JP6489017B2 - Nucleic acid amplification method - Google Patents

Description

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

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

  On the other hand, since PCR is a very sensitive detection method, false positives caused by carry-over of previously performed PCR amplification products become a problem. Therefore, PCR is performed using a substrate containing a base analog such as dUTP instead of dTTP, uracil base is incorporated into the amplification product, and uracil-N-glycosylase (UNG) treatment is performed in the next PCR. A technique (dUTP / UDG contamination removal method) for decomposing contaminated (carry over) amplification products has been used (Non-patent Document 1).

However, in the dUTP / UDG contamination removal method, when PCR is carried out using dUTP instead of dTTP as a substrate, there has been a problem that the reaction efficiency is lowered and the amount of amplified product is reduced. It is also known that when a template DNA containing uracil is amplified, the reaction efficiency is lowered and the amount of amplification product is reduced.
Therefore, even if dUTP is present or the template DNA contains uracil, it is sufficient when normal dTTP is used or when a template composed of normal adenine, cytosine, guanine, and thymine is amplified. There has been a need for a nucleic acid amplification method that exhibits a large amount of amplification.

Patent 4395377 Special table 2006-507012

Gene, Vol. 93 (1), 125-128 (1990)

  Accordingly, an object of the present invention is to provide an efficient method for amplifying a target nucleic acid in a reaction composition for nucleic acid amplification containing a base analog. Furthermore, the other object of this invention is to provide the reagent kit suitable for said objective. In summary, an object of the present invention is to provide an improved PCR method and a PCR reaction reagent suitable for amplification of a gene in the presence of a base analog.

  In order to achieve the above object, the nucleic acid amplification method of the present invention comprises a DNA belonging to Family B having a reduced base analog detection activity with respect to a base analog such as uracil in a reaction composition of nucleic acid amplification including a base analog. It is characterized by using a polymerase and PCNA.

  That is, the present inventor uses a DNA polymerase belonging to Family B having reduced base analog detection activity and PCNA in a nucleic acid amplification reaction composition containing a base analog, so that even if the base analog exists. As a result, it has been found that PCR can be performed with the same amount of amplification as in the presence of a normal base, and the present invention has been achieved.

The representative invention of the present application is as follows.
[1]
A method of amplifying a nucleic acid with a reaction composition comprising a base analog, the reaction composition comprising:
(A) a variant of a DNA polymerase belonging to family B having reduced base analog detection activity, and (b) PCNA
Including the nucleic acid amplification method.
[2]
The nucleic acid amplification method according to [1], wherein the mutant of DNA polymerase belonging to family B having reduced base analog detection activity is represented by any one of (a1) to (a3) below.
(A1) A polypeptide comprising an amino acid sequence having at least one amino acid modification among amino acid sequences 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.
(A2) In the DNA polymerase mutant represented by (a1), at least one amino acid is further modified at positions other than the 7, 36, 37, 90 to 97, and 112 to 119th positions. A polypeptide having 80% or more identity with No. 1 or 80% or more identity between its amino acid sequence and SEQ ID NO: 2 and having reduced base analog detection activity.
(A3) In the DNA polymerase mutant represented by (a1), one or several amino acids are further deleted, substituted, or added at sites other than positions 7, 36, 37, 90 to 97, and 112 to 119. And a polypeptide having reduced base analog detection activity.
[3]
A variant of a DNA polymerase belonging to Family B having reduced base analog detection activity is at least one amino acid among amino acids corresponding to the 7, 36, and 93rd amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2 The nucleic acid amplification method according to [2], which is a polypeptide consisting of an amino acid sequence having the modification of
[4]
Any one of [1] to [3], wherein the DNA polymerase belonging to family B is a mutant having at least one amino acid modification in any of the amino acids constituting the 3′-5 ′ exonuclease active region The nucleic acid amplification method described.
[5]
Any of the amino acids corresponding to D141, I142, E143, H147, N210 and Y311 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is altered to the 3′-5 ′ exonuclease active region of DNA polymerase belonging to family B The nucleic acid amplification method according to any one of [1] to [4], wherein the method is a modification of at least one amino acid.
[6]
The nucleic acid amplification method according to any one of [1] to [5], wherein the PCNA is represented by any of the following (b1) to (b3).
(B1) In the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 12, it has at least one modification among the amino acid residues present at the position of the group represented by (x) and (y) below, and is amplified A polypeptide exhibiting potentiating activity.
(X) In the PCNA represented by the 82, 84, 109th amino acid residue group (y), 139, 143, 147th amino acid residue group (b2) (b1), further in (x) and (y) A polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added at a site other than the group shown and having amplification enhancing activity.
(B3) It comprises an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 11 or 80% or more identity with the amino acid sequence represented by SEQ ID NO: 12, and has amplification enhancing activity Polypeptide.
[7]
PCNA, SEQ ID NO: 11 or SEQ ID NO: 12, amino acid corresponding to position 143 was changed to basic amino acid, both amino acid 82 and 143 were changed to neutral amino acid, and position 147 was changed to neutral amino acid Or the nucleic acid amplification method according to any one of [1] to [6], which is a mutant of any one of those obtained by altering both the 109th and 143rd positions to neutral amino acids.
[8]
The nucleic acid amplification method according to any one of [1] to [7], wherein a biological sample that has not undergone a purification step is added to a nucleic acid amplification reaction solution to amplify a target nucleic acid in the biological sample.
[9]
The nucleic acid amplification method according to any one of [1] to [8], wherein the biological sample is any of animal and plant tissues, body fluids, excreta, cells, bacteria, and viruses.
[10]
A reagent for performing a nucleic acid amplification reaction, comprising the following (a) to (e):
(A) a variant of a DNA polymerase belonging to family B having reduced base analog detection activity (b) PCNA
(C) Primer pair (d) DNA synthesis substrate (deoxynucleotide triphosphate (dNTP))
(E) Buffer solution containing Mg ions [11]
[10] A kit comprising the reagent according to [10].

  According to the present invention, in order to prevent contamination by the dUTP / UDG contamination removal method, not only in the research field, but also in the clinical field or forensic field such as genetic diagnosis in which the same sample is amplified many times, or in the microbial examination in food and the environment Can also be used widely

Evaluation of uracil detection activity of KOD DNA polymerase and its various variants Evaluation of uracil detection activity of KOD DNA polymerase and its various variants Evaluation of uracil detection activity of KOD DNA polymerase and its various variants Evaluation of uracil detection activity of KOD DNA polymerase and its various variants Evaluation of PCNA amplification enhancing activity Comparison of amplification of long-chain targets by adding PCNA Evaluation of amplification from blood by adding PCNA Evaluation of amplification from blood by adding PCNA Evaluation of amplification from blood by adding PCNA Amplification from plant lysate by addition of PCNA Amplification from nails, hair and oral mucosa by adding PCNA Amplification from DNA after bisulfite treatment by adding PCNA The figure which shows the amino acid region regarding multimer formation of PCNA Evaluation of amplification from the presence of feces using DNA polymerase used in the nucleic acid amplification method of the present invention

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
In this specification, the base sequence, amino acid sequence, and individual components thereof may be simplified by alphabetical symbols, but all follow the practices in the fields of molecular biology and genetic engineering. Further, in this specification, in order to simply show the mutation of the amino acid sequence, for example, a notation such as “D143A” is used. “D143A” indicates that the 143rd aspartic acid was substituted with alanine, that is, the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. Further, the sequence numbers correspond to the sequence numbers described in the sequence listing unless otherwise specified. In the case of multiple mutants, the above notation is connected by “/”. For example, “D143A / D147A” indicates that the 143rd aspartic acid was substituted with alanine and the 147th aspartic acid was substituted with alanine. For multiple mutants more than triple mutants, information on the mutation site such as “P36H” is added after the symbol “/”.
In the present specification, the term “mutant” in the case of “mutant PCNA” means that it has an amino acid sequence different from the conventionally known PCNA, and is caused by artificial mutation or natural variation. It does not distinguish whether or not.

(1) Nucleic acid amplification method One of the embodiments of the present invention is a method of amplifying a nucleic acid with a reaction composition containing a base analog, wherein the reaction composition comprises:
(A) a variant of a DNA polymerase belonging to family B having reduced base analog detection activity, and (b) PCNA
It is the said nucleic acid amplification method containing.

  In the method of the present invention, the nucleic acid amplification method is not particularly limited as long as it can be amplified by a DNA polymerase. Typically, PCR is used, but the present invention is not limited to PCR, and a DNA primer extension product is obtained by extending a primer by reacting with one kind of primer, dNTP (deoxyribonucleotide triphosphate), using DNA as a template. It is also used in the method of synthesizing. Specifically, a primer extension method, a sequencing method, a conventional method that does not perform temperature cycling, and a cycle sequence method are included.

  The base analog in the present invention refers to a base other than adenine (A), cytosine (C), guanine (G), and thymine (T), and examples thereof include uracil (U) and inosine (I). However, it is not limited to these.

  The reaction composition including a base analog in the present invention includes a base analog in the composition for nucleic acid amplification, and the base analog reacts as a substrate for DNA synthesis (deoxynucleotide triphosphate (dNTP)). It may be included in the composition, may be included in a primer used for nucleic acid amplification, or may be included in DNA to be amplified.

The nucleic acid applied to the nucleic acid amplification method of the present invention is not particularly limited as long as it can be amplified by DNA polymerase, and is not limited by its length, sequence, GC content, or the like.
The nucleic acid is typically DNA composed of adenine (A), cytosine (C), guanine (G), and thymine (T). However, in the nucleic acid amplification method of the present invention, the nucleic acid is “reduced”. Since a mutant having a base analog (in this specification, a base other than adenine, cytosine, guanine, and thymine is referred to as a base analog) is used, a base other than adenine, cytosine, guanine, and thymine, for example, It may contain uracil or inosine.
Hereinafter, unless otherwise specified in the present specification, the base constituting the DNA may include any of the above A, C, G, T, and base analogs.

  When the nucleic acid applied to the nucleic acid amplification method of the present invention incorporates dUTP, it is necessary that the nucleic acid contains T, and it is preferable that 10 or more T be contained.

[Biological sample]
The biological sample applied to the nucleic acid amplification method of the present invention is not particularly limited as long as it is a sample collected from a living body. For example, it refers to animal and plant tissues, body fluids, excreta, cells, bacteria, viruses and the like. Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood. The biological sample includes a processed product such as food, a natural environment such as soil, and a living environment such as a business establishment and a residence, and those derived from a living body.

  The biological sample applied to the nucleic acid amplification method of the present invention may not be subjected to a purification step. That is, the nucleic acid amplification method in the present invention may be a method in which a biological sample that has not undergone a purification step is added to a nucleic acid amplification reaction solution to amplify a target nucleic acid in the biological sample.

  Purification is a method for separating contaminants such as tissues and cell walls of biological samples from DNA in biological samples. Methods for separating DNA using phenol or phenol / chloroform, ion exchange resins, glass filters, etc. Alternatively, there is a method of separating DNA with a reagent having a protein aggregation action. In the nucleic acid amplification method of the present invention, a biological sample may be added to a nucleic acid amplification reaction solution and amplified without taking these purification methods.

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

The biological sample applied to the nucleic acid amplification method of the present invention may have undergone bisulfite treatment. That is, the nucleic acid amplification method in the present invention may be a method of amplifying DNA after bisulfite treatment.
As a technique for analyzing DNA methylation, a method of sequencing DNA after bisulfite treatment has been adopted. Bisulfite treatment converts unmethylated cytosine into uracil, while methylated cytosine is not converted. Therefore, methylated cytosine and unmethylated cytosine can be distinguished by confirming the sequence.

In the method of the present invention, typical conditions in the case of PCR are shown below, but are not limited thereto.
In the amplification target DNA,
(A) a variant of a DNA polymerase belonging to family B having reduced base analog detection activity (b) PCNA
Besides,
(C) a pair of primers in which one primer is complementary to the DNA extension product of the other primer (d) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)), and
(E) mixing a buffer solution containing magnesium ions, ammonium ions and / or potassium ions;
By raising or lowering the temperature of the reaction solution using a thermal cycler or the like, a specific DNA fragment is amplified by repeating (1) DNA denaturation, (2) primer annealing, and (3) primer extension thermal cycle.
In the PCR method, BSA and a nonionic surfactant may be further used as necessary.

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

(1.1) Variant of DNA polymerase belonging to family B The mutant of DNA polymerase belonging to family B used in the method of the present invention has “reduced base analog detection activity”.
(1.1.1) Base analog detection activity The base analog detection activity is an activity that strongly binds to a base analog and inhibits the polymerase function. (The method for evaluating the base analog detection activity will be described later.)
Usually, a DNA polymerase belonging to Family B binds strongly when a base analog such as uracil or inosine is detected, and the polymerase function is almost completely inhibited. Is a variant obtained by modifying the wild-type amino acid sequence so as to reduce the base analog detection activity of DNA polymerase belonging to. Therefore, even if a base analog such as uracil or inosine is present, DNA synthesis (DNA polymerase elongation reaction) proceeds without completely inhibiting the polymerase function.

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

In this specification, the measurement of the base analog detection activity follows the following method.
(Preparation of reaction solution)
DNA polymerase 1U to be measured for activity (a method for measuring the activity of DNA polymerase will be described later in (1.1.3)) and a base buffer (the activity to be measured is Thermococcus kodakaraensis (SEQ ID NO: 1) or a mutation thereof) 10 × PCR Buffer attached to KOD-Plus-Ver.2 (manufactured by Toyobo) and Pfu DNA Polymerase (manufactured by Agilent) when the activity measurement target is Pyrococcus furiosus (SEQ ID NO: 2) or a mutant thereof. Use the attached 10 × PCR Buffer, or substitute the 10 × PCR Buffer attached to the optimal reaction Buffer or KOD-Plus-Ver.2 (Toyobo) in the case of other DNA polymerases or mutants thereof Used Te. Indicating that it is concentrated 10 times the concentration used in the reaction to herein as 10 ×.
In the PCR reaction, 1 × PCR Buffer is added with 1.5 mM MgSO ₄ ,
0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP),
15 pmol of the primer pair set forth in SEQ ID NOs: 13 and 14 for amplifying about 1.3 kb, 10 ng of human genomic DNA (Roche),
Prepare 50 μl of the reaction solution.
(PCR)
DUTP (Roche) is added to the reaction solution to a final concentration of 0.5, 5, 50, 100, and 200 μM. After the pre-reaction at 94 ° C. for 30 seconds, PCR is performed on a schedule of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds repeated 30 cycles.
(Detection of amplification products)
After 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 ultraviolet irradiation.

(1.1.2) Determination of whether the base analog detection activity is “decreased” The base analog detection activity of the mutant DNA polymerase is “decreased”. Says that it is reduced compared to that of the wild type.
In the present specification, the base analog detection activity is compared between the mutant type and the wild type without mutation according to <Method for measuring base analog detection activity> described in (1.1.1). If it can be confirmed that more PCR products were obtained than the wild type, it is determined that the mutant DNA polymerase has “reduced base analog detection activity”.
However, when it is difficult to compare with the wild type by the above method, a DNA polymerase mutant belonging to Family B that can amplify PCR even when dUTP is added at a concentration of 0.5 μM Presumed to have reduced base analog detection activity compared to wild type.

(1.1.3) DNA polymerase activity measurement method The DNA polymerase used in the nucleic acid amplification method of the present invention measures the activity as follows.
If the enzyme activity is strong, the sample is stored in a storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). Dilute and measure.
(1) 25 μl of the following solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterilized water, and 5 μl of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes.
(2) Then, ice-cool, add 50 μl of E solution and 100 μl of D solution, and stir on ice for 10 minutes after stirring.
(3) This solution is filtered through a glass filter (GF / C filter manufactured by Whatman) and thoroughly washed with 0.1N hydrochloric acid and ethanol.
(4) The radioactivity of the filter is measured with a liquid scintillation counter (manufactured by Packard), and the incorporation of nucleotides into the template DNA is measured. One unit of enzyme activity is defined as the amount of enzyme that takes 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction (that is, the fraction insolubilized when solution D is added) under these conditions.
A: 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / mL BSA (bovine serum albumin)
B: 1.5 μg / μl activated calf thymus DNA
C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
E: 1 mg / mL calf thymus DNA

(1.2) DNA polymerase modification site (1.2.1)
The DNA polymerase used in the nucleic acid amplification method of the present invention is a mutant in which a DNA polymerase belonging to Family B, preferably a DNA polymerase derived from Archaea, has been modified to reduce the base analog detection activity.

Examples of DNA polymerases derived from archaea belonging to family B include DNA polymerases isolated from bacteria of the genera Pyrococcus and Thermococcus. Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus (SEQ ID NO: 2), Pyrococcus sp. Including, but not limited to, DNA polymerases isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii. Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaaraensis (SEQ ID NO: 1), Thermococcus gonorarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including but not limited to DNA polymerase isolated from KS-1, Thermococcus celler, or Thermococcus sicili. PCR enzymes using these DNA polymerases are commercially available, Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England) , Tgo (Roche), Pwo (Roche).
Among these, from the viewpoint of PCR efficiency, KOD DNA polymerase excellent in extensibility and thermal stability is preferable.

  Specifically, the archaebacteria-derived DNA polymerase mutant belonging to family B specifically includes the 1st to 40th and 78th to 130th amino acid sequences of the DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis KOD1 strain. A DNA polymerase variant in which at least one amino acid sequence (uracil-binding pocket) relating to uracil binding formed by the method is modified, and the ability to bind to uracil or inosine is reduced compared to wild-type DNA polymerase Illustrated.

  The amino acid sequence for uracil binding is highly conserved in DNA polymerases from Pyrococcus and DNA polymerases from Thermococcus. As described above, the DNA polymerase (SEQ ID NO: 1) derived from Thermococcus kodakaraensis is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus furiosus (SEQ ID NO: 2), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus gorgonarius (SEQ ID NO: 3), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus litoralis (SEQ ID NO: 4), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus sp. GB-D (SEQ ID NO: 5), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp. JDF-3 (sequence number 6), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus sp 9 ° N-7 (SEQ ID NO: 7), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp KS-1 (SEQ ID NO: 8), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus cellar (SEQ ID NO: 9), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus cyclis (SEQ ID NO: 10), it is formed by amino acids 1 to 40 and amino acids 78 to 130.

(1.2.2)
More preferred as a DNA polymerase variant belonging to Family B having reduced base analog detection activity for use in the nucleic acid amplification method of the present invention is a sequence that is assumed to be directly related to interaction with uracil. A variant of a DNA polymerase belonging to Family B, wherein at least one of the 7, 36, 37, 90 to 97 and 112 to 119 of the amino acid sequence shown in No. 1 or SEQ ID No. 2 is modified,
(A1) Poly consisting of an amino acid sequence having at least one amino acid modification among amino acids corresponding to the seventh, thirty-sixth, thirty-seventh, ninety-seventh to ninety-seventh and ninety-first to ninety-first amino acids of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 It is a peptide.

The mutant of the DNA polymerase belonging to the above family B may be one represented by the following amino acid sequence (a2).
(A2) In the DNA polymerase mutant represented by (a1), at least one amino acid is further modified at positions other than the 7, 36, 37, 90 to 97, and 112 to 119th positions. 80% or more (preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more). More preferably, it is 99% or more) and has a reduced base analog detection activity.

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

The mutant of the DNA polymerase belonging to the above family B may be one represented by the following amino acid sequence (a3).
(A3) In the DNA polymerase mutant represented by (a1), one or several amino acids are further deleted, substituted, or added at sites other than positions 7, 36, 37, 90 to 97, and 112 to 119. And a polypeptide having reduced base analog detection activity.

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

  The “amino acids corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 1” are amino acid sequences that are not completely identical to the amino acid sequence shown in SEQ ID NO: 1. In the DNA polymerase having the sequence of SEQ ID NO: 1, the expression includes amino acids assumed to be related to uracil binding, corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 of SEQ ID NO: 1.

  In the present specification, a position (order) on SEQ ID NO: 1 in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 corresponds to a position corresponding to the position corresponding to the primary structure of the sequence (alignment). , A position corresponding to the position of SEQ ID NO: 1.

Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In this specification, by using default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php/lang=ja) of DNA Database of Japan (DDBJ), Compare the primary structure of the sequences.

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

(1.2.3)
More preferably, the variant of DNA polymerase belonging to Family B having reduced base analog detection activity used in the nucleic acid amplification method of the present invention is 7, 36 of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or It is a polypeptide comprising an amino acid sequence having at least one amino acid modification among amino acids corresponding to the 93rd amino acid.

  In the polypeptide, when the amino acid is modified at one position, it is preferable that tyrosine (Y) as the seventh amino acid is substituted with a nonpolar amino acid, specifically, Y7A, Y7G, Y7V, Y7L. An amino acid substitution selected from the group consisting of Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C is exemplified. As another example, proline (P), which is the 36th amino acid, is preferably substituted with a positively charged polar amino acid, and specific examples include P36H, P36K, or P36R amino acid substitution. As another example, valine (V), which is the 93rd amino acid, is preferably substituted with a polar amine acid having a positive charge. Specifically, amino acid substitution of V93Q, V93K, or V93R is exemplified. .

  More preferably, it is any one selected from the group consisting of Y7A, P36H, P36K, P36R, V93Q, V93K, and V93R.

  In the polypeptide, when there are two amino acid modifications, preferred mutants include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K. Preferred examples include Y7A / P36H or Y7A / V93K, but are not limited thereto.

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

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

  Said modification to the 3'-5 'exonuclease region can consist of substitutions, deletions or additions. Modifications to amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 1 are shown. More preferably, those obtained by modifying at least one of amino acids corresponding to positions 141, 142, 143, 147, 210, 311 in SEQ ID NO: 1 are included.

Specific examples of the modification include any one of amino acid modifications selected from D141A / E143A, I142R, N210D, and Y311F. These modified forms are deficient in 3′-5 ′ exonuclease activity due to the modification.
In addition, (exo (−)) DNA polymerase deficient in 3′-5 ′ exonuclease activity includes a complete lack of activity, for example, 50% (preferably 20%, preferably Preferably 10%, more preferably 5%, more preferably 1%, more preferably 0.1%, more preferably 0.05%, more preferably 0.03%) or less exonuclease activity Refers to the DNA polymerase produced.

  Another specific example of the modification includes any one in which the amino acid modification is selected from H147E and H147D. These modified forms are DNA polymerases that have improved PCR efficiency while maintaining exonuclease activity by the modification.

Methods for producing 3′-5 ′ exonuclease DNA polymerase and methods for analyzing 3′-5 ′ exonuclease activity 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.
For example, it is disclosed in US Pat. No. 6,946,273. A DNA polymerase with improved PCR efficiency refers to a modified DNA polymerase in which the amount of PCR product is increased compared to the parent enzyme. A method for analyzing whether the amount of the PCR product is increased as compared with the parent enzyme is described in Japanese Patent No. 3891330.

(1.3) PCNA
PCNA is used in the nucleic acid amplification method of the present invention.
PCNA is an abbreviation for Proliferating Cell Nuclear Antigen (Proliferation Nuclear Antigen), and its origin is not particularly limited, and examples include PCNA isolated from bacteria of the genus Pyrococcus and Thermococcus.
As PCNA derived from the genus Pyrococcus, Pyrococcus furiosus, Pyrococcus sp. Including, but not limited to, PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii.
PCNA derived from the genus Thermococcus includes Thermococcus kodakaraensis, Thermococcus gorgonaris, Thermococcus literalis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, PCNA isolated from KS-1, Thermococcus celler, or Thermococcus sicili.
Known amino acid sequences include those derived from Pyrococcus furiosus (SEQ ID NO: 12) (hereinafter also referred to as Pfu-PCNA), and those derived from Thermococcus kodakaraensis KOD-1 (SEQ ID NO: 11) ( Hereinafter, it is also expressed as KOD-PCNA).

  DNA replication is initiated when the double-stranded structure at the origin of replication is broken by a DNA helicase. Single-stranded DNA-binding protein binds to the unfolded DNA to stabilize the single strand, and primase for synthesizing a primer on each strand works. Next, replication factor Replication Factor C (abbreviated as RFC) recognizes and binds to the primer, and induces PCNA on the DNA strand. PCNA serves as a clamp to keep the DNA polymerase on the DNA strand, and the DNA polymerase complexed with PCNA synthesizes the nascent strand.

  PCNA used in the present invention preferably exhibits amplification enhancing activity. PCNA usually forms a multimer and has a ring-like structure. Passing DNA through the ring structure of a PCNA multimer is referred to as “loading DNA”, and usually in conjunction with RFC, PCNA can only be loaded into DNA. A mutant that exhibits amplification enhancing activity refers to a mutant that loads DNA alone, alters the site involved in PCNA multimer formation, and destabilizes multimer formation. Mutants that are easy to pass inside.

  The site where PCNA is related to multimer formation is PCNA (SEQ ID NO: 11) derived from Thermococcus kodakaraensis, and PCNA of Pyrococcus furiosus (SEQ ID NO: 12) is an N consisting of amino acids 82, 84 and 109. Examples thereof include a terminal region and a C-terminal region consisting of amino acids 139, 143 and 147. The N-terminal region is positively charged, the C-terminal region is negatively charged, and multimers are formed by interaction.

(1.3.1) Amplification enhancing activity The amplification enhancing activity can be evaluated by PCR. By adding PCNA to be evaluated to a normal PCR reaction solution containing DNA as a template, buffer material, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, and comparing the amount of amplification with that without PCNA The amplification enhancing activity can be confirmed. This makes it possible to confirm whether PCNA can be loaded into DNA alone.

In the present specification, the amplification enhancing activity is evaluated using a mutant of a DNA polymerase belonging to Family B having a reduced base analog detection activity in a reaction system containing dUTP.
Specifically, the following method is used.
(I) PCR
KOD-Plus-Ver. 2 (manufactured by Toyobo) 10 × PCR Buffer attached, 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, SEQ ID NO: 15 and 16, for 2.8 kb amplification, SEQ ID NO: 17 and 18 for amplification of 3.6 kb), 10 ng of human genomic DNA (Roche 250 μg of PCNA to be evaluated in a 50 μl reaction solution containing 1U KOD DNA polymerase V93K mutant (archaebacterium DNA polymerase mutant having reduced base analog detection activity) mixed with 0.8 μg of KOD antibody. And after a pre-reaction at 94 ° C. for 30 seconds, 98 ° C., 10 seconds → 6 ℃, 30 seconds → 68 ℃, 1 min / kb (1 minute 30 seconds for amplification of 1.3 kb, 3 minutes for amplification of 2.8 kb, 4 minutes for amplification of 3.6 kb) Perform PCR.
(Ii) Analysis of PCR product After completion of the reaction, 5 μl of the reaction solution is subjected to agarose electrophoresis, stained with ethidium bromide, and amplified by comparing the amplified DNA fragment with that without addition of PCNA under ultraviolet irradiation. Activity can be evaluated. Addition of PCNA with high amplification enhancing activity increases the amplification amount.

  Even if wild-type PCNA or other PCNA that cannot be loaded into DNA alone is added, the PCR amplification amount does not change, but rather the amplification amount tends to decrease. On the other hand, a mutant that can be loaded into DNA alone can obtain an amplification amount superior to that without PCNA addition or with wild-type PCNA addition.

(1.4) PCNA mutation site (1.4.1)
PCNA used in the nucleic acid amplification method of the present invention may be a mutant exhibiting amplification enhancing activity. Examples of such mutants include mutants represented by any of the following (b1) to (b3).
(B1) In the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 12, it has at least one modification among the amino acid residues present at the position of the group represented by (x) and (y) below, and is amplified A polypeptide exhibiting potentiating activity.
(X) In the PCNA represented by the 82, 84, 109th amino acid residue group (y), 139, 143, 147th amino acid residue group (b2) (b1), further in (x) and (y) A polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added at a site other than the group shown and having amplification enhancing activity.
(B3) It comprises an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 11 or 80% or more identity with the amino acid sequence represented by SEQ ID NO: 12, and has amplification enhancing activity Polypeptide.

In the polypeptide (b2), one or several amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 11 or 12 as long as the amplification enhancing activity is retained. (Hereinafter, these are collectively referred to as “mutation”.) A polypeptide comprising an amino acid sequence.
One or several mutations include restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New (York) can be used by introducing a mutation into the DNA encoding the PCNA of the present invention described later. Variant PCNA can also be obtained by other methods such as ultraviolet irradiation. Variant PCNA includes naturally occurring variants (for example, single nucleotide polymorphisms) such as cases based on individual differences of microorganisms holding PCNA, differences in species or genera.

  The polypeptide (b3) is a polypeptide comprising an amino acid sequence having an identity of 80% or more compared to the amino acid sequence shown in SEQ ID NO: 11 or 12 as long as the amplification enhancing activity is retained. Preferably, the identity between the amino acid sequence of the PCNA of the present invention and the amino acid sequence shown in SEQ ID NO: 11 or 12 is 85% or more, more preferably 88% or more, still more preferably 90% or more, and even more. Preferably it is 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Such a polypeptide comprising an amino acid sequence having a certain identity or more can be prepared based on the known genetic engineering techniques as described above. For example, in the PCNA represented by the above (b1), those further modified at sites other than the groups represented by (x) and (y) and retaining amplification enhancing activity can be mentioned.

(1.4.2)
More preferred as PCNA for use in the nucleic acid amplification method of the present invention is that the amino acid corresponding to position 143 of SEQ ID NO: 11 or 12 is changed to a basic amino acid, and both positions 82 and 143 are changed to neutral amino acids. Or the 147th modified to a neutral amino acid, or the 109th and 143th modified to a neutral amino acid.
Examples of neutral amino acids of the present invention include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine. Preferably, alanine has the least influence on the three-dimensional structure of the peripheral site of the substitution site. Examples of basic amino acids include arginine, histidine and lysine as long as they are natural. Arginine is preferable.

  Further, PCNA used in the nucleic acid amplification method of the present invention may be a modified methionine corresponding to the 73rd position in order to increase the expression level. More preferably, it is modified to M73L, but is not limited thereto.

(1.4.3)
The PCNA used in the nucleic acid amplification method of the present invention is also applicable to PCNA other than the PCNA whose sequence is specifically presented herein. For example, as shown in FIG. 13, in PCNA other than PCNA shown in SEQ ID NOs: 11 and 12, a region relating to multimer formation consisting of amino acids 82, 84, 109, 139, 143, and 147 of SEQ ID NO: 11 Indicates the corresponding area.

More preferably, the mutant that exhibits amplification enhancing activity (PCNA mutant that is loaded alone into DNA) is involved in PCNA multimer formation.
(A) N-terminal region consisting of amino acids corresponding to positions 82, 84 and 109, or (b) C-terminal region consisting of amino acids corresponding to positions 139, 143 and 147, having at least one modification and no RFC Both include mutants that load into DNA and promote the elongation reaction of DNA polymerase.

  More preferably, the PCNA variant described in WO2007 / 004654, a sequence in which the 147th amino acid residue is changed to alanine (D147A), a sequence in which the 82nd and 143rd amino acid residues are changed to alanine ( R82A / D143A or R82A / E143A), the 109th and 143rd amino acid residues are changed to alanine (R109A / D143A, or R109A / E143A), and the like. It is not a thing.

  The PCNA used in the nucleic acid amplification method of the present invention may be either a monomer or a multimer composed of the monomer, and both forms may be mixed.

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

(1.5) Method for Introducing Amino Acid Modification A method for modifying DNA polymerase and / or PCNA used in the nucleic acid amplification method of the present invention has already been established in the art. Therefore, the modification can be performed according to a known method, and the mode is not particularly limited.

As one embodiment of a method for introducing an amino acid modification, a site-directed mutagenesis method based on the inverse PCR method can be used. Hereinafter, the modification of DNA polymerase will be described as an example.
KOD-Plus-Mutageness Kit (manufactured by Toyobo) denatures a plasmid into which a target gene (wild-type DNA polymerase gene) has been inserted, anneals a mutation primer to the plasmid, and then KOD DNA polymerase (2) repeat the cycle of (1) 15 times, (3) selectively cleave only the plasmid as a template using the restriction enzyme DpnI, (4) newly synthesized (5) A kit that can transform a cyclized gene into Escherichia coli and obtain a transformant having a plasmid introduced with the target mutation. is there.
In the above method, the wild-type DNA polymerase gene can be prepared by synthesizing DNA corresponding to the amino acid sequence when the amino acid sequence is known. Alternatively, it can be obtained by cloning from a biological species derived from the DNA polymerase and / or PCNA.

  Next, the modified DNA polymerase gene obtained by the above method or the like is transferred to an expression vector as necessary. For example, Escherichia coli as a host is transformed with the expression vector and then contains a drug such as ampicillin. Apply to agar medium to form colonies. The colony is inoculated in a nutrient medium such as LB medium or 2 × YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted. A vector derived from pBluescript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the host-derived polymerase, and the DNA polymerase activity is measured.

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

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

(2) Nucleic Acid Amplification Reagent One embodiment of the present invention is a reagent for performing a nucleic acid amplification reaction, including the following (a) to (e). In this specification, the aspect of the reagent is not particularly limited, and may be in the form of a kit.
(A) a variant of a DNA polymerase belonging to family B having reduced base analog detection activity (b) PCNA
(C) Primer pair (d) DNA synthesis substrate (deoxynucleotide triphosphate (dNTP))
(E) Buffer solution containing Mg ions If necessary, other reagents may be included.
This reagent can be used to amplify nucleic acids with a reaction composition that includes a base analog.

  In the nucleic acid amplification reagent of the present invention described above, the DNA synthesis substrate is typically composed of four types of deoxynucleotide triphosphates, dATP, dCTP, dGTP, and dTTP. , DCTP, dGTP, and dTTP may also contain deoxynucleotide triphosphates such as dUTP and dITP.

  In the nucleic acid amplification reagent of the present invention described above, the primer is typically composed of nucleotides consisting of four bases of adenine, cytosine, guanine, and thymine. However, in the nucleic acid amplification method of the present invention, adenine, cytosine, guanine, It may contain nucleotides other than thymine, such as uracil and inosine.

(2.1) dUTP / UDG contamination removal method In the dUTP / UDG contamination removal method, dUTP (deoxyuridine) is used as a DNA synthesis substrate. The route for obtaining dUTP is not particularly limited, but commercially available products can be used. Although the concentration of dUTP in the reaction solution is not particularly limited, the lower limit is preferably 0.5 μM or more, more preferably 50 μM or more, 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 preferable upper limit is 1 mM or less, more preferably 0.6 mM or less.
Moreover, dTTP and dUTP may be mixed from the viewpoint of PCR efficiency. The ratio of dTTP and dUTP is preferably 100: 1 to 1: 100. More preferably, it is 10: 1 to 1:10, and further preferably 1: 1, but is not limited thereto.

Hereinafter, the present invention will be specifically described by way of examples. The description of the following examples does not limit the invention in any way.
Example 1
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: 25) (pKOD) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBluescript was used. The mutation was introduced by using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.

(Example 2)
Preparation of Pfu mutant A plasmid containing a modified thermostable DNA polymerase gene derived from Pyrococcus furiosus was prepared.
As a DNA template used for mutagenesis, a modified thermostable DNA polymerase gene (SEQ ID NO: 26) (pPfu) derived from Pyrococcus furiosus cloned in pBluescript was used. For mutation introduction, KOD-Plus-Mutageness
Using Kit (manufactured by Toyobo), the method was performed according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.

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

(Example 3)
(Example 3-1)
Production of modified thermostable DNA polymerase The cells obtained in Examples 1 and 2 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin in advance in this medium. Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and heparin sepharose chromatography were performed. Finally, storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% nonidet P40, 50% glycerin) to obtain a modified thermostable DNA polymerase.

(Example 3-2)
Evaluation of decreased base analog detection activity of modified thermostable DNA polymerase The detection activity of uracil was measured according to <Method for measuring base analog detection activity> described in (1.1.1) above.

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

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

FIG. 2 shows that wild type (KOD), 3′-5 ′ exonuclease was deleted by mutation of N210D, KOD (KOD N210D) and I142R were deleted by mutation of N210D, and 3′-5 ′ exonuclease was deleted. Three types of KOD (KOD I142R) were prepared, and each of KOD and KOD N210D was added with any of the mutations of V93K, V93R, V93Q, and Y7A (8 types in total). Those with any mutation were prepared (two types). In addition, KOD in which 3′-5 ′ exonuclease was deleted by double mutation of D141A / E143A and further, any one of mutations of V93K and Y7A (2 types) were prepared. Using these 15 species in total, water or dUTP (Roche) prepared to a final concentration of 0.5, 5, 50, 100, and 200 μM was added to the reaction system, and PCR reaction was performed. It is the result of electrophoresis of the product.
In the wild type, when dUTP was added, PCR products could not be confirmed (lanes 2-6), and when dUTP was not added (lane 1), amplification products were confirmed. In contrast, in the mutant type, amplification products were also confirmed when dUTP having a final concentration of 0.5 μM was added. It was shown that the mutant type lacking the 3′-5 ′ exonuclease (N210D, I142R) has a higher dUTP resistance concentration than the mutant type lacking the 3′-5 ′ exonuclease. In addition, even mutants having the same Y7A mutation showed resistance to higher dUTP concentrations in combination with mutations lacking the 3′-5 ′ exonuclease (N210D, I142R). Similarly, with respect to V93Q and V93Q / N210D, V93Q / N210D lacking the 3′-5 ′ exonuclease was shown to be resistant to higher dUTP concentrations. From these facts, it can be seen that, in addition to the mutation to the uracil-binding pocket, the resistance to dUTP is improved by adding a mutation that gives a deficiency to the 3′-5 ′ exonuclease.

Similar experiments were performed with other KOD mutants, and the concentrations of dUTP in which amplification was observed in the column of dUTP resistance in Table 3 were summarized. 0 indicates that PCR was not possible with 0.5 μM dUTP. Similarly, 0.5, 5, and 100 were amplified with 0.5, 5, and 100 μM dUTP, respectively, but one step higher each. No amplification was seen with 5, 100, 200 μM dUTP. > 200 indicates that amplification was observed even when 200 μM was added.
As a result, uracil-binding pocket mutations of Y7, P36, and V93 tend to increase the dUTP resistance concentration, and it was confirmed that exo (-) tends to have a higher dUTP resistance concentration than exo (+).

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

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

(Example 3-3)
Evaluation of long-chain DNA amplification using a modified thermostable DNA polymerase Whether long-chain DNA exceeding 1.3 kB can be amplified even under the condition of dUTP alone without dTTP using the KOD mutant described in Table 2 Examined. Table 2 shows both amino acid mutations in the exo region and amino acid mutations related to uracil bonds. Regarding amino acid mutations in the exo region, exo (+) is a mutation that retains 3'-5 'exonuclease including wild type, and exo (-) is missing 3'-5' exonuclease. It shows that it is a lost mutation.
In PCR containing dUTP, 1.3 kbp and 2.8 kbp and 3.6 kbp amplification of Human β-globin were compared. At this time, each enzyme used was a KOD mutant mixed with 1 μg of KOD antibody per 1U.

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

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

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

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

As for the amount of amplification, a comparison between V93K and P36H, a comparison between Y7A / V93K and Y7A / P36H, a comparison between V93K and P36R, and a comparison between Y7A / V93K and Y7A / P36R, respectively, It was confirmed that the mutation to position P36 has a larger amount of amplification and can be amplified up to a long target. In addition, the amount of amplification was higher in the double mutation into the uracil binding pocket than in the single mutation. These mutants were able to amplify long chain lengths that could not be amplified with Taq (FIG. 3).
In addition, wild-type DNA polymerase subjected to mutations V93K, Y7A / V93K, P36R, Y7A / P36R, P36H and Y7A / P36H and exo (−) mutants N210D, I142R, and D141A / E143A Comparing the mutants with V93K, Y7A / V93K, P36R, Y7A / P36R, P36H and Y7A / P36H mutations, the amount of amplification was greater when the exo (-) DNA polymerase was mutated. Results were obtained (FIGS. 3 and 4).
Furthermore, V93K, Y7A / V93K, V93K, Y7A / V93K, P36R, Y7A / P36R, P36H and Y7A / P36H mutants with improved PCR efficiency, and V147K, Y7A / V93K, When the P36R, Y7A / P36R, P36H and Y7A / P36H mutations were compared, the H147E mutant showed a higher amplification amount. This is because the modification effect of H147E is independent of the modification to the uracil-binding pocket, and it is considered that the amount of amplification increased due to the effect of modification of H147E. (FIGS. 3 and 4)

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

Example 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, PCNA (SEQ ID NO: 27) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBluescript was used. The mutation was introduced by using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5α was transformed with the obtained plasmid and used for enzyme preparation.

(Example 4-2)
Preparation of Pfu-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Pyrococcus furiosus was prepared.
As a DNA template used for mutagenesis, PCNA (SEQ ID NO: 28) (pPfuPCNA) derived from Pyrococcus furiosus cloned in pBluescript was used. The mutation was introduced by using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5α was transformed with the obtained plasmid and used for enzyme preparation.

  The plasmids prepared in Example 4 are shown in Table 4.

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

(Example 4-4)
Evaluation of PCNA To confirm the effect of PCNA, KOD-PCNA mutants (M73L, M73L / E143R, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, M73L / E143F, M73L / E143A) were used. 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. At this time, the DNA polymerase used was the KOD V93K mutant among the mutants prepared in Example 3. For PCR, KOD-Plus-Ver. 2 (manufactured by Toyobo Co., Ltd.) using attached Buffer, MgSO ₄ , 1 × PCR Buffer, and dNTPs (dATP, dUTP, dCTP, dGTP) in which 0.2 mM dTTP is replaced with dUTP, 1.5 mM MgSO ₄ , 15 pmol Primer (SEQ ID NOs: 13 and 14), 10 ng of human genomic DNA (Roche), 50 μl of a reaction solution containing 1 U of enzyme mixed with an antibody was reacted at 94 ° C. for 2 minutes, followed by 98 ° C., 10 seconds → PCR was performed using PCR system GeneAmp9700 (Applied Biosystem) on a schedule of 35 cycles of 60 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds. After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

  FIG. 5 shows the results of electrophoresis of the products obtained by adding 250 ng of various PCNA mutants and performing PCR reaction. The PCNA mutants used were 7 types in total: M73L, M73L / E143R, M73L / E143A, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, and M73L / E143F.

In the case where no PCNA was added (lane 8) or when the KOD-PCNA M73L mutant was used as PCNA (lane 1), only a small band was found, but other KOD-PCNA mutants. When was added, the amount of amplification increased and a clearer band was confirmed.
PCNA forms a multimer and promotes a nucleic acid synthesis reaction. Usually, however, the reaction cannot proceed without loading into DNA without the action of RFC. M73L / E143R, M73L / E143A, M73L / R109A / E143A, M73L / D147A, M73L / R82A / E143A, and M73L / E143F are changes to sites involved in multimer formation, and multimer formation is moderately weakened. Therefore, it is conceivable that PCNA can be loaded onto DNA and the amount of PCR amplification has been improved (FIG. 5).

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

(Example 5)
Amplification evaluation of long-chain target using mutant PCNA Long-chain target amplification (HBg 8.5 kb) was performed in a PCR reaction system containing dUTP and evaluated with and without mutant PCNA.
As the enzyme, KOD Y7A / V93K mutant mixed with 0.8 μg of KOD antibody per U and KOD Y7A / P36H / N210D mutant were used, and 8.5B PCR of HBg was performed to compare the difference in amplification.
As above, KOD PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo) attached buffer, MgSO ₄ , 1 × PCR buffer, 1.5 mM or 2.0 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 19 and 20), 1 U each enzyme mixed with antibody In each of 50 μl of the reaction solution, dNTPs in which dTTP is replaced with dUTP (dATP, dUTP, dCTP, dGTP) was added to a concentration of 0.2 mM, and 50 ng of human genome (Roche) was used as a template. Using. Moreover, what added 250 ng of KOD-PCNA D147A mutant was also implemented. PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 94 cycles of 94 ° C., 2 minutes, and 98 cycles of 10 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 9 minutes.

FIG. 6 shows the result of electrophoresis of a product obtained by amplifying a 8.5 kb long-chain target containing dUTP with / without mutant PCNA (KOD-PCNA M73L / D147A). The first and second lanes of each photograph show no PCNA, and the third and fourth lanes show that PCNA was added. The first and third lanes show amplification of 1.5 mM MgSO ₄ , and the second and fourth lanes show amplification of 2 mM MgSO ₄ .
As a result, there was no mutant PCNA, and although KOD N210D Y7A / P36H did not show amplification even when MgSO ₄ was 1.5 mM or 2.0 mM, a firm band of 8.5 kb was amplified by adding mutant PCNA. (Fig. 6).

(Example 6)
Evaluation of amplification from blood using mutant PCNA Amplification of targets of various lengths was performed from blood in a PCR reaction system containing dUTP, and evaluation was performed with and without mutant PCNA.
For the enzyme, KOD Y7A / V93K mutant mixed with 0.8 μg of KOD antibody per 1 U, KOD Y7A / P36H / N210D mutant, antibody mixed Taq DNA polymerase (Taq DNA polymerase (manufactured by Toyobo)) and Anti-Taq HBg 482 bp, 1.3 kb, 2.8 kb, 3.6 kb, and 5.7 kb were used to compare the differences in amplification using High (mixed in equal amounts of Toyobo).
As above, KOD PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer, MgSO ₄ attached thereto, 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 21 and 22 for amplification of 482 bp, SEQ ID NO: 13 for amplification of 1.3 bp) And 14, 2.8 kb amplification is SEQ ID NO: 15 and 16, 3.6 kb amplification is SEQ ID NO: 17 and 18, 5.7 kb amplification is SEQ ID NO: 23 and 24), each containing 1 U of enzyme mixed with antibody In a 50 μl reaction solution, normal dNTPs (dATP, dTTP, dCTP, dGTP) or dNTPs in which dTTP is replaced with dUTP (dATP, dUTP, dCTP, dGTP) added to 0.2 mM are used. As a template, 1 μl of blood was used. Moreover, what added 250 ng of KOD-PCNA D147A mutant was also implemented. 94 ° C., 2 minutes pre-reaction, 98 ° C., 10 seconds → 65 ° C., 10 seconds → 68 ° C., 1 minute / kb (482 bp amplification is 1 minute, 1.3 bp amplification is 1.5 minutes, 2 minutes PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 35 cycles of 3 minutes for .8 kb amplification, 4 minutes for 3.6 kb amplification, and 6 minutes for 5.7 kb amplification).
Taq DNA polymerase PCR was performed by adding ordinary dNTPs (in a 50 μl reaction solution containing Buffer (Toyobo product) attached to 1 × BlendTaq, 10 pmol primer (same as above), 2.5 U enzyme mixed with antibody. dATP, dTTP, dCTP, dGTP) or dNTPs in which dTTP is replaced with dUTP (dATP, dUTP, dCTP, dGTP) were added to 0.2 mM, and 1 μl of blood was used as a template. Moreover, what added 250 ng of KOD-PCNA M73L / E143R mutant was also implemented. PCR at a schedule of 94 ° C, 2 minutes pre-reaction, followed by 35 cycles of 94 ° C, 30 seconds → 65 ° C, 30 seconds → 68 ° C, 1 minute / kb (same as above)
PCR was performed using system GeneAmp9700 (Applied Biosystem).
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 7 shows the results obtained by amplifying a target having various lengths with and without mutant PCNA (KOD-PCNA M73L / D147A) in a reaction solution containing dTTP or dUTP, and electrophoresing the obtained product. In each photograph,-indicates no PCNA, and + indicates that PCNA was added. The first lane is 482 bp, the second lane is 1.3 kb, the third lane is 2.8 kb, the fourth lane is 3.6 kb, and the fifth lane is 5.7 kb.
As a result, when the amount of amplification is compared between dTTP and dUTP, it can be seen that a significant inhibition is observed with dUTP. In Taq DNA polymerase, 482 bp was amplified with dTTP, but 482 bp was not confirmed with dUTP. In the KOD Y7A / V93K mutant and the KOD Y7A / P36H / N210D mutant, good amplification was observed for all targets with dTTP, but almost no amplification was observed with a target of 482 bp or more with dUTP. As a result of adding mutant PCNA there, there was not much difference in Taq, but KOD Y7A / V93K mutant and KOD Y7A / P36H / N210D mutant could not be amplified by adding mutant PCNA. A target of 8 kb or more could be amplified (FIG. 7). It was shown that the addition of the PCNA mutant can achieve the same amplification as dTTP in the presence of dUTP. It was also shown that PCR can be performed directly from a crude sample by adding PCNA.

(Example 7)
Amplification from blood by addition of PCNA using reaction solution containing dUTP It was evaluated whether crude (blood) resistance was improved by addition of PCNA in a PCR reaction system containing dUTP.
As the enzyme, the KOD Y7A / V93K mutant was used, and the difference in the amount of HBg 482 bp was compared. KOD-PCNA M73L / D147A was used as PCNA.

PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo) attached buffer, MgSO ₄ , 1 × PCR buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NOs: 21 and 22), dNTPs (dATP, dUTP with 2 mM dTTP replaced with dUTP) , DCTP, dGTP), in a 50 μl reaction solution containing 1 U enzyme mixed with KOD antibody, 0.002%, 0.02%, 0.2%, 2%, 5% In addition to 10%, no PCNA and 250 ng of KOD-PCNA M73L / D147A were compared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 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 amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

In FIG. 8, blood is used as a sample, and blood is added to a reaction solution containing dUTP in a proportion of 0.002%, 0.02%, 0.2%, 2%, 5%, 10%, The result of having electrophoresed the product obtained by performing PCR reaction with and without the addition of KOD-PCNA M73L / D147A is shown. In each photograph,-indicates no PCNA, and + indicates that PCNA was added.
As a result, in KOD Y7A / V93K without PCNA addition, when 10% of blood was added, inhibition was not observed and amplification was not confirmed. However, when PCNA mutant was added, a firm band was confirmed even if 10% of blood was contained. (FIG. 8). The PCNA mutant was also shown to be effective in PCR in the presence of dUTP.

(Example 8)
Amplification from blood by addition of PCNA using reaction solution containing dUTP As in FIG. 8, it was evaluated whether the crude (blood) resistance was improved by addition of PCNA in the PCR reaction system containing dUTP.
As the enzyme, KOD Y7A / V93K mutant, KOD Y7A / P36H / N210D was used, and the difference in the amount of 1.3 kb amplification of HBg was compared. PCNA has Pfu-PCNA
M73L / D147A was used.

PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer, MgSO ₄ attached, 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 13 and 14), dNTPs (dATP, dUTP in which 2 mM dTTP was replaced with dUTP) , DCTP, dGTP), 0.02%, 0.2%, 0.5%, 2%, 5% % And 10%, PCNA-free and Pfu-PCNA
What added 250 ng of M73L / D147A was compared. The reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1.5 minutes. It was.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

In FIG. 9, blood is used as a sample, and the reaction solution is prepared so that the ratio of blood in the reaction solution is 0.02%, 0.2%, 0.5%, 2%, 5%, 10%. The results of electrophoresis of the product obtained by adding Pfu-PCNA M73L / D147A to KOD Y7A / V93K mutant and KOD Y7A / P36H / N210D and performing PCR reaction are shown. In each photograph,-indicates no PCNA, and + indicates that PCNA was added.
As a result, inhibition was not achieved without addition of PCNA, and amplification was not confirmed from blood. However, a solid band was confirmed even when 10% of blood was added to the PCNA mutant (FIG. 9). Similar to FIG. 8, the PCNA mutant was shown to be effective in PCR in the presence of dUTP.

Example 9
It was also evaluated whether the crude (plant) tolerance was improved by adding PCNA even in a PCR reaction system containing amplified dUTP from plant lysate by adding PCNA.
For comparison, the KOD Y7A / P36H / N210D mutant and Taq polymerase were used, and the difference in amplification amount of rbcL 1.3 kb was compared. KOD-PCNA M73L / D147A was used as PCNA.

As a template, a rice leaf 3 mm square was added to 100 μl of Buffer A (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) and heat-treated at 95 ° C. for 10 minutes, and used as a lysate.
The PCR of KOD Y7A / P36H / N210D was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using Buffer, MgSO ₄ attached, 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NOs: 29 and 30), dNTPs (dATP, dUTP in which 2 mM dTTP was replaced with dUTP) , DCTP, dGTP), plant lysate in 50 μl reaction solution containing 1 U enzyme mixed with KOD antibody to 2%, 4%, 8%, 16% of the reaction solution, without PCNA And KOD-PCNA M73L / D147A added with 250 ng were compared. The reaction was carried out using PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 94 cycles at 94 ° C. for 2 minutes followed by 35 cycles of 98 ° C., 10 seconds → 65 ° C. 30 seconds → 68 ° C., 1.5 minutes. .
Taq DNA polymerase was manufactured by Toyobo, and mixed with Anti-Taq High (manufactured by Toyobo). The reaction was performed by adding 50 μl of Buffer attached to 1 × BlendTaq, 10 pmol primer (same as above), dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, and 2.5 U enzyme mixed with the antibody. In the reaction solution, plant lysate was added at 2%, 4%, 8% and 16% with respect to the reaction solution, and those without PCNA and 250 ng of KOD-PCNA M73L / D147A were compared. The reaction was performed at 94 ° C. for 2 minutes, followed by PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 ° C., 30 seconds → 65 ° C., 30 seconds → 68 ° C., and 1 and 5 minutes repeated 35 cycles. went.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

In FIG. 10, plant lysate was used as a sample, and the reaction solution was prepared so that the proportion of lysate in the reaction solution was 2, 4, 8, 16%, and KOD-PCNA M73L / D147A was added to various enzymes. The result of carrying out PCR reaction and electrophoresis of the obtained product is shown. The enzymes used were KOD Y7A / P36H / N210D mutant and Taq polymerase in total.
1, 2, 8, and 16 of each photograph show the percentage (%) of added plant lysate,-indicates no PCNA, and + indicates that PCNA was added.
As a result, in KOD Y7A / P36H / N210D without PCNA addition, when 4% or more of plant lysate was added, inhibition was not observed and amplification was not confirmed. Band was confirmed (FIG. 10). It was shown that the PCNA mutant is effective even in a reaction solution containing dUTP.
On the other hand, in Taq polymerase, which is not a DNA polymerase belonging to family B, no change in crude (plant) resistance was observed with the addition of PCNA. It is shown that the crude resistance does not change even when a PCNA mutant is added to Taq polymerase.

(Example 10)
PCR from body tissues (nail, hair, oral mucosa)
We examined whether PCR is possible using nails, hair, and oral mucosa as a template. A piece of nail cut with a nail clipper and one piece of hair were added to 180 μl of 50 mM NaOH, crushed by heat treatment at 95 ° C. for 10 minutes, and then neutralized by adding 20 μl of 1M Tris-HCl (pH 8.0). The supernatant was used as a template. The oral mucosa was obtained by suspending mucosa collected with a cotton swab in 200 μl of water as a template.
Using KOD (wild-type), KOD Y7A / V93K mutant, and KOD Y7A / P36H / N210D mutant mixed with 0.8 μg KOD antibody per 1 U per unit, PCR for HBg 482 bp was performed and the difference in amplification was compared. did.
As above, PCR was performed using KOD-Plus-Ver. 2 (manufactured by Toyobo), using the attached Buffer, MgSO ₄ , 1 × PCR Buffer, and 1.5 mM MgSO ₄ , 15 pmol primer (SEQ ID NO: 21 and 22), 50 μl containing 1 U of each enzyme mixed with the antibody In the reaction solution, 0.2 mM of normal dNTPs (dATP, dTTP, dCTP, dGTP) added and dNTPs (dATP, dUTP, dCTP, dGTP) in which dTTP is replaced with dUTP are added to 0.2 mM. 1 μl of the above sample was used as a template. Moreover, what added 250 ng of KOD-PCNA M73L / E143R mutant which is a PCR enhancement factor to each reaction liquid was also implemented. PCR was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of repeating 94 cycles of 94 ° C., 2 minutes and 98 cycles of 10 ° C., 10 seconds → 65 ° C., 10 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 amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 11 shows results obtained by performing PCR reaction with various DNA polymerases in the presence of KOD-derived PCNA mutant (M73L / E143R) using nail, hair and oral mucus as samples, and electrophoresis of the obtained products. Indicates. The DNA polymerase used was a total of three types: KOD (wild type) and two KOD mutants (Y7A / V93K, Y7A / P36H / N210D). The left side of each photograph uses dTTP, and the right side uses dUTP. When 1 lane is a nail sample, 2 lane is hair, 3 lane is oral mucosa. Each “+” lane is in the presence of KOD-derived PCNA mutant (M73L / E143R), and “−” indicates no PCNA.
As a result, wild-type KOD DNA polymerase was not amplified in the presence of dUTP, but a band was confirmed even in the presence of dUTP in the KOD Y7A / V93K and KOD Y7A / P36H / N210D mutants having decreased base analog detection activity. (FIG. 11). As a result of adding PCNA, it was confirmed that the amplification amount was improved as compared with the case where PCNA was not added.

  Other variants of KOD (P36H, P36K, P36R, V93K, V93R, Y7A / P36H, Y7A / P36R, Y7A / V93R, P36H / H147E, P36K / H147E, P36R / H147E, V93K / H147E, V93R / H147E, V93R / H147E / P36H / H147E, Y7A / P36R / H147E, Y7A / V93K / H147E, Y7A / V93R / H147E, P36H / N210D, P36K / N210D, P36R / N210D, V93K / N210D, V93R / N210D, Y7A / D36R, Y7A / P36R / V93K / N210D, Y7A / V93R / N210D, P36H / I142R, P36R / I142R, V93K / I142R, V93R / I142R, Y7A / P36H / I14 R, Y7A / P36R / I142R, P36H / D141A / E143A, P36R / D141A / E143A, V93K / D141A / E143A, V93R / D141A / E143A, Y7A / P36H / D141A / E143A, Y7A / P36R / 14) Under these reaction conditions, amplification from body tissues (nail, hair, oral mucosa) was confirmed, and a firm band could be confirmed even in the presence of dUTP. In addition, when KOD-PCNA mutants (M73L / D147A, M73L / R109A / E143A, M73L / E143R) and Pfu-PCNA mutants (M73L / D143R) are added to these, the amplification amount can be confirmed as described above. It was.

(Example 11)
Amplification evaluation of bisulfite- treated DNA After bisulfite treatment, it was examined whether amplification from DNA containing uracil was possible. In order to eliminate the influence of methylation, the DNA used for bisulfite was KOD-Plus-Neo (manufactured by Toyobo), and a 17.5 kb PCR product amplified with the primers of SEQ ID NOs: 31 and 32 was used. Bisulfite was MethylEasy Xceed Rapid DNA Bisulfite Modification Kit, and PCR and bisulfite treatment were performed according to the attached instruction manual. Amplification studies are described in KOD-Plus-Ver. 2 (Toyobo Co., Ltd.) attached Buffer, using MgSO _4, 1 × PCR Buffer, and 1.5 mM MgSO _4, amplification of 15pmol primers (574 bp is amplified for SEQ ID NO: 33 and 34,519bp SEQ ID NO: 35 and 36 , 1004 bp amplification is SEQ ID NO: 35 and 37, 1561 bp amplification is SEQ ID NO: 38 and 39, 1993 bp amplification is SEQ ID NO: 38 and 40), 0.2 mM dNTPs, KOD Y7A mixed with 0.8 μg KOD antibody per U 20 ng of the above-mentioned bisulfite-treated DNA was added to 50 μl of the reaction solution containing the / V93K mutant, and the PCR enhancement factor KOD-PCNA M73L / D147A mutant was not added and 250 ng was added. PCR is 94 ° C., after 2 minutes of pre-reaction, 98 ° C., 10 seconds → 60 ° C., 10 seconds → 68 ° C., 1 minute / kb (574 bp, 519 bp, 1004 bp amplification is 1 minute, 1561 bp, 1993 bp amplification is 2 minutes) was performed using a PCR system GeneAmp 9700 (Applied Biosystem) with a schedule of repeating 30 cycles.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

  FIG. 12 shows the results of amplifying targets of various lengths using DNA after bisulfite treatment as a template and comparing the amounts of amplification with and without the addition of KOD-PCNA mutant (M73L / D147A). The left side of each photograph shows the case where the reaction was carried out without adding KOD-PCNA (M73L / D147A), and the right side was the case where the reaction was carried out with addition of KOD-PCNA (M73L / D147A). 1 lane is 574 bp, 2 lane is 519 bp, 3 lane is 1004 bp, 4 lane is 1561 bp, 5 lane is the result of amplifying a 1993 bp target. As a result, there was a target that could not be amplified such as 574 bp in the reaction in which the KOD-PCNA mutant (M73L / D147A) was not added, but in the case where the KOD-PCNA mutant (M73L / D147A) was added, all targets were solid. Amplification was confirmed. Further, the amount of amplification was higher when the KOD-PCNA mutant (M73L / D147A) was added (FIG. 12).

(Example 12)
It was evaluated whether amplified dUTP from stool and gene amplification can be performed in the presence of stool.
As the enzyme, KOD Y7A / P36H / N210D mutant and Taq polymerase were used, and the difference in amplification of about 700 bp of Salmonella invA gene was compared by real-time PCR using SYBR GREEN I and melting curves. The KOD Y7A / P36H / N210D mutant was also added with KOD-PCNA M73L / D147A added as a PCR enhancing factor.

As the inhibitor, a 10% fecal suspension was heat-treated at 95 ° C. for 10 minutes.
For the PCR of KOD Y7A / P36H / N210D, using Buffer attached to KOD Dash (manufactured by Toyobo), 1 × PCR Buffer, 50 copies of Salmonella genome, 4 pmol primer (SEQ ID NO: 41 and 42), 2 mM dTTP to dUTP In 20 μl of a reaction solution containing 0.4 U of enzyme mixed with substituted dNTPs (dATP, dUTP, dCTP, dGTP), 1/30000 SYBR GREEN I, and KOD antibody, stool was added to the reaction solution at 0, 0. 1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5%, 95 ° C., 30 seconds pre-reaction, 98 ° C., 10 seconds → 60 ℃ 10 seconds → 68 ℃, 30 seconds is repeated using LightCycler 2.0 (Roche) on a schedule of 50 cycles . 100 ng of KOD-PCNA M73L / D147A was added to the above reaction system and compared with that without PCNA.
Taq DNA polymerase was manufactured by Toyobo, and mixed with Anti-Taq High (manufactured by Toyobo). The reaction is as follows: Buffer attached to 1 × Taq (Mg attachment type), 50 copies of Salmonella genome, 4 pmol of primer (same as above), 2 mM dTTP substituted with dUTP (dATP, dUTP, dCTP, dGTP), 4 mM In 20 μl of a reaction solution containing 1 U of enzyme mixed with MgSO ₄ , 1/30000 SYBR GREEN I, antibody, feces were added to the reaction solution at 0, 0.1, 0.25, 0.5, 1.0. , 1.5, 2.0, 2.5%, 95 ° C., 30 seconds pre-reaction, 98 ° C., 10 seconds → 60 ° C. 10 seconds → 68 ° C., 30 seconds repeated 50 cycles The schedule was performed using LightCycler 2.0 (Roche).
After completion of the reaction, a target peak appearing in the latter half of 80 ° C. was confirmed by melting curve analysis.

  Table 5 shows the Cq value of Example 16.

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

FIG. 14 shows the results of melting curve analysis of the amplification product obtained using PCR in the presence of dUTP and feces. The polymerases used were KOD Y7A / P36H / N210D mutant, KOD Y7A / P36H / N210D mutant with KOD-PCNA M73L / D147A added, and Taq polymerase in total.
1 shows the result of KOD Y7A / P36H / N210D, 2 shows the result of adding KOD-PCNA M73L / D147A to KOD Y7A / P36H / N210D, and 3 shows the result of Taq polymerase.
As a result, in KOD Y7A / P36H / N210D, amplification was confirmed even in the presence of dUTP and stool, and the peak decreased with the addition of stool, but the target peak could be confirmed even when 2.5% was added. . Similarly to the case of adding PCNA, the target peak could be confirmed even with the addition of 2.5%. However, with Taq polymerase, the target peak disappeared when 0.25% was added, suggesting that it was inhibited by the influence of feces (FIG. 14).

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

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

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

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

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

Claims (11)

A method for amplifying a nucleic acid in a reaction composition containing a base analog, comprising the following (a) and (b) in the reaction composition:
(A) a DNA polymerase variant belonging to Family B having reduced base analog detection activity, wherein the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is either of the following (a1) or (a2) A polypeptide which is a variant substituted with an amino acid.
(A1) A variant in which the seventh amino acid is substituted with alanine and the 93rd amino acid is substituted with lysine, histidine or arginine
(A2) a variant in which the seventh amino acid is substituted with alanine and the 36th amino acid is substituted with lysine, histidine or arginine (b) a PCNA (Proliferating Cell Nuclear Antigen) variant, wherein SEQ ID NO: 11 Or a polypeptide having at least one modification among amino acid residues present at the position of the group represented by the following (x) and (y) in the amino acid sequence represented by SEQ ID NO: 12 and exhibiting an amplification enhancing activity .
(X) 82th, 84th and 109th amino acid residue groups (y) 139th, 143th and 147th amino acid residue groups The DNA polymerase variant is an amino acid sequence corresponding to at least one amino acid corresponding to positions 141, 142, 143, 147, 210, and 311 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The nucleic acid amplification method according to claim 1, further comprising modification. The nucleic acid amplification method according to claim 1 or 2 , wherein the PCNA mutant is any one of the following mutants (b1) to (b4) in the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 12.
(B1) Mutant in which the amino acid corresponding to the 143rd position is replaced with a basic amino acid (b2) Mutant in which both the 82nd and 143rd positions are replaced with neutral amino acids (b3) The 147th position is replaced with a neutral amino acid Mutant (b4) Mutant in which 109th and 143rd positions are both substituted with neutral amino acids PCNA mutants in the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, the following (b1) or (b2) to any one of claims 1 to 3 which is a mutant substituted to any amino acid of The nucleic acid amplification method described.
(B1) Mutant in which the 143rd amino acid is replaced with alanine or arginine (b2) Mutant in which the 147th amino acid is replaced with alanine or arginine The nucleic acid amplification method according to any one of claims 1 to 4, wherein a biological sample that has not undergone a purification step is added to a nucleic acid amplification reaction solution to amplify a target nucleic acid in the biological sample. The nucleic acid amplification method according to claim 5 , wherein the biological sample is any one selected from the group consisting of animal and plant tissues, body fluids, excreta, cells, bacteria, and viruses. A nucleic acid amplification reaction reagent comprising the following (a) to (e):
(A) a DNA polymerase mutant comprising a polypeptide which is a mutant in which the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is substituted with any of the following amino acids (a1) or (a2):
(A1) A variant in which the seventh amino acid is substituted with alanine and the 93rd amino acid is substituted with lysine, histidine or arginine
(A2) A variant in which the seventh amino acid is substituted with alanine and the 36th amino acid is substituted with lysine, histidine or arginine (b) In the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 12, a PCNA variant comprising a polypeptide having at least one modification among the amino acid residues present at the position of the group represented by x) and (y) and exhibiting an amplification enhancing activity (x) 82, 84 and 109 (Y) 139, 143 and 147th amino acid residue group (c) primer pair (d) deoxynucleotide triphosphate (dNTP)
(E) Buffer solution containing magnesium ions The DNA polymerase variant is an amino acid sequence corresponding to at least one amino acid corresponding to positions 141, 142, 143, 147, 210, and 311 of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The nucleic acid amplification reaction reagent according to claim 7 , further comprising a modification. The nucleic acid amplification reaction reagent according to claim 7 or 8 , wherein the PCNA mutant is any one of the following mutants (b1) to (b4) in the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 12.
(B1) Mutant in which the amino acid corresponding to the 143rd position is replaced with a basic amino acid (b2) Mutant in which both the 82nd and 143rd positions are replaced with neutral amino acids (b3) The 147th position is replaced with a neutral amino acid Mutant (b4) Mutant in which 109th and 143rd positions are both substituted with neutral amino acids PCNA mutants in the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, the following (b1) or to any of the claims 7-9 is mutant substituted to any amino acid of (b2) The nucleic acid amplification reaction reagent described.
(B1) Mutant in which the 143rd amino acid is replaced with alanine or arginine (b2) Mutant in which the 147th amino acid is replaced with alanine or arginine A kit comprising the nucleic acid amplification reaction reagent according to any one of claims 7 to 10 .