform PCR amplification even when dUTP is added at a concentration of 0.5 μM has a decrease in the mutant compared to the wild-type. Presumed to have the activity of detecting the base analog.

The evaluation of the base analog detection activity in the present invention is performed according to the following method.
KOD-Plus-Ver. 2 (Toybo) and 10 × PCR Buffer attached to Pfu DNA Polymerase (Agilent) using 1 × PCR Buffer and 1.5 mM MgSO ₄ , 0.2 mM dNTPs (dATP, dUTP (dTTP, dCTP, dGTP), 15 pmol of the primers of SEQ ID NOs: 13 and 14 that amplify about 1.3 kb, 10 ng of human genomic DNA (Roche), and 1 U of each enzyme in a 50 μl reaction mixture containing dUTP ( Roche) is added 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 repeating 98 cycles of 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute and 30 seconds for 30 cycles. After the completion of the reaction, 5 μl of the reaction solution is subjected to agarose electrophoresis, stained with ethidium bromide, and an amplified DNA fragment of about 1.3 kb is confirmed under irradiation with ultraviolet light, so that it can be evaluated whether the activity of detecting the base analog is reduced. .

The higher the concentration of dUTP at which PCR can be amplified, the lower the activity of detecting base analogs. In this specification, this is also expressed as "the dUTP resistance is higher" as the concentration of dUTP at which PCR amplification can be performed is higher.

(11) [Method for measuring DNA polymerase activity]
The activity of the DNA polymerase used in the nucleic acid amplification method of the present invention 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). Measure after dilution.
(1) 25 μl of 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, the mixture was cooled on ice, 50 μl of Solution E and 100 μl of Solution D were added, and after stirring, the mixture was further cooled on ice for 10 minutes.
(3) This solution is filtered through a glass filter (GF / C filter manufactured by Whatman), and sufficiently 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 in the template DNA is measured. One unit of the enzymatic activity is the amount of the enzyme that takes in 10 nmol of nucleotides per 30 minutes into the acid-insoluble fraction (that is, the fraction that becomes insoluble when the 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.
The 3'-5 'exoase activity refers to the ability to remove incorporated nucleotides from the 3' end of the DNA polymer, and the above 3'-5 'exonuclease region is a DNA polymerase belonging to Family B that is highly DNA polymerases derived from Thermococcus kodakaraensis (SEQ ID NO: 1), DNA polymerases derived from Pyrococcus furiosus (SEQ ID NO: 2), DNAs derived from Thermococcus gorgonarius Polymerase (SEQ ID NO: 3), DNA polymerase derived from Thermococcus litoralis (SEQ ID NO: 4), DNA polymerase derived from Pyrococcus sp. GB-D (SEQ ID NO: 5), derived from Thermococcus sp. JDF-3 DNA polymerase (SEQ ID NO: 6), Thermoco DNA polymerase derived from Cas 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) ) Or DNA polymerases derived from Thermococcus siculi (SEQ ID NO: 10), which are amino acids 137 to 147, 206 to 222, and 308 to 318. The present invention also applies to DNA polymerases other than the DNA polymerase whose sequence is specifically displayed. In archaebacteria-derived DNA polymerases 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 in SEQ ID NO: 1 Indicates the region corresponding to the 5 'exonuclease region.

In addition, “amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 shown in SEQ ID NO: 1” refers to a DNA polymerase having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1. , SEQ ID NO: 1 and amino acid sequences corresponding to positions 137 to 147, 206 to 222, and 308 to 318.

The modification to the 3'-5 'exonuclease region described above may consist of substitution, deletion, or addition. 2 shows alterations to amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 1.

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

Another preferred form of the DNA polymerase having a modified 3'-5 'exonuclease active region is a modified DNA polymerase corresponding to the 147th amino acid in SEQ ID NO: 1 or SEQ ID NO: 2. 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) Any one of (A) H147E and (B) any of Y7A / V93K, Y7A / V93R, Y7A / V93Q, Y7A / P36H, Y7A / P36R, P36H / V93K, P36K, P36R, P36H, V93R or V93Q ) (A) N210D, (B) any of Y7A / V93K, Y7A / P36H, Y7A / P36R, P36K, P36R, P36H, V93Q, V93K or V93R, (3) (A) I142R, and (B) Y7A / Any of 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 producing a DNA polymerase having a modified 3′-5 ′ exonuclease active region and a method for analyzing the 3′-5 ′ exonuclease activity are known. It 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 as 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 and the like.

Methods for modifying the DNA polymerase used in the nucleic acid amplification method of the present invention have already been established in the art. Therefore, the modification can be performed according to a known method, and the mode is not particularly limited.

As one embodiment of the method for introducing amino acid modification, a site-specific mutagenesis method based on the Inverse PCR method can be used. For example, KOD-Plus-Mutogenesis Kit (Toyobo) (1) denatures a plasmid into which a gene of interest is inserted, anneals a mutant primer to the plasmid, and then performs an extension reaction using KOD DNA polymerase. (2) Repeat the cycle of (1) 15 times, (3) Selectively cut only the plasmid used as a template using the restriction enzyme DpnI, (4) Phosphorylate the newly synthesized gene, Ligation And (5) a kit capable of transforming the cyclized gene into Escherichia coli and obtaining a transformant having a plasmid into which the desired mutation has been introduced.

The modified DNA polymerase gene is transferred to an expression vector, if necessary, and, for example, Escherichia coli as a host is transformed using the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form a colony. The colony is inoculated into a nutrient medium, for example, an LB medium or 2 × YT medium, and cultured at 37 ° C. for 12 to 20 hours. Then, the cells are disrupted to extract a crude enzyme solution. The vector is preferably one derived from pBluescript. As a method for disrupting the cells, any known method may be used. For example, ultrasonic treatment, a physical disruption method such as French press or glass bead disruption, or a lytic enzyme such as lysozyme can be used. The 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.

As a method for obtaining a purified DNA polymerase from the strain selected by the above method, any method may be used, and for example, the following methods are available. After recovering the cells obtained by culturing in a nutrient medium, the cells are crushed and extracted by enzymatic or physical crushing to obtain a crude enzyme solution. The obtained crude enzyme extract is heat-treated, for example, treated at 80 ° C. for 30 minutes, and then a DNA polymerase fraction is recovered by ammonium sulfate precipitation. The 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, separation and purification are carried out 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 almost a single band.

(14) Nucleic acid amplification method The nucleic acid amplification method of the present invention is a method for amplifying a target nucleic acid in a biological sample by adding a biological sample that has not been subjected to a purification step to a nucleic acid amplification reaction solution, wherein an enzyme used for amplification is used. It is not particularly limited except that it is a DNA polymerase belonging to Family B and contains deoxyuridine (dUTP) in the reaction solution.

As a method that can be amplified with a DNA polymerase, typically, PCR can be mentioned. In the present invention, not only PCR but also reaction of one kind of primer, dNTP (deoxyribonucleotide triphosphate) using DNA as a template is described. The primers are also used to synthesize DNA extension products by extending the primers. Specifically, the method includes a primer extension method, a sequencing method, a method without performing a conventional temperature cycle, and a cycle sequence method.

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

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

(15) [PCR enhancing factor]
Furthermore, the nucleic acid amplification reaction in the present invention may include a PCR enhancing factor. The enhancer refers to a complex or protein having a polynucleotide polymerase enhancing activity, such as PEF, dUTPase, ssb, PCNA, RFC, helicase, and the like (Hogrefe et al., 1997, Strategies 10: 93-96; and U.S. Pat. 6,183,997). 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 a heat-resistant one that can withstand the thermal cycle of PCR, and preferably one that retains its activity even after PCR. More preferably, it is soluble even after a heat treatment at 80 ° C. for 30 minutes, and it is desired that 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 belonging to the genus Pyrococcus and the genus Thermococcus. Examples of PCNA derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. GB-D, including but not limited to Pyrococcus Wosei, Pyrococcus abyssi, PCNA isolated from Pyrococcus horikoshii. Examples of PCNAs derived from the genus Thermococcus include Thermococcus kodaraensis (SEQ ID NO: 21), 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, KS-1, Thermococcus celler, or PCNA isolated from Thermococcus siculi.

Further, in order to increase the expression level, PCNA used in the nucleic acid amplification method of the present invention may have a modified methionine corresponding to the 73rd position of SEQ ID NO: 21 or SEQ ID NO: 22. 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 multimers and takes a ring-like structure. Loading into DNA indicates that the DNA is passed inside the ring structure of the PCNA multimer, and PCNA can only be loaded into DNA in cooperation with factors usually referred to as RFCs. Mutants that are loaded into DNA alone are those that modify the site involved in PCNA multimerization and destabilize multimer formation, making it easier for DNA to pass through the PCNA multimer without RFC. Show.

In the region where PCNA is involved in multimer formation, the PCNA derived from Thermococcus kodakaraensis (SEQ ID NO: 21) and the PCNA of Pyrococcus furiosus (SEQ ID NO: 22) are composed of N, 82, and 109 amino acids. And the C-terminal region consisting of the terminal region and the amino acids at positions 139, 143, and 147. The N-terminal region is positively charged and the C-terminal region is negatively charged, and interacts to form multimers.

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

The PCNA variant that is alone loaded into DNA is more preferably a PCNA variant of the amino acid sequence represented by SEQ ID NO: 21 or SEQ ID NO: 22, which is involved in multimerization of PCNA.
(A) having at least one modification in the N-terminal region consisting of amino acids corresponding to positions 82, 84, and 109, or (b) the C-terminal region consisting of amino acids corresponding to positions 139, 143, and 147; Mutants that load both DNAs and promote the DNA polymerase elongation reaction can be mentioned.

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

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

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

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

"Evaluation method for amplification enhancing activity"
In this specification,
(I) PCR
KOD-Plus-Ver. 2 (manufactured by Toyobo) using the attached 10 × PCR Buffer, 1 × PCR Buffer and 1.5 mM MgSO ₄ ,
0.2 mM dNTPs containing dUTP instead of dTTP (dATP, dUTP, dCTP, dGTP),
15 pmol of primer (SEQ ID NOs: 13 and 14 for 1.3 kb amplification),
10 ng human genomic DNA (Roche),
To 50 μl of a reaction solution containing 1 U KOD DNA polymerase V93K mutant (archaeal DNA polymerase mutant having reduced base analog detection activity) mixed with 0.8 μg of KOD antibody, 250 ng of PCNA to be evaluated was added,
After the pre-reaction at 94 ° C. for 30 seconds, PCR is performed according to a schedule of repeating 35 cycles of 98 ° C., 10 seconds → 60 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds.
(Ii) Analysis of PCR product After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplified DNA fragment was compared with that to which PCNA had not been added under UV irradiation. It can be evaluated whether the DNA can be loaded (whether or not it has amplification enhancing activity). The amount of PCNA that can be loaded into DNA alone (having a high amplification enhancing activity) increases when added.

(16) Reagent and kit for performing nucleic acid amplification method The reagent and kit for performing the nucleic acid amplification method of the present invention contain a DNA polymerase belonging to Family B and deoxyuridine (dUTP) in a reaction solution. The other configurations are not particularly limited. The nucleic acid amplification method to be applied is not particularly limited.

When performing PCR as a nucleic acid amplification method, the reagents and kits of the present invention can include, but are not limited to, the following configurations (a) to (d).
(A) an archaeal DNA polymerase mutant with reduced base analog detection activity (b) a pair of primers wherein one primer is complementary to the DNA extension product of the other primer (c) DNA synthesis containing dUTP A substrate (deoxynucleotide triphosphate (dNTP)) and
(D) Buffer solution containing magnesium ion, ammonium ion and / or potassium ion

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

Hereinafter, the present invention will be described specifically with reference to Examples. The description of the following examples does not limit the invention in any way.
(Example 1)
Preparation of KOD mutant A plasmid containing a modified thermostable DNA polymerase gene derived from Thermococcus kodakaraensis KOD1 strain was prepared.
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. Mutation was introduced using a KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The confirmation of the mutant was performed by decoding the nucleotide 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 Pyrococcus furiosus-derived modified thermostable DNA polymerase gene (SEQ ID NO: 12) (pPfu) cloned into pBluescript was used. Mutation was introduced using a KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The confirmation of the mutant was performed by decoding the nucleotide sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.

The plasmids produced in Examples 1 and 2 are shown in Tables 1 and 2.

(Example 3)
Example 3-1
Preparation of Modified Thermostable DNA Polymerase The cells obtained in Examples 1 and 2 were cultured as follows. First, 80 mL of sterilized TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia C. previously cultured 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 this medium E. coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells were collected from the culture solution by centrifugation, suspended in 50 mL of a disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then sonicated. This was crushed to obtain a cell lysate. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin Sepharose chromatography were performed. 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 a modified heat-resistant DNA polymerase.

Example 3-2
Evaluation of reduced activity of detecting the base analog of modified thermostable DNA polymerase The detection activity of uracil was carried out in accordance with the above-mentioned "Evaluation method of base analog detection activity" described in (10).

  As a result, in the wild-type DNA polymerase of Thermococcus kodakaraensis (KOD), inhibition was added by the addition of 0.5 μM dUTP, and PCR products could not be confirmed. In the mutant to the uracil binding pocket, the PCR product could be confirmed even when a little dUTP was added. When P36K and P36K / Y7A, and P36R and P36R / Y7A, etc., were compared, it was found that those with a double mutation as compared to a single mutation were more tolerant to the addition of a high concentration of dUTP and had a larger amount of amplification (FIG. 1, FIG. 2).

FIG. 1 shows a reaction system using wild-type (KOD) and 7 kinds of KOD mutants of Y7A, P36K, P36R, Y7A / P36K, Y7A / P36R, P36H, V93Q, and Y7A / P36H in total. It is the result of performing a PCR reaction by adding water or dUTP (manufactured by Roche) adjusted to a final concentration of 0.5, 5, 50, 100, or 200 μM, and electrophoresing the obtained product.
In the wild type, when dUTP was added, no PCR product was confirmed (lanes 2-6), and only when dUTP was not added (lane 1), an amplification product was confirmed. On the other hand, in the seven mutants, amplification products were confirmed even when dUTP having a final concentration of 0.5 μM was added. Above all, amplification products were confirmed for all the P36K, P36R, Y7A / P36K, Y7A / P36R, P36H, V93Q, and Y7A / P36H in the final dUTP concentration range of 0.5 to 200 μM.

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

In the wild type, when dUTP was added, no PCR product was confirmed (lanes 2-6), and only when dUTP was not added (lane 1), an amplification product was confirmed. On the other hand, in the mutant type in which any one of V93K, V93R, V93Q, and Y7A was added to KOD, an amplification product was confirmed even when dUTP was added at a final concentration of 0.5 μM. 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 the dUTP resistance by adding any mutation of V93K, V93R, V93Q, and Y7A to KOD is more deficient in 3′-5 ′ exonuclease than that of adding mutation to wild-type KOD (N210D, I142R or D141A / It was shown that the mutation was higher in E143A) -KOD. From these results, it can be seen that, in addition to the mutation in the uracil binding pocket, the mutation that causes a defect in 3'-5 'exonuclease is added to improve the dUTP resistance performance.

Similar experiments were carried out with other KOD mutants, and the dUTP concentration at which amplification was observed was summarized in the column of dUTP resistance in Table 3. 0 indicates that PCR could not be performed at 0.5 μM dUTP, and similarly, at 0.5, 5, and 100, amplification was observed at 0.5, 5, and 100 μM dUTP, respectively, but each was higher by one step. This shows that no amplification was observed at 5, 100, and 200 μM of dUTP. > 200 indicates that amplification was observed even when 200 μM was added.
As a result, mutations to the uracil-binding pocket of Y7, P36 or V93 tend to increase dUTP-resistant concentrations, further increasing the 3′-5 ′ exonuclease-bearing mutants such as H147E (exo (+)). As compared with the case where a mutation is added, a dUTP-resistant concentration is higher when a mutation is further added to a mutant (exo (-)) lacking a 3′-5 ′ exonuclease such as N210D, I142R or D141A / E143A. A trend was confirmed.

As for Pfu, a single mutant of Y7A and P36H or V93K was compared with a multiple mutant of Y7A / P36H or Y7A / V93K, and similar results were obtained such that the amplification amount of the multiple mutant was larger.

Example 3-3
Evaluation of long-chain DNA amplification using modified thermostable DNA polymerase Using the KOD mutants shown in Table 2, long-chain DNA exceeding 1.3 kB was obtained even under the condition of dUTP alone without dTTP. It was checked whether it could be amplified. Table 2 shows both amino acid mutations in the exo region and amino acid mutations relating to uracil binding. Regarding amino acid mutations in the exo region, exo (+) indicates a mutation retaining 3'-5 'exonuclease including wild type, and exo (-) indicates a deletion of 3'-5' exonuclease. This indicates that the mutation was deleted.

The 1.3 kbp and 2.8 kbp, 3.6 kbp amplifications of Human β-globin were compared in a PCR containing dUTP. At this time, as each enzyme, a KOD mutant mixed with 1 μg of KOD antibody per 1 U was used.

For PCR, use KOD-Plus-Ver. 2 (manufactured by Toyobo) using 1 × PCR Buffer, 1.5 mM MgSO ₄ , dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, 15 pmol of primer (1.3 kbp of SEQ ID NOS: 13 and 14 for amplification, SEQ ID NOs: 15 and 16 for 2.8 kbp amplification, SEQ ID NOs: 17 and 18 for 3.6 kbp amplification), 10 ng of human genomic DNA (Roche), 1 U mixed with antibody 50 μl of the reaction solution containing the enzyme was used. After the pre-reaction at 94 ° C. for 2 minutes, 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., about 1 minute per 1 kbp (1 minute 30 seconds for 1.3 kbp amplification, 3 minutes for 2.8 kbp amplification) PCR was performed using a PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 35 cycles of the same cycle (amplification, 3.6 kbp amplification, 4 minutes).

As a control, amplification with Taq DNA polymerase was also performed. Taq DNA polymerase used was from Toyobo, and was mixed with Anti-Taq High (Toyobo). The reaction was performed using a buffer attached to 1 × BlendTaq, dNTPs in which 2 mM dTTP was replaced with dUTP (dATP, dUTP, dCTP, dGTP), 10 pmol of primer (as described above), 10 ng of human genomic DNA (Roche), and an antibody. After the pre-reaction at 94 ° C. for 2 minutes, 50 μl of the reaction solution containing 2.5 U of the enzyme was placed at 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., about 1 minute per 1 kbp (1.3 kbp). PCR was performed using a PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 35 cycles of 1 minute and 30 seconds for amplification, 3 minutes for 2.8 kbp amplification, and 4 minutes for 3.6 kbp amplification.
After each reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 3 shows 10 types of wild-type (KOD) and V93K, Y7A / V93K, P36R, Y7A / P36R, P36H, Y7A / P36H, P36R / V93K, Y7A / P36R / V93K, P36H / V93K, and Y7A / P36H / V93K. PCR was performed on Human β-globin DNAs having different lengths in a reaction system containing dUTP (Roche) instead of dTTP in a final concentration of 0.2 mM using a total of 11 types of KOD mutants. The 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. As a control, a similar study was performed using Taq. In addition, the results of PCR performed with normal dTTP using wild-type KOD are also shown.

FIG. 4 shows that the wild-type (KOD) exo region was mutated with any of H147E, N210D, I142R, and D141A / E143A, and was further respectively subjected to V93K, Y7A / V93K, P36R, Y7A / P36R, P36H, and Y7A. / P36H to which any one of the mutations was added (a total of 24 types) was prepared, and these mutants were used in a reaction system containing dUTP (manufactured by Roche) instead of dTTP in a final concentration of 0.2 mM to obtain a length. Is a result of performing a PCR reaction on different Human β-globin DNAs and electrophoresing the obtained products. 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 respective amplification amounts of V93K, P36H, and P36R, the mutants (P36R, P36H) having a mutation at the P36 position have a larger amplification amount than V93K, and can amplify long targets. Was confirmed.
Next, as a result of comparing the amplification amounts of V93K, P36H, and P36R, and the double mutants each having a further Y7A mutation, those having the double mutation in the uracil-binding pocket than those having the single mutation, The amount of amplification has increased. These mutants have been able to amplify long chain lengths that cannot be amplified by Taq.
On the other hand, when further studying various multiple mutants combined for V93K, P36H, and P36R, which were effective as described above, a synergistic effect was observed with P36H / V93K, but P36R, Y7A / P36R / V93K, and Y7A / No amplification was observed for P36H / V93K.

In FIG. 3 and FIG. 4, mutants of wild-type DNA polymerase such as V93K and P36H and mutants of N210D, I142R and D141A / E143A, which are exo (-) mutants, have V93K and P36H. As a result of comparison of the mutants, the mutation in the exo (-) DNA polymerase resulted in a larger amount of amplification.
Further, in FIGS. 3 and 4, mutations such as V93K and P36H were applied to wild-type DNA polymerase, and mutations such as V93K and P36H were added to H147E, an exo (+) mutant that improves PCR efficiency. As a result of comparison with the exo (+) H147E mutant, the amplification amount was larger.

Table 3 summarizes the evaluation results of the KOD mutant. In Table 3, the 11-step evaluation of dUTP resistance indicates that the closer to 0, the higher the activity of detecting the base analog, and the closer to 10, the lower the activity of detecting the base analog. In Table 3, ○ indicates that amplification was sufficiently performed, Δ indicates that amplification was performed to some extent, and X indicates that amplification was not performed.

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

Example 3-4
Comparison of synthesis rate The synthesis rate was determined according to the <Method for measuring DNA polymerase synthesis rate> described in (8) above.
FIG. 5 shows eight types of wild-type (WT) and seven types of KOD mutants of Y7A / V93K, P36H, Y7A / P36H, Y7A / V93K / N210D, P36H / N210D, Y7A / P36H / N210D, and N210D. Is a result of performing a PCR reaction for 30 seconds, 60 seconds, and 120 seconds using the Biotinylated P7 primer, and electrophoresing the products obtained in each case. 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 in the upper part of the electrophoresis photograph, the longer the amplification product is obtained and the faster the synthesis speed. The left side of the photograph shows that 7000 bp and 3000 bp amplification products are detected at the respective arrows. The number shown at the bottom of the photograph is the synthesis rate of each DNA polymerase calculated based on the result.

In FIG. 5, as compared with the wild type (WT), the results of the synthesis rate of the mutants of Y7A / V93K, P36H, and Y7A / P36H were faster.
In addition, the N210D mutant in which the exo region is modified has a higher synthesis rate than the wild type, and the mutant in which the N210D mutant is modified with Y7A / V93K, P36H, or Y7A / P36H 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 tolerance was evaluated by changing the amount of blood added to the reaction solution. For comparison, Taq DNA polymerase (Taq DNA polymerase (Toyobo) was prepared by mixing KOD (wild type), KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant mixed with 0.8 μg KOD antibody / U, and antibody. 482 bp PCR of HBg was used to compare the differences in amplification between Anti-Taq High (manufactured by Toyobo Co., Ltd.) and Anti-Taq High (manufactured by Toyobo).

As described above, the PCR of KOD is performed according to KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer and MgSO ₄ attached thereto, 1 × PCR Buffer, 1.5 mM MgSO ₄ , 15 pmol of primer (SEQ ID NOS: 19 and 20 for 482 bp amplification), and 1 U of each enzyme mixed with antibody In a 50 μl reaction solution containing the above, 0.2 mM of normal dNTPs (dATP, dTTP, dCTP, dGTP) was added, and 0.2 mM of dNTPs (dATP, dUTP, dCTP, dGTP) in which dTTP was replaced with dUTP was added. The blood was used as a template, and the blood was diluted with water. After the pre-reaction at 94 ° C. for 2 minutes, the PCR system GeneAmp 9700 (Applied) was repeated on a schedule of repeating 35 cycles of 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute per 1 kbp (1 minute for 482 bp amplification). PCR was performed using Biosystem).

PCR of Taq DNA polymerase is performed by adding a buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol of a primer (same as above), and 50 μl of a reaction solution containing 2.5 U of enzyme mixed with an antibody in a normal dNTPs (50 μl). dATP, dTTP, dCTP, dGTP) were added at 0.2 mM, and dNTPs (dATP, dUTP, dCTP, dGTP) obtained by replacing dTTP with dUTP were added at 0.2 mM. Blood itself and blood diluted with water were used. After pre-reaction at 94 ° C. for 2 minutes, PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 35 cycles of 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., about 1 minute per 1 kbp (same as above) for 35 cycles Was used to perform PCR.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amount of the amplified DNA fragment amplified under ultraviolet irradiation was confirmed.

FIG. 6 shows that various DNA polymerases were prepared by using blood as a sample and adjusting the reaction solution so that the proportion of blood in the reaction solution was 10, 5, 2, 0.2, 0.02, and 0.002%. Shows the results of performing a PCR reaction according to and electrophoresis of the obtained product. The DNA polymerases used were Taq, KOD (wild type), and two types of KOD mutants (Y7A / V93K, Y7A / P36H / N210D), for a total of four types. The left side of each photograph is a case where dTTP is used, and the right side is a case where dUTP is used. One lane corresponds to the case where the ratio of the blood in the reaction solution is 10%, and the following 2-6 lanes correspond to the cases of 5%, 2%, 0.2%, 0.02% and 0.002%, respectively.
As a result, amplification was not confirmed in the presence of dUTP for Taq DNA polymerase and wild-type KOD DNA polymerase, but firm bands were confirmed for KOD Y7A / V93K and KOD Y7A / P36H / N210D mutant even in the presence of dUTP.
The KOD Y7A / P36H / N210D mutant, which has a higher 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 / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, 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, and Y7A / P36E / A Under the above reaction conditions, amplification was confirmed from the addition of 5% of blood, and a firm band was confirmed even in the presence of dUTP.

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

(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.
As a DNA template used for mutagenesis, a PCNA (SEQ ID NO: 23) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 clone cloned in pBluescript was used. Mutation was introduced using a KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The confirmation of the mutant was performed by decoding the nucleotide 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. Mutation was introduced using a KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The confirmation of the mutant was performed by decoding the nucleotide 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
Preparation of modified heat-resistant PCNA The cells obtained in Example 4-1 were cultured as follows. First, 80 mL of sterilized TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia C. previously cultured 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 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 were collected from the culture solution by centrifugation, suspended in 50 mL of a disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then sonicated. This was crushed to obtain a cell lysate. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Further, nucleic acid removal 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, .1% Tween 20, 0.1% Nonidet P40, 50% glycerin) to obtain a modified heat-resistant PCNA.

Example 4-4
Evaluation of PCR enhancing factor (PCNA) In order to confirm the effect of PCNA, KOD-PCNA mutants (M73L, M73L / E143R, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, M73L / E143F, M73L / Using E143A), 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 “Amplification Enhancing Activity Measurement Method”.

FIG. 7 shows the results of performing a PCR reaction by adding 250 ng of various PCNA mutants and electrophoresing the resulting products. The PCNA mutants used were M73L, M73L / E143R, M73L / E143A, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, and M73L / E143F.
When no PCNA was added (lane 8) or when the KOD-PCNA M73L mutant was used as PCNA (lane 1), only a few bands were found, but other KOD PCNA mutants were used. When added, the amount of amplification was increased, and a clearer band was confirmed.

PCNA forms a multimer and promotes a nucleic acid synthesis reaction, but usually cannot be loaded onto DNA without the action of RFC and the reaction does not proceed. Modification of M73L / E143R, M73L / E143A, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, and M73L / E143F is a modification to a site involved in multimer formation, and moderately reduces multimer formation. Therefore, it is conceivable that PCNA could be loaded on DNA, and the amount of PCR amplification was improved (FIG. 7).
PCNA has also previously been reported to increase the synthesis rate and reduce the elongation time, but increasing the synthesis rate has the effect of shortening the interaction time between uracil and the binding pocket and weakening the uracil detection activity. It is possible.

Similar experiments were performed with the Pfu-PCNA mutants (M73L, M73L / D143R, M73L / R109A / D143A, M73L / D147A, M73L / R82A / D143A, M73L / D143A), and almost all bands were found in the Pfu-PCNA M73L mutant. Although it could not be confirmed, the addition of other Pfu-PCNA mutants (M73L / D143R, M73L / R109A / D143A, M73L / D147A, M73L / R82A / D143A, M73L / D143A) confirmed a firm band.

Example 4-5
Comparison of synthesis rate by adding PCNA Various PCNA mutants were added to the KOD V93K mutant, and the synthesis rates were measured. KOD V93K was used after dilution 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). Also, 250 ng of the PCNA mutant was added to each reaction system, and the synthesis rate was measured.
Reagents and methods were carried out according to the <Method for measuring DNA polymerase synthesis rate> described in (8) above.

FIG. 8 shows the results obtained by adding various PCNA mutants to the V93K mutant, performing a PCR reaction for 30, 60, and 120 seconds, and electrophoresing the resulting products. As the PCNA mutant, a KOD-derived PCNA mutant (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 amplification product is detected in the upper part of the electrophoresis photograph, the longer the amplification product is obtained and the higher the synthesis speed is. The left side of the photograph shows that 7000 bp and 3000 bp amplification products are detected at the respective arrows. The number shown at the bottom of the photograph is the synthesis rate of each DNA polymerase calculated based on the result.

As a result, it was confirmed that the addition of PCNA greatly increased the synthesis rate (FIG. 8). This is presumably because PCNA performed a clamping function to tightly bind DNA and polymerase.

Example 4-6
Amplification from blood in PCR in the presence of dUTP and effect of addition of PCNA The effect of adding PCNA to the evaluation of blood tolerance was confirmed. For comparison, Taq DNA polymerase (Taq DNA polymerase (Toyobo) was prepared by mixing KOD (wild type), KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D mutant mixed with 0.8 μg KOD antibody / U, and antibody. ) And Anti-Taq High (Toyobo) were mixed in equal amounts, and 250 ng of the PCNA D147A mutant was added, and 482 bp of HBg PCR was performed to compare differences in amplification.
As described above, the PCR of KOD is performed according to 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), of 50μl containing each enzyme 1U mixed with antibody 0.2 mM of normal dNTPs (dATP, dTTP, dCTP, dGTP) was added to the reaction solution, and 0.2 mM of dNTPs (dATP, dUTP, dCTP, dGTP) obtained by replacing dTTP with dUTP were added. 250 ng of the KOD-PCNA M73L / D147A mutant was added to each reaction solution. As the template, blood itself and blood diluted with water were used. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed using a PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 35 cycles of 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute.
PCR of Taq DNA polymerase is performed by adding a buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol of a primer (same as above), and 50 μl of a reaction solution containing 2.5 U of enzyme mixed with an antibody in a normal dNTPs (50 μl). dATP, dTTP, dCTP, dGTP) were added at 0.2 mM, and dNTPs (dATP, dUTP, dCTP, dGTP) obtained by replacing dTTP with dUTP were added at 0.2 mM. Blood itself and blood diluted with water were used. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed using a PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 35 cycles of 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amount of the amplified DNA fragment amplified under ultraviolet irradiation was confirmed.

FIG. 9 shows that various DNA polymerases were prepared by using blood as a sample and adjusting the reaction solution so that the proportion of blood in the reaction solution was 10, 5, 2, 0.2, 0.02, and 0.002%. Is performed in the presence of a PCNA mutant (D147A) derived from KOD and the results of electrophoresis of the obtained product are shown. The DNA polymerases used were Taq, KOD (wild type), and two types of KOD mutants (Y7A / V93K, Y7A / P36H / N210D), for a total of four types. The left side of each photograph is a case where dTTP is used, and the right side is a case where dUTP is used. One lane corresponds to the case where the ratio of the blood in the reaction solution is 10%, and the following 2-6 lanes correspond to 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 a polymerase belonging to Family B. Since polymerases belonging to Family A such as Taq do not have a PIP motif, no effect was observed when PCNA was added (FIG. 9).
On the other hand, it was found that in the KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants having a reduced activity of detecting the base analog, the reaction occurs even to more concentrated blood when PCNA is added.
In addition, the amount of amplification is increased in the order of increasing the synthesis rate of KOD Y7A / P36H / N210D to which PCNA was added and KOD Y7A / V93K to which PCNA was added, and it is considered that the synthesis rate is also important in 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 / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, 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, and Y7A / P36E / A Under the above reaction conditions, amplification was confirmed from the addition of 10% of blood, and a firm band was confirmed even in the presence of dUTP.

PCNA is also a mutant of KOD-PCNA M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / D147A, M73L / R82A / D143A, and M73L / R109A / D143A. A similar reaction was performed, and amplification was confirmed from the addition of 10% of blood, and a firm band was confirmed even in the presence of dUTP.

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

Example 4-7
PCR from body tissues (nail, hair, oral mucosa) in the presence of dUTP
Using nails, hair, and oral mucosa as templates, it was examined whether PCR could be performed in the presence of dUTP. One piece of nail was cut with a nail clipper, and one piece of hair was 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 1 M Tris-HCl (pH 8.0). The supernatant was used as a template. For the oral mucosa, a mucosa collected with a cotton swab and suspended in 200 μl of water was used as a template.
Taq DNA polymerase (Taq DNA polymerase (Toyobo Inc.) containing KOD (wild type), KOD Y7A / V93K mutant, and KOD Y7A / P36H / N210D mutant mixed with 0.8 μg KOD antibody / U, and antibody were used. And Anti-Taq High (manufactured by Toyobo) in equal amounts), and 482 bp of HBg PCR was performed to compare differences in amplification.
As described above, the PCR of KOD is performed according to 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), of 50μl containing each enzyme 1U mixed with antibody 0.2 mM of normal dNTPs (dATP, dTTP, dCTP, dGTP) was added to the reaction solution, and 0.2 mM of dNTPs (dATP, dUTP, dCTP, dGTP) obtained by replacing dTTP with dUTP were added. Each sample was used, and 1 μl of the sample was used as a template. In addition, each reaction solution was supplemented with 250 ng of a KOD-PCNA E143R mutant which is a PCR enhancing factor. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed using a PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 35 cycles of 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute.
PCR of Taq DNA polymerase is performed by adding a buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol of a primer (same as above), and 50 μl of a reaction solution containing 2.5 U of enzyme mixed with an antibody in a normal dNTPs (50 μl). dATP, dTTP, dCTP, dGTP) were added at 0.2 mM, and dNTPs (dATP, dUTP, dCTP, dGTP) obtained by replacing dTTP with dUTP were added at 0.2 mM. 1 μl of the above sample was used. In addition, a reaction solution in which 250 ng of a KOD-PCNA M73L / E143R mutant, which is a PCR enhancing factor, was added to each reaction solution was also carried out. After a pre-reaction at 94 ° C. for 2 minutes, PCR was performed using a PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 35 cycles of 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amount of the amplified DNA fragment amplified under ultraviolet irradiation was confirmed.

FIG. 10 shows the results of performing a PCR reaction using various DNA polymerases in the presence of a PCNA mutant (E143R) derived from KOD using nails, hair, and oral mucus as a sample, and electrophoresing the obtained product. . The DNA polymerases used were Taq, KOD (wild type), and two types of KOD mutants (Y7A / V93K, Y7A / P36H / N210D), for a total of four types. The left side of each photograph is a case where dTTP is used, and the right side is a case where dUTP is used. One lane is a nail sample, two lanes are hair, and three lanes are oral mucosa. Each "+" lane is in the presence of the KOD-derived PCNA variant (E143R), and "-" indicates no PCNA.
As a result, almost no amplification was confirmed in the presence of dUTP for Taq DNA polymerase and wild-type KOD DNA polymerase, while a firm band was confirmed in the presence of dUTP for KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants ( (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 / 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, and Y7A / P36E / A Under the above reaction conditions, amplification from body tissues (nail, hair, oral mucosa) was confirmed, and a firm band was confirmed even in the presence of dUTP. When KOD-PCNA mutants (M73L / D147A, M73L / R109A / E143A, M73L / E143R) and Pfu-PCNA mutants (M73L / D143R) are added to these, an increase in the amount of amplification can be confirmed as described above. Was.

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

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

FIG. 11 shows that a plant lysate was used as a sample, a reaction solution was prepared so that the ratio of the lysate in the reaction solution was 2%, and a KOD Y7A / P36H / N210D mutant and a KOD Y7A / P36H / N210D mutant were prepared. -The result of performing a PCR reaction with a total of three kinds, to which PCNA M73L / D147A was added and Taq polymerase, and electrophoresing the obtained product is shown.
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 KOD Y7A / P36H / N210D added with KOD-PCNA M73L / D147A.
As a result, in KOD Y7A / P36H / N210D, amplification was confirmed even from an unpurified plant lysate. Further, when the mutant of PCNA was added, a firm band having a larger amplification amount than that without the addition was confirmed.
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 / H93E, V93K / H147E / H147E, Y7A / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210 / 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, E7A / E14A, Y7A) However, under the same reaction conditions, amplification was confirmed from the plant lysate, and a firm band was confirmed even in the presence of dUTP.

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

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

Example 4-9
Amplification from feces dUTP and whether the gene could be amplified in the presence of feces was evaluated.
KOD Y7A / P36H / N210D mutant and Taq polymerase were used as enzymes, 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 curve. The KOD Y7A / P36H / N210D mutant was also supplemented with KOD-PCNA M73L / D147A as a PCR enhancing factor.

As the inhibitor, a 10% fecal suspension heat-treated at 95 ° C. for 10 minutes was used.
For the PCR of KOD Y7A / P36H / N210D, 1 × PCR Buffer, 50 copies of Salmonella genome, 4 pmol of primers (SEQ ID NOs: 27 and 28), and 2 mM dTTP were converted to dUTP, using a buffer attached to KOD Dash (manufactured by Toyobo). In a 20 μl reaction mixture containing the substituted dNTPs (dATP, dUTP, dCTP, dGTP), 1/30000 SYBR GREEN I, and 0.4 U of enzyme mixed with KOD antibody, stool was added to the reaction solution at 0,0. 1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5%, and after a pre-reaction at 95 ° C. for 30 seconds, 98 ° C., 10 seconds → 60 Performed using LightCycler 2.0 (Roche) on a schedule of repeating 50 cycles of 10 ° C. → 68 ° C., 30 seconds for 30 seconds. . KOD-PCNA M73L / D147A was added to the above reaction system in an amount of 100 ng, and compared with that without PCNA.
Taq DNA polymerase used was from Toyobo, and was mixed with Anti-Taq High (Toyobo). The reaction was performed with Buffer (Mg attached type) attached to 1 × Taq, Salmonella genome of 50 copies, 4 pmol of primer (as above), dNTPs (dATP, dUTP, dCTP, dGTP) with 2 mM dTTP replaced with dUTP, 4 mM In a 20 μl reaction mixture containing MgSO ₄ , 1/30000 SYBR GREEN I, 1 U of enzyme mixed with antibody, feces were added to the reaction mixture at 0, 0.1, 0.25, 0.5, 1.0 , 1.5, 2.0 and 2.5%, and after a pre-reaction at 95 ° C. for 30 seconds, 50 cycles of 98 ° C., 10 seconds → 60 ° C. 10 seconds → 68 ° C., 30 seconds are repeated. Performed using LightCycler 2.0 (Roche) on a schedule.
After the 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 values of Example 4-9.

Table 5 shows Cq values (default setting 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 the Cq value was not determined.
As a result, amplification was not observed when Taq polymerase was added with 0.5% stool, but amplification was confirmed with KOD Y7A / P36H / N210D even when 2.5% stool was added. Further, when comparing the presence and absence of PCNA, it was found that the one to which PCNA was added had a smaller Cq value and exhibited excellent PCR efficiency.

FIG. 12 shows the results of melting curve analysis of the amplification product obtained using PCR in the presence of dUTP and feces. Polymerases used were KOD Y7A / P36H / N210D mutant, KOD Y7A / P36H / N210D mutant added with KOD-PCNA M73L / D147A, and Taq polymerase in total.
1 shows the result of KOD Y7A / P36H / N210D, 2 shows the result of KOD Y7A / P36H / N210D added with KOD-PCNA M73L / D147A, 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 feces, and although the peak was lowered by the addition of feces, the target peak could be confirmed even when 2.5% was added. . Similarly, the target peak could be confirmed in the case of adding PCNA even in the case of adding 2.5%. However, with Taq polymerase, the target peak disappeared when 0.25% was added, suggesting that the Taq polymerase was inhibited by the effect 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 / H93E, V93K / H147E / H147E, Y7A / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210 / 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, E7A / E14A, Y7A) However, similarly, amplification was confirmed in a reaction system containing 2.5% feces, and a firm band was confirmed even in the presence of dUTP.

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

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

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

PCR of the KOD mutant was performed according to 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: 13 and 14), dNTPs substituted with 2 mM dTTP to dUTP (dATP, dUTP , DCTP, dGTP), and 50 μl of a reaction solution containing 1 U of each enzyme mixed with KOD antibody, blood was added to 2% of the reaction solution, and 250 ng of no PCNA and Pfu-PCNA M73L / D147A was added. The additions were compared.
Taq DNA polymerase PCR was performed by mixing Buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol of primer (same as above), dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, and an antibody. To 50 μl of a reaction solution containing 2.5 U of enzyme, blood was used so as to be 2% of the reaction solution.
The reaction was carried out using a PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 35 cycles of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1.5 minutes after the pre-reaction at 94 ° C. for 2 minutes. Was.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amount of the amplified DNA fragment amplified under ultraviolet irradiation was confirmed.

FIG. 13 shows a reaction mixture containing 2% blood, prepared by adding Pfu-PCNA M73L / D147A to the KOD Y7A / P36H / N210D mutant, and the KOD Y7A / P36H / N210D mutant, and using Taq polymerase. The result of performing a PCR reaction and electrophoresing the obtained product is shown. In each photograph,-indicates no PCNA, and + indicates that PCNA was added.
As a result, it was shown that the use of the polymerase of the present invention can amplify even a relatively long amplification of 1.3 kb from crude conditions containing blood components. In addition, a firm band was confirmed in the case where the mutant of PCNA was added as compared with the case where no mutant was 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 / E141A / E93A / D141A / E143A, Y7A / P36R / D141A / E143A), the amplification was confirmed under the same reaction conditions, and a firm band was observed even in the presence of dUTP. It was confirmed.

PCNA is also KOD-PCNA M73L / D147A, M73L / E143R, M73L / R82A / E143A, M73L / R109A / E143A, Pfu-PCNA M73L / D143R, M73L / R82A / D143A, and mutants of M73L / R109A / D143A. The same reaction was carried out, and it was confirmed that the amount of amplification was improved as compared with the case without PCNA addition.

The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art.
The contents of articles, published patent gazettes, and patent gazettes specified in this specification are all incorporated by reference.

According to the present invention, the risk of loss and carryover during DNA purification can be eliminated, and the time and cost can be further reduced. In addition, to prevent contamination due to the dUTP / UDG contamination removal method, it can be used not only in the research field, but also in the clinical field or forensic field such as genetic diagnosis where the same sample is amplified many times, or in microbiological tests in food and the environment. Can also be widely used

Claims (5)

A method of adding a biological sample selected from the group consisting of animal and plant tissues, body fluids, excreta, cells, bacteria, and viruses, which has not been subjected to a purification step, to a nucleic acid amplification reaction solution to amplify a target nucleic acid in the biological sample. The enzyme used for the amplification is a DNA polymerase derived from Thermococcus kodakaraensis or Pyrococcus furiosus, wherein in the SEQ ID NO: 1 or SEQ ID NO: 2, (I) P36K, It has any modification selected from the group consisting of P36R and P36H , and (II) any amino acid modification selected from the group consisting of Y7A, Y7G, Y7V, Y7L and Y7I (provided that Y7A / P36H Or Y7A / P36R A nucleic acid amplification method, characterized in that the method is a DNA polymerase mutant having a combination of amino acid modifications (excluding combinations of amino acid modifications) and contains deoxyuridine (dUTP) in a reaction solution.   The nucleic acid amplification method according to claim 1, wherein the biological sample is any one selected from the group consisting of blood, saliva, leukocytes, plant lysate, feces, nails, hair, and oral mucosa. The nucleic acid amplification method according to claim 1 or 2 , wherein the mutant of the DNA polymerase has a modification of Y7A / P36K in SEQ ID NO: 1 or SEQ ID NO: 2. The DNA polymerase mutant according to any one of claims 1 to 3 , further comprising 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 according to the above. The nucleic acid amplification method according to any one of claims 1 to 4 , wherein the mutant of the DNA polymerase has any one of the following modifications (1) or (2) in SEQ ID NO: 1 or SEQ ID NO: 2.
(1) (A) H147E, (B) Y7A / P36K,
(2) (A) N210D and (B) Y7A / P36K.