JP6950184B2 - PCR method - Google Patents

Description

The present invention relates to the field of nucleic acid amplification, particularly PCR (Polymerase chain reaction). More specifically, it relates to a method of shortening the PCR reaction time.

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

Although PCR has high detection sensitivity, it is necessary to carry out a thermal cycle for the reaction, and the problem is that the reaction time is long. Recently, a thermal cycler that changes the temperature at high speed has been sold, and a device for completing the reaction in a shorter time is being studied. Also, in terms of reaction, the composition and method of PCR are being studied in order to cope with high-speed cycles.

In terms of reaction, the performance of the DNA polymerase used for PCR is important for high-speed PCR. We have found that DNA polymerases belonging to Family B are more suitable for high-speed PCR than polymerases belonging to Family A such as Taq DNA polymerase (Patent Document 1). It has also been reported that the reaction time can be shortened by adding a cofactor of DNA polymerase, particularly PCNA (Proliferating Cell Nuclear Antigen) (Proliferating Cell Nuclear Antigen) (Patent Document 2).

However, despite the progress of these studies, DNA polymerase did not work well in extremely high-speed cycles, and amplification defects were occasionally found. In addition, for diagnostic purposes, the measurement sample may contain a substance that inhibits PCR, and high-speed PCR could not be performed under conditions containing such a substance. In particular, for diagnostic purposes, rapid result reporting is required, and a method for high-speed and high-efficiency DNA synthesis has been strongly demanded.

Japanese Unexamined Patent Publication No. 2010-239880 International Publication No. 2007/004654 Pamphlet

It is an object of the present invention to provide a method for shortening a reaction time and a reagent composition in DNA synthesis using PCR.

As a result of diligent research in view of the above circumstances, the present inventors have used DNA polymerase and PCNA belonging to Family B in high-speed PCR with a short reaction time, and further increased the primer concentration to make a more efficient nucleic acid. We have found that amplification can be obtained, and have completed the present invention.
A typical invention is as follows.

Item 1. It contains DNA polymerase belonging to Family B, Proliferating Cell Nuclear Antigen (PCNA), and primers at a concentration of 0.6 μM or more, and has a composition for carrying out 30 to 50 cycles within 40 minutes. A PCR method composition characterized by being present.
Item 2. The PCR method composition according to Item 1, which is a composition for carrying out 30 to 50 cycles within 20 minutes.
Item 3. The PCR method composition according to Item 1 or 2, which is a composition for carrying out 30 to 50 cycles within 12 minutes.
Item 4. Item 4. The PCR composition method according to any one of Items 1 to 3, wherein the primer concentration is 0.8 μM or more.
Item 5. Item 4. The PCR composition method according to any one of Items 1 to 4, wherein the primer concentration is 1.0 μM or more.
Item 6. Item 2. The PCR method composition according to any one of Items 1 to 5, wherein the composition is for carrying out PCR in a temperature range of 0.1 ° C./sec to 20 ° C./sec.
Item 7. Item 2. The PCR method composition according to any one of Items 1 to 6, which is a composition for carrying out PCR in a temperature range of 5 ° C./sec to 20 ° C./sec at a rate of temperature increase or decrease.
Item 8. Item 2. The PCR method composition according to any one of Items 1 to 7, which is a composition for carrying out PCR in a temperature range of 10 ° C./sec to 20 ° C./sec at a rate of temperature increase or decrease.
Item 9. Item 4. The PCR composition according to any one of Items 1 to 8, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from archaea.
Item 10. Item 4. The PCR composition according to any one of Items 1 to 9, which is an archaeal DNA polymerase variant having a reduced base analog detection activity in which DNA polymerase belonging to Family B is reduced.
Item 11. Item 4. The PCR composition according to any one of Items 1 to 10, wherein the PCNA is a PCNA derived from archaea.
Item 12. Item 6. The PCR composition according to any one of Items 1 to 11, wherein the PCNA is a mutant PCNA having a DNA polymerase amplification enhancing activity.
Item 13. A reagent for PCR, which comprises the PCR composition according to any one of Items 1 to 12.
Item 14. Item 3. A PCR reagent kit comprising the PCR reagent according to Item 13.
Item 15. A PCR method according to any one of Items 1 to 12, wherein the PCR method is performed by 15 to 50 thermal cycles, and the PCR reaction time is within 60 minutes.
Item 16. Item 2. The PCR method according to Item 15, wherein the PCR reaction time is 30 minutes or less.
Item 17. Item 15. The PCR method according to Item 15 or 16, wherein the biological sample is directly added to the reaction solution containing the PCR composition according to any one of Items 1 to 12.

According to the present invention, the reaction time of PCR can be shortened. The and composition in the present invention enable high-speed PCR even under conditions containing an inhibitor such as blood, and are very useful especially in diagnostic applications where rapidity is required.

Examination of primer concentration and magnesium concentration Ct value Examination of primer concentration and magnesium concentration Melting curve Examination of primer concentration Melting curve Confirmation of synergistic effect of PCNA and high-concentration primer Examination of fast PCR in the presence of PCR inhibitors Examination of high-speed PCR in the presence of PCR inhibitor / dUTP PCNA sequence comparison

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
The PCR method in the present invention is characterized in that a DNA polymerase belonging to Family B, PCNA and a high-concentration primer are contained in the reaction solution, and PCR is performed at high speed.

(1) Notation of Amino Acids In this specification, abbreviated symbols in alphabetical notation may be used for the base sequence, amino acid sequence and their individual constituent factors, but all of them are in the fields of molecular biology and genetic engineering. Follow the practice. Further, in the present specification, in order to briefly indicate the mutation of the amino acid sequence, for example, a notation such as "D143A" is used. "D143A" indicates that the 143rd aspartic acid was replaced with alanine, that is, the type of amino acid residue before the substitution, its location, and the type of the amino acid residue after the substitution. The SEQ ID NOs correspond to the SEQ ID NOs listed in the sequence listing unless otherwise specified. In the case of multiple mutants, the above notation is connected by "/". For example, "D143A / D147A" indicates that the 143rd aspartic acid was replaced with alanine and the 147th aspartic acid was replaced with alanine.
Further, in the present specification, the terms "mutant" and "mutant type" mean that the protein has an amino acid sequence different from that of a conventionally known protein, and distinguishes between an artificial mutation and a mutation in the natural world. It's not something to do.

The "142nd" corresponding "amino acid in the amino acid sequence shown in SEQ ID NO: 1" is 142 of SEQ ID NO: 1 in PCNA having an amino acid sequence that is not exactly the same as the amino acid sequence shown in SEQ ID NO: 1. It is an expression containing the second corresponding amino acid sequence. In the present specification, the meaning of "corresponding" in the same form of notation as described above is the same as that of the above examples.

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

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

(2) High-speed PCR
The PCR in the present invention is a high-speed PCR in which the reaction time is 40 minutes or less when 30 thermal cycles are performed, preferably 30 minutes or less, more preferably 20 minutes or less, and further preferably 10 minutes or less. Fast PCR is within. The reaction time indicates the time of a thermal cycle in which denaturation, annealing, and elongation are repeated. With the thermal cycle setting of a normal thermal cycler, the reaction time is rarely less than 60 minutes. For example, in order to complete the reaction within 60 minutes with the PCR system GeneAmp® 9700 (manufactured by Applied Biosystem), the time of each step of the thermal cycle needs to be extremely short. In fact, if the time of each step is extremely shortened and 40 cycles of 98 ° C., 0 seconds → 60 ° C., 0 seconds → 68 ° C., 0 seconds are carried out after the pre-reaction at 94 ° C. for 2 minutes, the reaction time is about 60. It will be a minute. Further, in order to achieve a reaction time of 30 minutes or less, it is necessary to use a thermal cycler that changes the temperature at a high speed and further shorten the time of each step of the thermal cycle. For example, when Light cycler (registered trademark) 2.0 (Roche) capable of changing the temperature at high speed is used, after a pre-reaction at 94 ° C. for 2 minutes, 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68. When 50 cycles of 0 minutes at ° C. are carried out, the reaction time becomes about 20 minutes. The number of heat cycles of PCR in the present invention can be set and carried out in the range of 15 to 50 times. It is preferably 25 to 45 times, more preferably 30 to 40 times.

In the high-speed thermal cycle setting in the present invention, it is preferable to shorten the annealing step. This is because it is necessary to increase the concentration of the primer in order to increase the PCR reaction rate, but in general, when the concentration of the primer is increased, non-specificity and primer dimer are likely to occur. By shortening the annealing step, it is considered that non-specificity and primer dimer are less likely to occur even with a high concentration of primer. The preferred annealing step time is 10 seconds. It is more preferably 5 seconds, and particularly preferably 0 seconds.

(3) DNA polymerase belonging to family B The DNA polymerase used in the nucleic acid amplification method of the present invention is a DNA polymerase belonging to family B. In the present invention, the DNA polymerase belonging to Family B refers to a DNA polymerase having 3'-5'exonuclease activity and not 5'-3'exonuclease activity. A DNA polymerase derived from archaea is preferred.

(3.1) Archaeal-derived DNA polymerase Examples of archaeal-derived DNA polymerases belonging to family B include DNA polymerases isolated from bacteria of the genus Pyrococcus and Thermococcus. Examples of the DNA polymerase derived from the genus Pyrococcus include Pyrococcus furiosus and Pyrococcus sp. DNA polymerases isolated from GB-D, Pyrococcus Womenei, Pyrococcus abyssi, Pyrococcus horikoshii, but not limited to these. Examples of the DNA polymerase derived from the genus Thermococcus include Thermococcus kodakaraensis, Thermococcus gogonarius, Thermococcus litoralis, and Thermococcus sp. JDF-3, Thermococcus sp. 9degres North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Includes, but is not limited to, DNA polymerases isolated from KS-1, Thermococcus celer, or Thermococcus siculi. PCR enzymes using these DNA polymerases are commercially available, and are commercially available: Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England) Roche), Pwo (Roche) and the like.

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

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

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

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

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

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

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

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

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

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

(3.3) Modification of DNA polymerase (II) Modification to prepare a DNA polymerase mutant having reduced base analog detection activity (3.3.1)
The DNA polymerase belonging to Family B used in the present invention may be a mutant having reduced base analog detection activity. Base analogs indicate bases other than adenine, cytosine, guanine, and thymine, and examples thereof include uracil and inosine. Normally, DNA polymerases belonging to Family B bind strongly when they detect base analogs such as uracil and inosine, and inhibit the DNA polymerase function. The base analog detection activity indicates an activity that strongly binds to a base analog and inhibits the DNA polymerase function. The DNA polymerase variant belonging to Family B having reduced base analog detection activity is a DNA polymerase variant belonging to Family B characterized by a low binding ability to uracil and inosine.

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

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

(3.3.2)
More preferred as the DNA polymerase variants with reduced base analog detection activity used in the present invention are 7, 36, 37, 90-97 and 112, which are assumed to be directly related to interaction with uracil. Paleobacterial DNA polymerase variants in which at least one of the 119th amino acids has been modified, such as (a) 7, 36, 37, 90-97 and the amino acid sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 2. It is an paleobacterial DNA polymerase variant represented by an amino acid sequence having a modification of at least one amino acid among the amino acids corresponding to positions 112 to 119.

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

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

The archaeal DNA polymerase variant described above may be represented by the amino acid sequence of (c) below.
(C) In the amino acid sequence shown in (a), 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 , An amino acid sequence encoding a DNA polymerase with reduced base analog detection activity.

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

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

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

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

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

(3.3.3)
The archaeal DNA polymerase variant with reduced base analog detection activity used in the present invention is more preferably at least one selected from the amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. Has one amino acid modification. Here, for example, Y7 means a tyrosine (Y) residue which is the seventh amino acid, and one letter of the alphabet represents an abbreviation of a commonly used amino acid. In a preferred example, the Y7 amino acid is selected from the group consisting of tyrosine (Y) substituted with a non-polar amino acid, specifically Y7A, Y7G, Y7V, Y7L, Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C. Amino acid substitution. In another preferred example, the P36 amino acid is a proline (P) substituted with a positively charged polar amino acid, specifically an amino acid substitution of P36H, P36K, or P36R. In another preferred example, the V93 amino acid is valine (V) substituted with a positively charged polar amine acid, specifically an amino acid substitution of V93H, V93K, or V93R.

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

The archaeal DNA polymerase variant with reduced base analog detection activity used in the present invention is two or more amino acids selected from the amino acids corresponding to amino acids Y7, P36, or V93 in SEQ ID NO: 1 or SEQ ID NO: 2. May be modified. Specific examples thereof include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K, and preferably Y7A / P36H or Y7A / V93K. It is not limited to.

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

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

Paleobacterial DNA polymerase variants with reduced base analog detection activity are mutated as a result of performing an optimal thermal cycle using arbitrary primers and template DNA in an enzyme-optimal reaction buffer. It refers to a DNA polymerase that does not inhibit the elongation reaction even when a high concentration of dUTP is added, and a PCR product can be confirmed, as compared with the wild type without DNA. However, if it is difficult to compare with the wild type, the number of archaeal DNA polymerase mutants that can amplify PCR even if dUTP is added at a concentration of 0.5 μM is reduced compared to the wild type. It is presumed that it has the activity of detecting base analogs.

The evaluation of the base analog detection activity in the present invention follows the following method.
KOD-Plus-Ver. Using the 10 × PCR Buffer attached to 2 (manufactured by Toyobo) or the 10 × PCR Buffer attached to Pfu DNA Polymerase (manufactured by Agent), 1 × PCR Buffer, and 1.5 _{mM sulfonyl 4} , 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), the primer set forth in SEQ ID NOs: 11 and 12 of 15 pmol that amplifies about 1.3 kb, 10 ng of human genomic DNA (manufactured by Roche), and dUTP (manufactured by Roche) in 50 μl of a reaction solution containing 1 U of each enzyme. ) Is added to a final concentration of 0.5, 5, 50, 100, 200 μM. After a pre-reaction at 94 ° C. for 30 seconds, PCR system GeneAmp® 9700 (Applied Biosystem) was used on a schedule of repeating 30 cycles of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds. Perform PCR. After completion of the reaction, 1% agarose gel electrophoresis was performed on 5 μl of the reaction solution, stained with ethidium bromide, and an amplified DNA fragment of about 1.3 kb was confirmed under ultraviolet irradiation to see if the base analog detection activity was reduced. Can be evaluated.

(3.4) Method for introducing amino acid modification A method for modifying the DNA polymerase used in the present invention has already been established in the art. Therefore, the modification can be performed according to a known method, and the mode thereof is not particularly limited.

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

(3.5)
The modified DNA polymerase gene is transferred to an expression vector as needed, and Escherichia coli, for example, as a host is transformed with the expression vector and then applied to an agar medium containing a drug such as ampicillin to form colonies. The colonies are inoculated into a nutrient medium, for example, LB medium or 2 × YT medium, cultured at 37 ° C. for 12 to 20 hours, and then the cells are disrupted to extract a crude enzyme solution. The vector is preferably derived from pBluescript. Any known method may be used for crushing the cells, and for example, sonication, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes to inactivate the host-derived DNA polymerase, and the DNA polymerase activity is measured.

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

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

(4) PCNA
(4.1)
The PCNA used in the present invention is a kind of PCR enhancer. The PCNA is not particularly limited, but a heat-resistant one that can withstand the heat cycle of PCR is desirable, and one that remains active even after PCR is preferably desired. More preferably, it is soluble even after heat treatment at 80 ° C. for 30 minutes, and it is desirable that the activity remains 50% or more, more preferably 70% or more, and particularly preferably 90% or more.

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

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

Furthermore, the PCNA used in the present invention may be a mutant (having DNA polymerase amplification enhancing activity) that is loaded into DNA alone. PCNAs usually form multimers and have a ring-like structure. Loading into DNA means passing DNA through the structure of the ring of PCNA multimers, and PCNA can only be loaded into DNA in collaboration with a factor, usually called RFC. A mutant that loads into DNA alone is a mutant that makes it easier for DNA to pass through the PCNA multimer without RFC by modifying the site involved in PCNA multimer formation and destabilizing the multimer formation. show.

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

What has been described above and below by exemplifying SEQ ID NO: 13 or SEQ ID NO: 14 also applies to PCNAs other than the PCNA specifically presented with the sequence herein. For example, in PCNAs other than the PCNAs shown in SEQ ID NOs: 13 and 14, as shown in FIG. 7, the region relating to multimeric formation consisting of the 82, 84, 109, 139, 143, and 147th amino acids of SEQ ID NO: 13 Indicates the corresponding area. Here, when there are two different amino acid sequences, the fact that the amino acid or region "corresponds" in one of the reference amino acid sequences means that the reference sequence corresponds to the reference sequence when the primary structures of the amino acid sequences are compared (aligned). The position corresponds to the position.

PCNA variants that load DNA alone are more preferably involved in PCNA multimer formation.
(A) N-terminal region consisting of amino acids corresponding to the 82nd, 84th, and 109th positions, or
(B) Examples thereof include mutants having at least one modification in the C-terminal region consisting of amino acids corresponding to positions 139, 143 and 147, loading into DNA even in the absence of RFC, and promoting the elongation reaction of DNA polymerase.

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

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

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

(4.1.2) Mja-PCNA
The PCNA used in the present invention is a PCNA monomer shown in any of the following (A) to (E).
(A) From a polypeptide consisting of an amino acid sequence in which the 142nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 23 is replaced with any amino acid residue selected from the group consisting of lysine, arginine, histidine, and tryptophan. A polypeptide having a DNA polymerase amplification enhancing activity.
(B) In the PCNA monomer represented by (A), the 142nd amino acid residue in the amino acid sequence set forth in SEQ ID NO: 23 is further selected from the group consisting of lysine, arginine, histidine, and tryptophan. It consists of a polypeptide consisting of an amino acid sequence substituted with an amino acid residue, and any of the following (a) to (e) substitutions, deletions, insertions and / or additions (collectively referred to as "mutations"). A polypeptide comprising, and having DNA polymerase amplification enhancing activity.
(A) Substitute the 80th amino acid residue with one selected from the group consisting of lysine, histidine and tryptophan (b) Any one selected from the group consisting of arginine, histidine and tryptophan with the 82nd amino acid residue Substituted with (c) Replaced the 108th amino acid residue with one selected from the group consisting of lysine, histidine and tryptophan (d) Replaced the 138th amino acid residue with glutamic acid (e) 146th amino acid residue In the PCNA monomer represented by (C) and (A), the 142nd amino acid residue in the amino acid sequence set forth in SEQ ID NO: 23 is further selected from the group consisting of lysine, arginine, histidine, and tryptophan. Consists of a polypeptide consisting of an amino acid sequence substituted with any of the amino acid residues that are A polypeptide consisting of an amino acid sequence in which individual amino acid residues are substituted, deleted, inserted or added, and having DNA polymerase amplification enhancing activity.
(D) In the PCNA monomer represented by (A), only the 142nd amino acid residue of the amino acid sequence set forth in SEQ ID NO: 23 is selected from the group consisting of lysine, arginine, histidine, and tryptophan. A polypeptide consisting of an amino acid sequence substituted with the amino acid residue.
(E) In the PCNA monomer represented by (A), a poly consisting of an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 23 and having a DNA polymerase amplification enhancing activity. peptide.

In SEQ ID NO: 23, the 142nd amino acid residue is one of the amino acid residues involved in PCNA multimer formation. Amino acid residues involved in PCNA multimer formation are present in the N-terminal region and the C-terminal region of each monomer. The PCNA multimer is formed by joining the N-terminal region of one monomer and the C-terminal region of the other monomer as an interface. In eukaryotic cells and archaea, PCNAs often form trimers. In the amino acid sequence shown in SEQ ID NO: 23, the N-terminal region corresponds to the position of the group shown in (a) below, and the C-terminal region corresponds to the position of the group shown in (b) below.
(A) Amino acid residue group at positions 80, 82 and 108 (b) Amino acid residue group at positions 138, 142 and 146

In the above-mentioned polypeptide (2), one or several amino acid residue is substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 23 as long as it retains the DNA polymerase amplification enhancing activity. (Hereinafter, these are collectively referred to as "mutation".) It is a polypeptide consisting of an amino acid sequence. One or several mutations can be treated with restriction enzymes, treatment with exonuclease, DNA ligase, etc., position-specific mutation introduction method or random mutation introduction method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New). It can be carried out by introducing a mutation into the DNA encoding the PCNA monomer of the present invention described later by using a known method such as York). The variant PCNA monomer can also be obtained by other methods such as ultraviolet irradiation. Variant PCNA monomers also include naturally occurring variants (eg, single nucleotide polymorphisms), such as when based on individual differences in microorganisms carrying PCNA, differences in species or genera.

The polypeptide of [3] above is a polypeptide consisting of an amino acid sequence having 80% or more identity as compared with the amino acid sequence shown in SEQ ID NO: 23, as long as it retains the DNA polymerase amplification enhancing activity. Preferably, the amino acid sequence of the PCNA monomer of the present invention has an identity of 85% or more, more preferably 88% or more, still more preferably 90% or more, and more. It is more preferably 93% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. A polypeptide consisting of an amino acid sequence having such an identity of a certain level or more can be prepared based on a known genetic engineering technique as described above.

As the PCNA as described in [2] or [3] above, preferably, an affinity tag such as a His tag inserted at the N-terminal is added to facilitate the purification of the PCNA, and this is particularly preferable. Not limited to.

In the above PCNA monomer, the 142nd amino acid residue in SEQ ID NO: 23 is replaced with a basic amino acid residue, but the type of basic amino acid to be replaced is not particularly limited. Examples of basic amino acids include arginine, histidine, lysine and tryptophan if they are naturally occurring. It is preferably arginine or lysine.

(4.2)
In the method for obtaining PCNA, the PCNA gene is transferred to an expression vector as needed, Escherichia coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin to obtain colonies. To form. The colonies are inoculated into a nutrient medium, for example, LB medium or 2 × YT medium, cultured at 37 ° C. for 12 to 20 hours, and then the cells are disrupted to extract a crude enzyme solution. The vector is preferably derived from pBluescript. Any known method may be used for crushing the cells, and for example, sonication, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. The expression of the target protein can be confirmed by heat-treating this crude enzyme solution at 80 ° C. for 30 minutes, centrifuging it to remove the protein derived from the host, and subjecting it to SDS-PAGE.

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

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

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

In order to quantitatively evaluate the increase in the amplification amount, in the present invention, the amplification amount is quantified by using analysis software called Gel Pro Analyzer (Media Cybernetics). When the amplification amounts are compared by such a method, the amplification amount when PCNA is added is 1.0 times (preferably 1.2 times, more preferably 1.) the amplification amount when PCNA is not added. 5 times, more preferably 2 times, 3 times). Alternatively, if the unamplified target is amplified, it is determined that the PCNA "has DNA polymerase amplification enhancing activity".

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

(5) High-concentration primer The primer concentration in the present invention is 0.6 μM or more. In general, high-concentration primers cause non-specificity and primer dimers, so it is generally said that 0.1 to 0.5 μM is used in ordinary PCR. It has been found that under the fast PCR conditions of the present invention, the priming efficiency is reduced unless the concentration is high because the annealing step of the cycle is short. On the contrary, since the annealing step is short under high-speed PCR conditions, non-specificity and primer dimers are unlikely to occur even with a high concentration of primers. The primer concentration in the present invention is more preferably 0.8 μM or more, still more preferably 1.0 μM or more. It is preferable that it does not exceed 4.0 μM, preferably 2.0 μM, and more preferably 1.4 μM.

In the present specification, when the term "primer concentration of 0.6 μM or more" is used, the primer concentrations of forward (F) and reverse (R) in a set of primer pairs may be the same, or one of the primers may be the same. The concentration may exceed the other concentration (even if the primer concentration is asymmetric). Further, in the case of multiplex PCR, in addition to the above, the concentration between all the primer pairs may be the same, or the concentration may be different between each primer pair.

(6) Heat cycle rate In the PCR in the present invention, it is preferable that the rate of temperature rise or fall in the heat cycle is 0.1 ° C./sec to 20 ° C./sec. It is more preferably in the range of 5 ° C./sec to 20 ° C./sec, and even more preferably in the range of 10 ° C./sec to 20 ° C./sec. In order to carry out a thermal cycle at such a rate of temperature rise or fall, it is particularly preferable to use a thermal cycler whose temperature can be controlled by heating and cooling with air.

(7) PCR containing an inhibitor
The PCR method of the present invention is suitable for carrying out in a state containing an inhibitor. The inhibitor in the present invention indicates, for example, a biological sample, but is not particularly limited as long as it is a substance that inhibits PCR. The biological sample 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 such as body hair, nails and oral mucosa, body fluids such as blood, excrement such as feces and urine, cells, bacteria, viruses and the like. Body fluids include blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.

(8) Antibody In the present invention, if necessary, an antibody having an activity of further suppressing the polymerase activity and / or the 3'-5'exonuclease activity of the heat-resistant DNA polymerase may be used. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective in increasing the sensitivity of PCR and reducing non-specific amplification.

(9) Reagent for Performing Nucleic Acid Amplification Method The reagent for carrying out the PCR of the present invention contains DNA polymerase belonging to Family B, PCNA and a primer having a concentration of 0.6 μM or more in the reaction solution. The configuration other than is not particularly limited. The reagent also includes the form of a kit.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not particularly limited by the following examples.

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

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

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

(Example 4)
Preparation of Modified Heat-Resistant PCNA The cells obtained in Example 3 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. Eshericia was cultured in 3 mL of LB medium (1% plasmid, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin in advance at 37 ° C. for 16 hours. Cory DH5α (plasmid transformant) (using a test tube) was inoculated, and aerated culture was performed at 37 ° C. for 16 hours. The cells were collected from the culture medium by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then the cells were subjected to sonication treatment. It was crushed to obtain a cell crushed solution. Next, the cell disruption solution was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Further, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and Q-sepharose chromatography were performed, and finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithioslateol, 0) was performed. .1% Tween20, 0.1% nonidet P40, 50% glycerin) was substituted to obtain a modified heat-resistant PCNA.

(Example 5)
Examination of primer concentration and magnesium concentration Using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, high-speed PCR was performed to confirm the effects of primer concentration and magnesium concentration. ..
For PCR, 0.2 mM dNTPs, 1 U KOD DNA polymerase mutant, 250 ng KOD PCNA were _{added to 1 × PCR Buffer (including 1.2 mM sulfonyl 4} ) obtained by diluting 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10 times. The following (a) or (b) was added to 50 μl of the reaction solution containing the mutant and SYBR® Green I diluted to 1 / 30,000, and the Ct values of each were compared.
(A) Primers set forth in SEQ ID NOs: 19 and 20 for amplifying the salmonella invA gene (about 700 bp), and SEQ ID NOs: 21 and 22 for amplifying the Salmonella genome (b) actin gene (about 550 bp) equivalent to 50 copies. The concentrations of the above-mentioned primers and the human genome primer corresponding to 50 copies were set at three levels of 0.2, 0.4, and 0.6 μM. Further, _{Prevent 4} was added to set the Mg concentration at 5 levels of 1.2, 2, 4, 6, and 8 mM, and the combination with the primer concentration of 3 levels was examined.
PCR is performed by using Light cycler 2.0 with a schedule of repeating a high-speed cycle of 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68 ° C., 0 seconds for 50 cycles after a prereaction at 94 ° C. for 30 seconds. And the rate of temperature lowering was 20 ° C./sec. The reaction time was about 20 minutes.

FIG. 1 shows the Ct values when amplification was compared by high-speed PCR from the Salmonella genome or the human genome corresponding to 50 copies by changing the primer concentration and Mg concentration, and FIG. 2 shows their melting curves. Primer concentrations were 0.2, 0.4, 0.6 μM, Mg concentrations were 1.2, 2, 4, 6, and 8 mM, and if non-specific amplification was seen on the melting curve, the Ct value was Depending on the amount of non-specific amplification, circle marking (some objective amplification but non-specific amplification) and square marking (only non-specific amplification is seen).

In the amplification of the Salmonella invA gene, Ct tended to be improved by increasing the primers and Mg concentrations, respectively.
On the other hand, in the amplification of the actin gene, when the Mg concentration was low, the Ct was improved by increasing the primer concentration, but when the Mg concentration was increased, non-specific amplification appeared. It is known that increasing the concentration of the primer and Mg has the function of increasing the efficiency of PCR, while increasing the concentration too much leads to non-specific amplification. However, in high-speed PCR such as this time, high-concentration Mg increases the possibility of increasing non-specific amplification as in normal PCR, but high-concentration primers make it difficult for non-specific amplification to occur. rice field.
Under high-speed PCR conditions, the cycle annealing step is short, so non-specific amplification and primer dimers are unlikely to occur even when high-concentration primers are used, and there is a function to improve only PCR efficiency while maintaining specificity. Conceivable.

(Example 6)
Examination of primer concentration As in Example 5, high-speed PCR was performed using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above to confirm the effect of the primer concentration.
here. It was also confirmed whether the primer concentration had an effect even in high-speed PCR in the presence of dUTP.

_{For PCR, 1 × PCR Buffer (including 1.2 mM Then 4} ) obtained by diluting 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10-fold, 0.2 mM dNTPs, or 2 mM dNTPs in which dTTP was replaced with dUTP ( Amplify about 550 bp of the actin gene in 50 μl of reaction solution containing dATP, dUTP, dCTP, dGTP), 1 U KOD DNA polymerase variant, 250 ng KOD PCNA variant, and SYBR GreenI diluted to 1 / 30,000. The primers set forth in SEQ ID NOs: 21 and 22 and the human genome equivalent to 50 copies were added, and the Ct values were compared for each. Primer concentrations were 0.2, 0.6, 0.8, 1.0, 1.2 and 1.4 μM, respectively. PCR is performed by using Light cycler 2.0 with a schedule of repeating a high-speed cycle of 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68 ° C., 0 seconds for 50 cycles after a prereaction at 94 ° C. for 30 seconds. And the rate of temperature lowering was 20 ° C./sec. The reaction time was about 20 minutes.

Table 1 shows the Ct value when the amplification from 50 copies by high-speed PCR was compared by changing the primer concentration, and FIG. 3 shows the melting curve. Primer concentrations were 0.2, 0.6, 0.8, 1.0, 1.2, 1.4 μM in the presence of dTTP (normal dNTPs) and in the presence of dUTP (dNTPs in which dTTP was replaced with dUTP). It was carried out in.

As a result, it was shown that the Ct value was improved by increasing the primer concentration to 0.6 μM or more in both the presence of dTTP and the presence of dUTP. In addition, no non-specific amplification or primer dimer was observed in this experiment.

(Example 7)
Confirmation of synergistic effect of PCNA and high-concentration primer Using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, high-speed PCR was performed to confirm the synergistic effect of PCNA and high-concentration primer. .. For PCR, 0.2 mM dNTPs, 1 U KOD DNA polymerase variant, 250 ng KOD PCNA were _{added to 1 × PCR Buffer (including 1.2 mM sulfonyl 4} ) obtained by diluting 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10 times. In 50 μl of the reaction solution containing the mutant and SYBR GreenI diluted to 1 / 30,000, the actin gene, the primers set forth in SEQ ID NOs: 21 and 22 for amplifying about 550 bp, and human genomes of various concentrations were added. Then, the Ct values were compared for each.
The human genome was ^{run on 5 samples: 10 4} , 10 ³ , 10 ² , 10 ¹ copy, non-template control (NTC). This time, PCR was performed and compared even with a composition containing no PCNA mutant. Primer concentrations were 0.2 and 1.0 μM, respectively, and PCR was performed with 50 cycles of 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68 ° C., 0 seconds after the prereaction of 94 ° C. and 30 seconds. Two types of comparisons were made: a high-speed PCR schedule in which cycles were repeated, and a normal PCR schedule in which cycles of 98 ° C., 10 seconds → 60 ° C., 10 seconds → 68 ° C., and 30 seconds were repeated for 50 cycles after a prereaction at 94 ° C. for 30 seconds. .. Light cycler (registered trademark) 2.0 was used as the thermal cycler, and the rate of temperature rise and fall was performed at 20 ° C./sec.

FIG. 4 shows Ct values by comparing amplification by high-speed PCR from human genomes of various copy numbers in order to confirm the synergistic effect between the primer concentration and the PCNA mutant. The primer concentrations were 0.2 and 1.0 μM, and the Ct was shown with and without PCNA. In addition, in the primer 0.2 μM, Ct in each case with and without PCNA in the normal cycle is also shown. Here, when non-specific amplification is seen in the melting curve, it is marked with a circle (there is amplification of the target gene but also non-specific amplification) and a square mark according to the amount of non-specific amplification. (Only non-specific amplification is seen).

When the primer concentration was 0.2 μM, no significant difference was observed with the addition of PCNA in the normal cycle. It is shown that in a normal cycle, the significant effect of the addition of PCNA is not seen. On the other hand, in the high-speed cycle, although Ct was significantly delayed without the addition of PCNA, the result was that Ct was improved by the addition of PCNA. However, when the primer concentration is 0.2 μM, even if PCNA is added, the Ct is inferior to that of the normal cycle. When the primer concentration is 1.0 μM, Ct is improved even without PCNA, but Ct is still delayed by about 1 in the normal cycle. When PCNA was added thereto, Ct almost equivalent to that in the normal cycle was obtained. It has been shown that both increasing the primer concentration and adding PCNA are required to make the fast cycle as efficient as the normal cycle.

(Example 8)
Examination of high-speed PCR in the presence of a PCR inhibitor Using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, high-speed PCR was performed under conditions containing an inhibitor.
The inhibitor was prepared as follows.
Feces: Suspend 2 g of feces in 8 ml of water to make a 20% fecal suspension. The mixture was heat-treated at 95 ° C. for 5 minutes, centrifuged at 12,000 rpm for 1 minute, and the supernatant was made into 20% feces.
Urine: Urine collected from humans was defined as 100% urine.
Oral mucosa: The oral mucosa collected with a cotton swab was dropped into 200 μl of water to prepare 100% oral mucosal fluid.
Blood: 100% blood was taken from the blood collected by the EDTA blood collection tube.

For PCR, 0.2 mM dNTPs, 1 U KOD DNA polymerase mutant, 250 ng KOD PCNA were _{added to 1 × PCR Buffer (including 1.2 mM ● 4} ) obtained by diluting 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10 times. In 50 μl of the reaction solution containing the mutant and SYBR GreenI diluted to 1 / 30,000, the Salmonella invA gene, the primers set forth in SEQ ID NOs: 19 and 20 for amplifying about 700 bp, and the Salmonella genome equivalent to 50 copies. Was added, and the Ct values were compared for each.
Primer concentrations were 0.2 and 1.0 μM. The sample prepared above was used as the inhibitor, 2.5, 0.5, 0.1% feces, 5, 1, 0.2, 0.04% urine, 25, 5, 1, 0.2%. Oral mucosa, 1.6, 0.32, 0.06% blood was added. PCR is performed with Light cycler (registered trademark) 2.0 on a schedule of repeating a high-speed cycle of 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68 ° C., 0 seconds for 50 cycles after a prereaction at 94 ° C. for 30 seconds. The temperature was raised and lowered at 20 ° C./sec. The reaction time was about 20 minutes.

FIG. 5 shows Ct when high-speed PCR was performed from a composition containing an inhibitor and 50 copies of Salmonella were amplified. Primer concentrations were 0.2, 1.0 μM, with no inhibitors and 2.5, 0.5, 0.1% feces, 5, 1, 0.2, 0.04% urine, 25. The effects of addition of 5, 1, 0.2% oral mucosal fluid or 1.6, 0.32, 0.06% blood were confirmed. If non-specific amplification is seen on the melting curve, circled markings (some target gene amplifications but also non-specific amplifications) and square markings (non-specific amplifications), depending on the amount of non-specific amplifications. Only specific amplification is seen). In addition, the sample that is not amplified at all is N.I. D. It was written as.

This system is a system that does not amplify without PCNA even in the absence of inhibitors.
Regarding feces, when the primer concentration was 0.2 μM, amplification was only up to 0.5%, but by increasing the primer concentration to 1.0 μM, amplification was confirmed even if 2.5% was contained.
Regarding urine, although the rise was delayed when the primer concentration was 0.2 μM, the rise was improved by increasing the primer concentration to 1.0 μM, and amplification was confirmed even when brought in at 5%. Regarding the oral mucosa, when the primer concentration was 0.2 μM, the amplification was only up to 5%, but by increasing the primer concentration to 1.0 μM, amplification was confirmed even if the primer concentration was 25%.
Regarding blood, when the primer concentration was 0.2 μM, amplification was only up to 0.32%, but by increasing the primer concentration to 1.0 μM, amplification was confirmed even if 1.6% was contained.
It is considered that high-speed PCR became possible even if an inhibitor was contained by increasing the primer concentration in addition to the addition of PCNA.

(Example 9)
Examination of high-speed PCR in the presence of PCR inhibitor / dUTP Using the KOD Y7A / P36H / N210D and KOD PCNA D147A obtained above, high-speed PCR was performed under conditions containing the inhibitor and dUTP. ..
The inhibitor was prepared as follows.
Feces: 2 g of feces was suspended in 8 ml of water to prepare a 20% fecal suspension. The mixture was heat-treated at 95 ° C. for 5 minutes, centrifuged at 12,000 rpm for 1 minute, and the supernatant was made into 20% feces.

For PCR, 2 mM dNTPs (dATP, dUTP, dCTP) in which dTTP was replaced with dUTP in _{1 × PCR Buffer (including 1.2 mM transferase 4} ) obtained by diluting 10 × PCR Buffer attached to KOD Dash (manufactured by Toyobo) 10 times. SEQ ID NO: 21 that amplifies the actin gene, 1.0 μM, about 550 bp in 50 μl of reaction solution containing dGTP), 1 U KOD DNA polymerase variant, 250 ng KOD PCNA variant, and SYBR GreenI diluted to 1 / 30,000. And 22 and the human genome equivalent to 50 copies were added to obtain Ct values of 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% in the presence of stool. Compared.
A similar study was performed on the Taq reaction system as a control. As the Taq DNA polymerase, one manufactured by Toyobo was used, and one mixed with Anti-Taq High (manufactured by Toyobo) was used. The reaction was mixed with Buffer attached to 1 × BlendTaq, dNTPs (dATP, dUTP, dCTP, dGTP) in which 2 mM dTTP was replaced with dUTP, 0.2, and 1.0 μM primers (similar to the above), and an antibody. 50 copies of human genome, 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% stool were added to 50 μl of the reaction solution containing 5 U of enzyme, and the Ct values were compared. .. PCR was elevated using Light cycler 2.0 on a schedule of repeating 50 cycles of high-speed cycles of 98 ° C., 0 seconds → 55 ° C., 0 seconds → 68 ° C., 0 seconds after prereaction at 94 ° C. and 30 seconds, respectively. The heating and lowering rates were 20 ° C./sec. The reaction time was about 20 minutes.

FIG. 6 shows Ct when high-speed PCR was performed from a composition containing feces and dUTP to amplify 50 copies of the human genome. In the system using the KOD mutant, the primer concentration was 1.0 μM, and it was carried out with and without the PCNA mutant. Ct in the absence of inhibitors and in 2.5, 2, 1.5, 1, 0.5, 0.25, 0.1% feces was confirmed. In the system using Taq DNA polymerase, the primer concentrations were 0.2 and 1.0 μM, and similarly, when there was no inhibitor and 2.5, 2, 1.5, 1, 0.5, 0.25, 0. Ct in 1% feces was confirmed. If non-specific amplification is seen on the melting curve, circled markings (some target gene amplifications but also non-specific amplifications) and square markings (non-specific amplifications), depending on the amount of non-specific amplifications. Only specific amplification is seen). In addition, the sample that is not amplified at all is described as "ND".

At the inspection site, it is necessary to confirm the amplification in the presence of 1% feces. As a result of the Taq system, in the presence of 1% stool, even if the primer concentration was high, no amplification was observed by high-speed PCR in the first place. On the other hand, in the KOD system, amplification was confirmed up to 0.5% even without the addition of PCNA, but inhibition began to occur at 0.5%, indicating that non-specific amplification appeared. In the system containing PCNA, amplification was confirmed even when 1.5% feces were added. From these results, it can be seen that the DNA polymerase belonging to Family B is suitable for high-speed PCR. However, even if a high-concentration primer and a DNA polymerase belonging to Family B were used, the rise of Ct was not so good, and the result was that even stool of 1% or less was greatly affected. The system that combines the high-concentration primer, the DNA polymerase belonging to Family B, and PCNA is also the most efficient, and in the high-speed PCR containing 1% feces, the result that amplification cannot be obtained without this combination was obtained.

The present invention is useful in biotechnology-related industries involved in DNA synthesis, and is particularly useful in diagnostic applications.

Claims (12)

It contains DNA polymerase belonging to Family B, Proliferating Cell Nuclear Antigen (PCNA), and primers at a concentration of 0.8 μM or more, and at a rate at which the rate of temperature rise or fall is 10 ° C./sec or more. A composition for PCR, which is a composition for carrying out 30 to 50 cycles within 20 minutes. The composition according to claim 1, wherein the concentration of the primer is 0.8 μM to 1.0 μM. The composition according to claim 1, wherein the concentration of the primer is 1.0 μM or more. The composition according to any one of claims 1 to 3 , which is a composition for carrying out PCR in a temperature range of 10 ° C./sec to 20 ° C./sec. The composition according to any one of claims 1 to 4 , wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from archaea. The composition according to any one of claims 1 to 5 , which is an archaeal DNA polymerase variant having a base analog detection activity in which DNA polymerase belonging to family B is reduced. The composition according to any one of claims 1 to 6 , wherein the PCNA is a PCNA derived from archaea. The composition according to any one of claims 1 to 7 , wherein the PCNA is a mutant PCNA having a DNA polymerase amplification enhancing activity. A reagent for PCR, which is configured to contain the composition according to any one of claims 1 to 8 in a reaction solution. A PCR reagent kit comprising the PCR reagent according to claim 9. PCR using the composition according to any one of claims 1 to 8 by performing 30 to 50 thermal cycles within 20 minutes at a rate of heating or lowering at a rate of 10 ° C./sec or higher. mETHODS. The PCR method according to claim 11 , wherein a biological sample containing an inhibitor is added to the reaction solution containing the composition according to any one of claims 1 to 8.

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