Method for reducing amplification bias in nucleic acid amplification

Abstract

The present invention addresses the problem of providing a method for reducing amplification bias in multiplex PCR.　The present invention has found that amplification bias is reduced by amplifying a nucleic acid using a proliferating cell nuclear antigen. The present invention relates to: a method for reducing amplification bias using a proliferating cell nuclear antigen; a reagent and a kit for use in the method; and others.

Description

Method for reducing amplification bias in nucleic acid amplification

[0001] The present invention relates to a method for reducing amplification bias in nucleic acid amplification, etc. The present invention can be used not only in the field of research but also in clinical diagnosis, etc.

[0002] PCR (polymerase chain reaction) is a method for amplifying a target nucleic acid in a sample by repeating three cycles: (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase. PCR can amplify the target nucleic acid hundreds of thousands of times even from a trace sample of only a few copies, and has come to be widely used not only in research fields but also in forensic fields such as genetic diagnosis and clinical diagnosis, as well as in microbial testing of food and the environment.

[0003] In PCR reactions, it is possible to amplify multiple target nucleic acids at once by using multiple primers or by identifying regions of homology between multiple target nucleic acids and designing primers that correspond to these regions. PCR that amplifies multiple target nucleic acids in a single reaction in this way is called multiplex PCR, and because it can detect multiple target nucleic acids simultaneously, it is used not only for research purposes but also in infectious disease diagnosis. In recent years, with the spread of next-generation sequencers (NGS), multiplex PCR is also being used to amplify sequence libraries.

[0004] In such multiplex PCR, it is expected that all regions will be amplified uniformly. However, due to influences such as PCR reaction conditions and bias in DNA sequences, some regions may be amplified more efficiently than other regions, i.e., amplification bias may occur. When such amplification bias occurs, only the regions that are easily amplified are overdetected, while the detection of other regions is suppressed, leading to the presence of other regions being overlooked. Particularly in NGS, this amplification bias reduces quantitativeness and reduces sequencing accuracy. Furthermore, if the amplification bias is large, more sequence reads are required to confirm the entire gene sequence, which has been a problem in terms of significantly affecting costs.

[0005] In fact, PCR reaction conditions and additives have been investigated to reduce amplification bias during NGS library preparation (Non-Patent Document 1). However, the performance has not yet been sufficient for practical use, and further improvement is needed.

[0006] Genome Biology volume 12, Article number: R18 (2011), Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries, Daniel Aird et al..

[0007] An object of the present invention is to provide a method for reducing amplification bias in multiplex PCR, etc. Another object of the present invention is to provide a reagent kit, etc. suitable for the above-mentioned object.

[0008] The method of the present invention to achieve the above object is a method for reducing amplification bias in multiplex PCR, which is characterized by amplifying nucleic acids using proliferating cell nuclear antigen. That is, the present inventors discovered that the use of proliferating cell nuclear antigen in multiplex PCR reduces amplification bias, and have completed the present invention.

[0009] Representative aspects of the present invention are as follows: [Item 1] A method for reducing amplification bias in multiplex PCR, comprising a step of amplifying nucleic acids in a reaction solution containing a proliferating cell nuclear antigen. [Item 2] The proliferating cell nuclear antigen is: (a) a polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the amino acids at positions 82, 84, and 109 of the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated, (b) a polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the amino acids at positions 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated, or (c) a polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the amino acids at positions 82, 84, and 109 of the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated, and at least one amino acid selected from the group consisting of the amino acids at positions 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated. (d) a polypeptide comprising an amino acid sequence in which one to several amino acids have been mutated in any of the amino acid sequences contained in the polypeptides (a) to (c) other than the amino acids corresponding to positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2, or (e) a polypeptide having 80% or more identity to any of the amino acid sequences contained in the polypeptides (a) to (c) and comprising an amino acid sequence in which amino acids corresponding to one or more positions selected from the group consisting of positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 have been mutated. [Item 3] The method of Item 2, wherein the amino acid corresponding to position 143 is a basic amino acid, or the amino acid corresponding to position 147 is a neutral amino acid, or the amino acid corresponding to position 143 is a basic amino acid and the amino acid corresponding to position 147 is a neutral amino acid.[Item 4] The method of Item 2 or 3, wherein the amino acid corresponding to position 143 is arginine or lysine, the amino acid corresponding to position 147 is alanine or glycine, or the amino acid corresponding to position 143 is arginine or lysine and the amino acid corresponding to position 147 is alanine or glycine. [Item 5] The method of any one of Items 2 to 4, wherein, in the amino acid sequence contained in any one of the polypeptides (a) to (e), the amino acid corresponding to position 73 of the amino acid sequence of SEQ ID NO: 1 or 2 is leucine. [Item 6] The method of any one of Items 2 to 5, wherein the polypeptide (d) is a polypeptide containing an amino acid sequence contained in any one of the polypeptides (a) to (c), in which one to three amino acids have been mutated, other than the amino acids corresponding to positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2. [Item 7] The method according to any one of Items 2 to 6, wherein the mutation is an amino acid substitution, deletion, insertion, and / or addition. [Item 8] The method according to any one of Items 2 to 7, wherein the polypeptide (e) is a polypeptide having an amino acid sequence that is 90% or more identical to any one of the amino acid sequences contained in the polypeptides (a) to (c), and in which amino acids corresponding to one or more positions selected from the group consisting of positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 have been mutated. [Item 9] The method according to any one of Items 1 to 8, wherein at least one of the nucleic acids amplified in the multiplex PCR has a GC ratio of 80% or more. [Item 10] The method according to any one of Items 1 to 9, wherein the reaction solution further comprises a DNA polymerase belonging to Family B. [Item 11] The method according to Item 10, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from Archea.[Item 12] The method according to Item 10, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from a bacterium of the genus Pyrococcus or Thermococcus. [Item 13] A method for improving amplification bias in multiplex PCR for preparing a library for use in a next-generation sequencer, comprising a step of amplifying nucleic acids in a reaction solution containing proliferating cell nuclear antigen. [Item 14] A method for preparing a library for use in a next-generation sequencer, comprising a step of performing multiplex PCR in a reaction solution containing proliferating cell nuclear antigen. [Item 15] A method for improving amplification of a target nucleic acid having a GC ratio of 80% or more in multiplex PCR, comprising a step of amplifying nucleic acids in a reaction solution containing proliferating cell nuclear antigen. [Item 16] Multiplex PCR, comprising a step of amplifying nucleic acids with a GC ratio of 80% or more in a reaction solution containing proliferating cell nuclear antigen.[Item 17] (a) A polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the amino acids at positions 82, 84, and 109 in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (b) A polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the amino acids at positions 139, 143, and 147 in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (c) A polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the amino acids at positions 82, 84, and 109 in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated, and at least one amino acid selected from the group consisting of the amino acids at positions 139, 143, and 147 in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (d) a polypeptide comprising an amino acid sequence in which one to several amino acids have been mutated in any of the amino acid sequences contained in the polypeptides (a) to (c) other than the amino acids at positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2, except for those at positions 82, 84, 109, 139, 143, and 147; or (e) a polypeptide having 80% or more identity to any of the amino acid sequences contained in the polypeptides (a) to (c) and comprising an amino acid sequence in which amino acids at one or more positions selected from the group consisting of 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 have been mutated. [Item 18] A reagent for performing the method of any one of Items 1 to 15 or the multiplex PCR of Item 16, comprising the proliferating cell nuclear antigen, the reagent comprising the reagent of Item 17.

[0010] The present invention can reduce amplification bias in multiplex PCR. This reduces the oversight of regions that are difficult to amplify, enabling more accurate amplification. It also improves sequencing accuracy and cost performance in NGS.

[0011] Normalized coverage of KOD (with and without PCNA addition) is shown. Normalized coverage of KOD (with and without PCNA addition) is shown. Normalized coverage of PS (with and without PCNA addition) is shown. An alignment of the amino acid sequences of various PCNAs is shown.

[0012] The present invention will be described in further detail below with reference to embodiments of the present invention.

[0013] In this specification, simplified alphabetical symbols may be used for nucleotide sequences, amino acid sequences, and their individual components, all of which follow the conventions of molecular biology or genetic engineering. Furthermore, in this specification, a notation such as "D143A" is used to concisely indicate mutations in amino acid sequences. "D143A" indicates that aspartic acid at position 143 has been substituted with alanine, i.e., it indicates the type and location of the amino acid before the substitution, and the type of amino acid after the substitution. Furthermore, unless otherwise specified, the sequence numbers described in this specification correspond to the sequence numbers listed in the sequence listing. Furthermore, in the case of multiple mutants, the above notations are connected with a " / ". For example, "D143A / D147A" indicates that aspartic acid at position 143 has been substituted with alanine, and that aspartic acid at position 147 has been substituted with alanine. Furthermore, in this specification, the term "mutant PCNA" refers to an amino acid sequence that is different from that of conventionally known PCNAs, and does not distinguish between artificial and natural mutations. Furthermore, in the present specification, "corresponding" in the phrase "an amino acid corresponding to position ..." refers to an amino acid that corresponds to the position in the amino acid sequence of SEQ ID NO: 1 or 2 when the amino acid sequence of a proliferating cell nuclear antigen having an amino acid sequence that is not completely identical to the amino acid sequence of SEQ ID NO: 1 or 2 is aligned with the amino acid sequence of SEQ ID NO: 1 or 2.

[0014] (1) Method for Reducing Amplification Bias of the Present Invention The method for reducing amplification bias of the present invention is a method for reducing amplification bias in multiplex PCR, characterized in that a reaction solution contains proliferating cell nuclear antigen (PCNA). Here, reducing the amplification bias means that the amplification bias of multiplex PCR using a reaction solution containing PCNA is lower than that of multiplex PCR using a reaction solution not containing PCNA.

[0015] (2) Multiplex PCR In the methods of the present invention (as used herein, the term "methods of the present invention" is intended to encompass the methods, multiplex PCRs, reagents, kits, and the like described in Sections 1 to 18), multiplex PCR refers to PCR that amplifies multiple target nucleic acids in a single reaction. Multiplex PCR may be multiplex PCR that includes multiple primers and simultaneously amplifies multiple target nucleic acids, or multiplex PCR in which primers corresponding to regions of homology among multiple target nucleic acids are designed and multiple target nucleic acids are amplified with a single primer pair. Furthermore, PCR in which adapters are ligated to multiple gene fragments and primers that hybridize to the adapter regions, such as in NGS library amplification, can also be included in multiplex PCR because it can simultaneously amplify multiple target nucleic acids. Multiplex PCR is not limited to these exemplified multiplex PCRs.

[0016] (3) PCNA The proliferating cell nuclear antigen (also referred to herein as PCNA or Proliferating Cell Nuclear Antigen) used in the methods of the present invention is a type of PCR enhancing factor. The PCNA is preferably one that can be loaded onto DNA alone, is heat-resistant enough to withstand the thermal cycle of PCR, and preferably remains active after PCR. It is more preferably one that remains active even after heat treatment at 80°C for 30 minutes, and retains 50% or more, more preferably 70% or more, and even more preferably 90% or more of its activity. Furthermore, PCNA may be a known wild-type PCNA or a mutant of a known wild-type PCNA.

[0017] PCNA typically forms multimers and forms a ring-like structure. Loading onto DNA refers to passing DNA through the ring-like structure formed by PCNA multimers. PCNA can typically only load onto DNA in cooperation with a factor called RFC (replication factor C). PCNA that loads onto DNA alone refers to wild-type or mutant PCNA that has the property of easily passing DNA through the ring-like structure of PCNA multimers even without RFC, achieved by mutating the site involved in PCNA multimer formation and destabilizing multimer formation. PCNA that can be used in the methods of the present invention is preferably one that can load onto DNA even without RFC and promote the elongation reaction of DNA polymerase.

[0018] More preferably, the PCNA used in the methods of the present invention is derived from bacteria of the genus Pyrococcus or Thermococcus. PCNA derived from the genus Pyrococcus includes, but is not limited to, PCNA isolated from Pyrococcus furiosus (SEQ ID NO: 2), Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii. PCNA derived from the genus Thermococcus includes, but is not limited to, PCNA isolated from Thermococcus kodakaraensis (SEQ ID NO: 1), Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9°N-7), Thermococcus sp. KS-1, Thermococcus celeri, or Thermococcus siculi.

[0019] PCNA is preferably: (a) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 2 in which at least one amino acid selected from the group consisting of the amino acids at positions 82, 84, and 109 has been mutated (hereinafter also referred to as polypeptide A); (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 2 in which at least one amino acid selected from the group consisting of the amino acids at positions 139, 143, and 147 has been mutated (hereinafter also referred to as polypeptide B); (c) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 2 in which at least one amino acid selected from the group consisting of the amino acids at positions 82, 84, and 109 and at least one amino acid selected from the group consisting of the amino acids at positions 139, 143, and 147 has been mutated (hereinafter also referred to as polypeptide C); (d) a polypeptide comprising an amino acid sequence in which one to several amino acids have been mutated among the amino acids at positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 in any of the amino acid sequences contained in the polypeptides (a) to (c); or (e) a polypeptide having an amino acid sequence that has 80% or more identity with any of the amino acid sequences contained in the polypeptides (a) to (c) and in which amino acids at one or more positions selected from the group consisting of 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 have been mutated.

[0020] The 82nd amino acid in the amino acid sequence of SEQ ID NO: 1 or 2 is arginine, and examples of the amino acid substituting for the 82nd arginine include cysteine, alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, serine, threonine, aspartic acid, glutamic acid, histidine, and lysine. Cysteine, alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, serine, threonine, aspartic acid, and glutamic acid are preferred, and cysteine, alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, serine, threonine, aspartic acid, and glutamic acid are more preferred.

[0021] The 84th amino acid in the amino acid sequence of SEQ ID NO: 1 or 2 is lysine, and examples of the amino acid substituting the 84th lysine include alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, aspartic acid, glutamic acid, arginine, and histidine. Alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, aspartic acid, and glutamic acid are preferred, and alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, aspartic acid, and glutamic acid are more preferred.

[0022] The 109th amino acid in the amino acid sequence of SEQ ID NO: 1 or 2 is arginine. Examples of the amino acid substituting for arginine at position 109 include glutamic acid, alanine, aspartic acid, proline, glycine, leucine, isoleucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, histidine, and lysine, and glutamic acid, alanine, aspartic acid, proline, glycine, leucine, isoleucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, and threonine are preferred.

[0023] The 139th amino acid in the amino acid sequence of SEQ ID NO: 1 or 2 is glutamic acid, and examples of the amino acid substituting for the 139th glutamic acid include alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, and aspartic acid. Alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, and lysine are preferred, and alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, and threonine are more preferred.

[0024] The 143rd amino acid in the amino acid sequence of SEQ ID NO: 1 is glutamic acid, and examples of amino acids that can be substituted for the 143rd glutamic acid include arginine, lysine, histidine, alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, and aspartic acid, with basic amino acids such as arginine, lysine, and histidine, or alanine being preferred.

[0025] The 143rd amino acid in the amino acid sequence of SEQ ID NO: 2 is aspartic acid, and examples of amino acids that substitute for the 143rd aspartic acid include arginine, lysine, histidine, alanine, proline, glycine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, and glutamic acid. For example, arginine, lysine, histidine, and alanine are preferred, and arginine and lysine are more preferred.

[0026] The 147th amino acid in the amino acid sequence of SEQ ID NO: 1 or 2 is aspartic acid, and examples of amino acids substituting the 147th aspartic acid include alanine, glycine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, lysine, and glutamic acid. For example, alanine, glycine, proline, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, cysteine, asparagine, glutamine, serine, threonine, arginine, histidine, and lysine are preferred, alanine, glycine, valine, leucine, isoleucine, and histidine are more preferred, and alanine or glycine is even more preferred.

[0027] More preferably, PCNA is one in which the amino acid corresponding to position 143 of the amino acid sequence of SEQ ID NO: 1 has been mutated to a basic amino acid, or one in which the amino acid corresponding to position 147 has been mutated to a neutral amino acid. Examples of neutral amino acids include naturally occurring glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine. Glycine, alanine, valine, leucine, and isoleucine are preferred, glycine and alanine are more preferred, and alanine is even more preferred, as it has the least effect on the conformation of the region surrounding the substitution site. Examples of naturally occurring basic amino acids include arginine, histidine, and lysine. Arginine and lysine are preferred, and arginine is more preferred.

[0028] In PCNA derived from Thermococcus kodakaraensis (SEQ ID NO: 1) and PCNA from Pyrococcus furiosus (SEQ ID NO: 2), the sites involved in PCNA multimerization include the 82nd, 84th, and 109th amino acids in the N-terminal region and the 139th, 143rd, and 147th amino acids in the C-terminal region (hereinafter, these six amino acids are also referred to as amino acids at predetermined positions). The N-terminal region is positively charged, and the C-terminal region is negatively charged, and it is thought that they interact to form multimers.

[0029] PCNA may be a polypeptide consisting of an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, and / or added (hereinafter, these are also collectively referred to as "mutations") in the amino acid sequence of any one of the above polypeptides A to C, as long as the mutations of the amino acids corresponding to the predetermined positions are preserved, and PCNA is preferably loaded alone into DNA. The number of mutated amino acids may be 1 to 49, 1 to 24, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, etc., with 1 to 4 being preferred and 1 to 3 being more preferred. One to several mutations can be introduced into the DNA encoding the PCNA of the present invention using known techniques such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Variant PCNA monomers can also be obtained by other methods, such as ultraviolet irradiation. Variant PCNA monomers also include naturally occurring variants (e.g., single nucleotide polymorphisms) that arise from individual differences, species, or genus differences among microorganisms that possess PCNA.

[0030] PCNA may be a polypeptide consisting of an amino acid sequence that has 80% or more identity to the amino acid sequence of any one of the above polypeptides A to C, so long as the amino acid mutations corresponding to the specified positions are preserved. PCNA is preferably loaded onto DNA alone. Preferably, the identity between the amino acid sequence of PCNA and the amino acid sequence of any one of the above polypeptides A to C is 85% or more, more preferably 88% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Polypeptides consisting of amino acid sequences with a certain level of identity can be prepared based on known genetic engineering techniques, as described above.

[0031] It is particularly preferred that the proliferating cell nuclear antigen consists of a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1, 2, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27.

[0032] Various methods are known for calculating the identity of amino acid sequences. For example, it can be calculated using an analytical tool that is commercially available or available via an electronic communication line (Internet). In this specification, the identity of amino acid sequences is calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / with default (initial setting) parameters.

[0033] Furthermore, in order to increase the expression level, PCNA used in the methods of the present invention may consist of a polypeptide containing an amino acid sequence in which the methionine at position 73 in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated. More preferably, PCNA consisting of a polypeptide containing an amino acid sequence mutated to M73L is exemplified, but is not limited to this.

[0034] More specifically, PCNA that can be used in the methods of the present invention includes PCNA mutants described in WO2007 / 004654 and Japanese Patent No. 6741061, as well as sequences in which the amino acid corresponding to the 147th position in the amino acid sequence of SEQ ID NOs: 1 and 2 is substituted with alanine or glycine (D147A, D147G), sequences in which the amino acid corresponding to the 143rd position is substituted with arginine or lysine (E143R, E143K or D143R, D143K), sequences in which the amino acids corresponding to the 82nd and 143rd positions are changed to alanine (R82A / E143A, or R82A / D143A), sequences in which the amino acid residues corresponding to the 109th and 143rd positions are changed to alanine (R109A / E143A, or R109A / D143A), and PCNAs consisting of polypeptides containing, but not limited to, these.

[0035] In a preferred embodiment, the amino acid sequence contained in the polypeptide constituting PCNA may have one or more amino acid mutations selected from M73L, R82C, R82A, R109E, R109A, E139R, E139A, D143R, D143A, D143H, D143K, E143R, E143A, E143H, E143K, D147R and D147A, preferably one or more amino acid mutations selected from M73L, D143A, D143A / D147A, D143R, R109A / D143A, R82A / D143A and D147A.

[0036] The above and below explanations using the amino acid sequence of SEQ ID NO: 1 or 2 as an example also apply to PCNAs other than those specifically presented in this specification. For example, as shown in Figure 4, for the mutation positions of PCNAs other than PCNAs consisting of polypeptides containing the amino acid sequences of SEQ ID NO: 1 and 2, the positions of amino acids corresponding to amino acids involved in multimer formation, such as the 82nd, 84th, 109th, 139th, 143rd, and 147th amino acids in the amino acid sequence of SEQ ID NO: 1, are shown.

[0037] The method for producing PCNA that can be used in the methods of the present invention is not particularly limited, and PCNA can be produced using methods known to those skilled in the art. For example, PCNA can be produced by expressing a vector incorporating DNA having a base sequence encoding the amino acid sequence of the above-mentioned polypeptide in cells such as Escherichia coli, and purifying and isolating the resulting PCNA, if necessary, using standard protein purification methods. Such DNA also includes DNA that differs due to codon degeneracy.

[0038] The above DNA has a base sequence that is 80% or more, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more homologous to the base sequence shown in SEQ ID NO: 3 or 4, as long as the amino acid mutation corresponding to a specific position in the protein it encodes is preserved, and it is preferable that the protein encoded by the base sequence is PCNA, which is loaded onto DNA alone.

[0039] Various methods are known for calculating the identity of nucleotide sequences. For example, calculations can be performed using analytical tools that are commercially available or available via telecommunications lines (Internet). In this specification, the homology value (%) of nucleotide sequences is calculated by performing a search using the Advanced BLAST 2.1 homology algorithm of the National Center for Biotechnology Information (NCBI) using the blastn program with various parameters set to default values.

[0040] The above DNA may be DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4, as long as the amino acid mutation corresponding to a predetermined position in the protein it encodes is preserved, and it is preferable that the protein encoded by the base sequence is PCNA that is loaded onto the DNA alone.

[0041] An appropriate vector can be selected taking into consideration the type of host cell, and examples thereof include plasmid vectors, cosmid vectors, phage vectors, viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, herpes virus vectors, etc.), etc. It is also possible to use vectors suitable for use in filamentous fungi as hosts, or vectors suitable for self-cloning.

[0042] The host cells are not particularly limited as long as they are capable of expressing DNA encoding PCNA to produce PCNA. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, fungi, insect cells, cultured plant cells, and mammalian cells can be used.

[0043] Any method may be used to purify PCNA expressed by host cells, but examples include the following: Host cells are cultured in a nutrient medium, harvested, and then disrupted or extracted by enzymatic or physical disruption to obtain a crude enzyme solution. The resulting crude enzyme extract is heat-treated, for example, at 80°C for 30 minutes, followed by ammonium sulfate precipitation to recover the PCNA fraction. This crude enzyme solution can be desalted by gel filtration using Sephadex G-25 (Amersham Pharmacia Biotech). Following this procedure, the crude enzyme solution can be purified by Q Sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified to the extent that it exhibits an almost single band on SDS-PAGE.

[0044] When obtaining PCNA, it is preferable to mutate the methionine corresponding to position 73 in the amino acid sequence of SEQ ID NO: 1 or 2 to leucine (M73L) in order to increase the expression level of PCNA. When PCNA is prepared as a recombinant protein using Escherichia coli as a host, in addition to the original translation protein from the initiation Met, a protein of approximately 20 kDa whose N-terminus starts at residue 73 may be generated as a by-product. However, M73L suppresses the production of this approximately 20 kDa protein, and the PCNA (M73L) prepared in this manner has properties identical to those of normal PCNA. Therefore, proteins starting from M73L are also included in the PCNA referred to in the present invention.

[0045] PCNA may be modified in various ways. For example, by inserting DNA encoding PCNA and other appropriate DNA into the same vector and using the vector to produce a recombinant protein, PCNA can be obtained as a recombinant protein linked to any peptide or protein. Furthermore, PCNA may be modified by the addition of sugar chains and / or lipids, or by N- or C-terminal processing. These modifications can simplify the extraction and purification of recombinant proteins, or can impart biological functions.

[0046] PCNA can form multimers by bonding at an interface between the N-terminal region of one monomer and the C-terminal region of the other monomer. Because this bonding is reversible, at least a portion of the PCNA expressed as a monomer may form multimers.

[0047] Whether PCNA can be loaded onto DNA alone can be evaluated by PCR. For example, the target PCNA is added to a standard PCR reaction solution containing template DNA, buffer, magnesium, dNTPs, primers, and DNA polymerase, and the amplification amount is compared with that of a PCR reaction solution without PCNA or with PCNA, which is known not to load onto DNA alone, to confirm whether PCNA can be loaded onto DNA alone. The addition of PCNA that does not load onto DNA alone does not change the PCR amplification amount, but rather tends to reduce it. On the other hand, mutants that can be loaded onto DNA alone can achieve superior amplification amounts compared to those without PCNA or those with PCNA, which is known not to load onto DNA alone.

[0048] In the present invention, evaluation of "whether PCNA can be loaded onto DNA by itself" (evaluation of amplification enhancing activity) is performed according to the following method: 10x PCR Buffer included with KOD-Plus-Ver. 2 (manufactured by Toyobo Co., Ltd.) was used, and 1x PCR Buffer and 1.5 mM MgSO were used. ４, 0.2 mM dNTPs (dATP, dUTP, dCTP, dGTP) containing dUTP instead of dTTP, 15 pmol of primers described in SEQ ID NOs: 5 and 6 that amplify approximately 1.3 kb, 10 ng of human genomic DNA (manufactured by Roche), 1U KOD-Plus-, 250 ng of PCNA to be evaluated was added to a 50 μl reaction solution containing 94 ° C, 30 seconds of pre-reaction, followed by 35 cycles of 98 ° C, 10 seconds → 65 ° C, 30 seconds → 68 ° C, 1 minute 30 seconds. After the reaction is complete, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplified DNA fragment of approximately 1.3 kb under ultraviolet irradiation was compared with that containing wild-type PCNA or that not containing PCNA to evaluate whether PCNA can be loaded onto DNA alone. PCNA, which can be loaded onto DNA by itself, increases the amount of amplification when added.

[0049] (4) Amplification Bias In the present invention, amplification bias refers to regions not being amplified uniformly in multiplex PCR. Such evaluation can be performed by confirming the uniformity of amplification in multiplex PCR, but is not limited thereto. Methods for confirming the uniformity of amplification in multiplex PCR include, for example, comparing band intensities by electrophoresis or subjecting amplified products to NGS and analyzing sequence information. A method for analyzing GC bias in sequence information includes, for example, using an analysis tool called Picard. In this method, a graph is created with the GC ratio on the horizontal axis and the normalized coverage on the vertical axis. The closer the vertical axis is to 1, the less amplification bias there is. When the vertical axis approaches 1 in the GC ratio range of approximately 40% to 90%, particularly in the range of 80% to 90%, this indicates that an ideal reduction in amplification bias has been achieved. The graph may be trapezoidal.

[0050] When multiplex PCR is performed according to the present invention, the GC content of the nucleic acid region to be amplified is not particularly limited, but may be 80% or more, 85% or more, 50 to 90%, 60 to 90%, 60 to 80%, 80 to 90%, etc.

[0051] (5) DNA Polymerase The DNA polymerase used in the method of the present invention is not particularly limited as long as it can be used for PCR, but is preferably a DNA polymerase belonging to Family B. The DNA polymerase belonging to Family B is preferably a DNA polymerase derived from Archea.

[0052] (6) DNA polymerases derived from archaea Examples of DNA polymerases derived from archaea belonging to Family B include DNA polymerases derived from bacteria of the genus Pyrococcus and Thermococcus. DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii. DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9°N-7), Thermococcus sp. KS-1, Thermococcus celeri, or Thermococcus siculi. PCR enzymes using these DNA polymerases are commercially available, including Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), Pwo (Roche), and PrimeSTAR (TaKaRa).

[0053] Among these, KOD DNA polymerase, which has excellent extensibility and thermostability, is preferred from the viewpoint of PCR efficiency.

[0054] Furthermore, the DNA polymerase used in the method of the present invention may be a mutant of the above-mentioned DNA polymerase, and may be a mutant that has been subjected to a mutation in the 3'-5' exonuclease region and / or a mutation that has reduced base analog detection activity, and is not particularly limited.

[0055] (7) Method for Reducing Amplification Bias In the method of the present invention, as long as the reaction solution or reagent contains PCNA, the reaction solution or reagent may contain other components that can be used for multiplex PCR, such as a DNA polymerase, preferably a DNA polymerase belonging to family B, but the other components are not limited to these.

[0056] In the method of the present invention, typical conditions for multiplex PCR are shown below, but are not limited to these. (a) DNA polymerase, preferably a DNA polymerase belonging to Family B; (b) a pair of primers, one of which is complementary to the DNA extension product of the other primer; (c) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)); (d) a buffer solution containing magnesium ions, ammonium ions, and / or potassium ions; and (e) PCNA are mixed. The temperature of the reaction solution is raised and lowered using a thermal cycler or the like, thereby repeating the thermal cycle of (1) DNA denaturation, (2) primer annealing, and (3) primer extension, thereby amplifying a specific DNA fragment. In the above PCR method, a PCR enhancer, BSA, a nonionic surfactant, or the like may also be used, if necessary.

[0057] In the above PCR method, an antibody having activity to inhibit the polymerase activity and / or 3'-5' exonuclease activity of the thermostable DNA polymerase may be further used, if necessary. Examples of the antibody include monoclonal antibodies and polyclonal antibodies. This reaction composition is particularly effective in increasing PCR sensitivity and reducing nonspecific amplification.

[0058] A feature of the present invention is that amplification bias can be suppressed by using PCNA in multiplex PCR. Therefore, the present invention may include a method for improving amplification bias in multiplex PCR for preparing a library for use in a next-generation sequencer, comprising the step of amplifying nucleic acids in a reaction solution containing proliferating cell nuclear antigen. The above-described description of the method for reducing amplification bias of the present invention also applies to this method. Furthermore, the present invention may include a method for preparing a library for use in a next-generation sequencer, comprising the step of performing multiplex PCR in a reaction solution containing proliferating cell nuclear antigen. The above-described description of the method for reducing amplification bias of the present invention also applies to this method. Furthermore, the present invention may include a method for improving amplification of a target nucleic acid having a GC ratio of 80% or more in multiplex PCR, comprising the step of amplifying nucleic acids in a reaction solution containing proliferating cell nuclear antigen. The above-described description of the method for reducing amplification bias of the present invention also applies to this method. The improvement here may be a greater amount of target nucleic acid amplified by multiplex PCR using PCNA than by multiplex PCR without PCNA. The present invention also encompasses multiplex PCR, which includes a step of amplifying nucleic acids with a GC ratio of 80% or more in a reaction solution containing a proliferating cell nuclear antigen, etc. The above description of the method for reducing amplification bias of the present invention also applies to this multiplex PCR.

[0059] (8) Reagents and Kits for Carrying Out the Methods of the Present Invention The reagents and kits for carrying out the methods of the present invention contain a proliferating cell nuclear antigen, preferably a proliferating cell nuclear antigen to be loaded onto DNA alone, and other components are not particularly limited. The reagents and kits of the present invention may also contain a DNA polymerase, preferably a DNA polymerase belonging to Family B, dNTPs, primers, and other additives commonly known in PCR reactions. The above description of the method for reducing amplification bias of the present invention also applies to these reagents and kits.

[0060] The reagent and kit of the present invention may, for example, be configured to include the following (a) to (e), but is not limited to these: (a) an archaeal DNA polymerase mutant having reduced base analog detection activity; (b) a pair of primers, one of which is complementary to the DNA extension product of the other primer; (c) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)); (d) a buffer solution containing magnesium ions, ammonium ions, and / or potassium ions; and (e) PCNA.

[0061] The reagent and kit may further contain other reagents, such as BSA and non-ionic surfactants, as needed.

[0062] Example 1: Preparation of KOD-PCNA Mutant A plasmid containing a mutant heat-stable PCNA gene derived from Thermococcus kodakaraensis KOD1 strain was prepared. The DNA template used for mutagenesis was PCNA (SEQ ID NO: 3) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 strain cloned into pBluescript. Mutagenesis was performed using the KOD-Plus-Mutagenesis Kit (Toyobo) according to the instruction manual. The mutant was confirmed by sequencing. The resulting plasmid was transformed into Escherichia coli DH5α and used for enzyme preparation.

[0063] (Example 2) Preparation of Pfu-PCNA Mutant A plasmid containing a mutant thermostable PCNA gene derived from Pyrococcus furiosus was prepared. The DNA template used for mutagenesis was PCNA (SEQ ID NO: 4) (pPfuPCNA) derived from a Pyrococcus furiosus strain cloned into pBluescript. Mutagenesis was performed using the KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by sequencing. Escherichia coli DH5α was transformed with the obtained plasmid and used to prepare the enzyme.

[0064] The plasmids prepared in Examples 1 and 2 are shown in Table 1.

[0065]

[0066] Example 3: Preparation of mutant thermostable PCNA The bacterial cells obtained in Example 1 were cultured as follows. First, 80 mL of sterilized TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. This medium was inoculated with Escherichia coli DH5α (plasmid-transformed strain) (test tube) that had been previously cultured in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin at 37°C for 16 hours, and the culture was aerated at 37°C for 16 hours. The cells were collected from the culture medium by centrifugation and suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA). The cells were then disrupted by sonication to obtain a cell lysate. The cell lysate was then treated at 80°C for 15 minutes, and the insoluble fraction was removed by centrifugation. The cell lysate was then subjected to nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and Q-Sepharose chromatography. Finally, the solution was replaced with a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin) to obtain a mutant heat-resistant PCNA.

[0067] (Example 4) Thermus thermophilus Library Amplification A library derived from Thermus thermophilus with a GC rate of 70% was amplified using various enzymes, and the sequencing data was compared. The Thermus thermophilus library is a Thermus thermophilus genome fragment with a consensus sequence at the end. The Thermus thermophilus genome was fragmented and tagmented (a consensus sequence was added to the end) using a KAPA HyperPlus Kit (KAPA) and a KAPA Unique Dual-Indexed Adapter Kit (KAPA), and purified using APMure (Beckman Coulter). In amplifying the library, KOD polymerase was evaluated using KOD-Plus- (Toyobo), and PrimeSTAR polymerase was evaluated using PrimeSTAR HS DNA Polymerase (TaKaRa). Primers used for amplification hybridized to the common sequences added to the ends of the library, allowing for multiplex PCR. The mutant heat-stable PCNA prepared in Example 3 was used as PCNA. The reaction solution preparation is shown in Table 2.

[0068]

[0069] Amplification was performed for 14 cycles: 72°C for 3 minutes, 95°C for 30 seconds, 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The amplified products purified with AMPure were sequenced using an Illumina MiSeq. The sequencing data was aligned to 200,000 reads and evaluated using Picard (Collect GC matrix) and FastQC.

[0070] The results of GC bias evaluation using Picard (normalized coverage relative to GC rate) are shown in Figures 1 to 3. The horizontal axis of the figure shows the GC rate, and the vertical axis shows normalized coverage. The closer the normalized coverage is to 1, the more reduced the bias is. In Figures 1 and 2, PCNA1 (KOD-PCNA M73L / D147A) and PCNA2 (Pfu-PCNA M73L / D143R) are added to the KOD reaction system, respectively. Without the addition of PCNA, the normalized coverage deteriorates dramatically when the GC rate exceeds 80%, but the addition of PCNA improves this deterioration. Figure 3 similarly shows the results when PCNA2 (Pfu-PCNA M73L / D143R) is added to the PrimeSTAR (PS) reaction system. For PrimeSTAR, the addition of PCNA was also confirmed to improve normalized coverage at high GC ratios.

[0071] Similarly, KOD-PCNA M73L / D147G, KOD-PCNA M73L / E143R, KOD-PCNA M73L / E143K, Pfu-PCNA M73L / D143K, Pfu-PCNA M73L / D147A, and Pfu-PCNA M73L / D147G were used in the KOD reaction system, and KOD-PCNA M73L / D147A, KOD-PCNA M73L / E143R, KOD-PCNA M73L / E143K, Pfu-PCNA M73L / D143K, Pfu-PCNA M73L / D147A, and Pfu-PCNA were used in the PrimeSTAR (PS) reaction system. Addition of M73L / D147G also resulted in an improvement in normalized coverage.

[0072] Table 3 shows the coverage rate of the Thermus thermophilus genome when the sequencing depth (depth) analyzed by FastQC is 1 to 5. X1 to X5 indicate the depth (the number of times the Thermus thermophilus genome has been sequenced), and the coverage rate indicates the proportion of sequenced regions in the entire length of the Thermus thermophilus genome. For example, X5 indicates the proportion of regions with five or more sequence reads in the entire length of the genome, and a higher percentage indicates a larger number of sequenced regions.

[0073]

[0074] The coverage rate obtained with the addition of PCNA was better. In this example, the test was conducted with a relatively small number of reads, 200,000, and it is believed that the addition of PCNA reduced the amplification bias and improved the coverage rate. The amount of sequencing (number of reads) is directly related to costs. The addition of PCNA resulted in a higher coverage rate even with the same number of reads, which also contributed to cost reduction.

[0075] The present invention provides a method for reducing amplification bias in multiplex PCR. The present invention makes it possible to reduce amplification bias, reducing oversights in multiplex PCR and improving accuracy. Furthermore, since the amplification bias is reduced by using the present invention, sequencing accuracy is improved in NGS, and a high coverage rate can be achieved even with a small number of sequence reads, contributing to cost reduction.

Claims

1. A method for reducing amplification bias in multiplex PCR, comprising the step of amplifying nucleic acid in a reaction solution containing proliferating cell nuclear antigen.

2. The proliferating cell nuclear antigen is: (a) a polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 82nd, 84th and 109th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (b) a polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 139th, 143rd and 147th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; or (c) a polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 82nd, 84th and 109th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated, and at least one amino acid selected from the group consisting of the 139th, 143rd and 147th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (d) a polypeptide comprising an amino acid sequence in which one to several amino acids have been mutated among the amino acids at positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 in any of the amino acid sequences contained in the polypeptides (a) to (c); or (e) a polypeptide comprising an amino acid sequence which has 80% or more identity with any of the amino acid sequences contained in the polypeptides (a) to (c) and comprises an amino acid sequence in which one or more amino acids have been mutated corresponding to positions selected from the group consisting of the 82nd, 84th, 109th, 139th, 143rd, and 147th of the amino acid sequence of SEQ ID NO: 1 or 2.

3. The method according to claim 2, wherein the amino acid corresponding to the 143rd position is a basic amino acid, the amino acid corresponding to the 147th position is a neutral amino acid, or the amino acid corresponding to the 143rd position is a basic amino acid and the amino acid corresponding to the 147th position is a neutral amino acid.

4. The method of claim 2, wherein the amino acid corresponding to the 143rd position is arginine or lysine, the amino acid corresponding to the 147th position is alanine or glycine, or the amino acid corresponding to the 143rd position is arginine or lysine and the amino acid corresponding to the 147th position is alanine or glycine.

5. The method according to claim 2, wherein the amino acid at position 73 of the amino acid sequence of SEQ ID NO: 1 or 2 in the amino acid sequence contained in any one of the polypeptides (a) to (e) is leucine.

6. The method according to claim 2, wherein the polypeptide (d) is a polypeptide containing an amino acid sequence in which one to three amino acids have been mutated among the amino acids other than those at positions 82, 84, 109, 139, 143 and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 in any of the amino acid sequences contained in the polypeptides (a) to (c).

7. The method of claim 2, wherein the mutation is an amino acid substitution, deletion, insertion and / or addition.

8. The method according to claim 2, wherein the polypeptide (e) is a polypeptide having an amino acid sequence that has 90% or more identity with any of the amino acid sequences contained in the polypeptides (a) to (c) and has a mutated amino acid sequence corresponding to one or more positions selected from the group consisting of the 82nd, 84th, 109th, 139th, 143rd and 147th positions of the amino acid sequence of SEQ ID NO: 1 or 2.

9. The method according to claim 1 or 2, wherein at least one of the nucleic acids amplified in the multiplex PCR has a GC ratio of 80% or more.

10. The method of claim 1, wherein the reaction solution further comprises a DNA polymerase belonging to family B.

11. The method according to claim 10, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from Archea.

12. The method according to claim 10, wherein the DNA polymerase belonging to family B is a DNA polymerase derived from a bacterium of the genus Pyrococcus or Thermococcus.

13. A method for improving amplification bias in multiplex PCR for preparing a library for use in a next-generation sequencer, comprising a step of amplifying nucleic acid in a reaction solution containing proliferating cell nuclear antigen.

14. A method for preparing a library for use in a next-generation sequencer, comprising a step of performing multiplex PCR in a reaction solution containing proliferating cell nuclear antigen.

15. A method for improving amplification of a target nucleic acid having a GC ratio of 80% or more in multiplex PCR, comprising the step of amplifying the nucleic acid in a reaction solution containing proliferating cell nuclear antigen.

16. A multiplex PCR comprising a step of amplifying a nucleic acid having a GC ratio of 80% or more in a reaction solution containing a proliferating cell nuclear antigen.

17. (a) A polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 82nd, 84th, and 109th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (b) A polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 139th, 143rd, and 147th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (c) A polypeptide comprising an amino acid sequence in which at least one amino acid selected from the group consisting of the 82nd, 84th, and 109th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated, and at least one amino acid selected from the group consisting of the 139th, 143rd, and 147th amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 has been mutated; (d) a polypeptide containing an amino acid sequence in which one to several amino acids are mutated among the amino acids at positions 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2 in any of the amino acid sequences contained in the polypeptides (a) to (c); or (e) a polypeptide containing an amino acid sequence that has 80% or more identity with any of the amino acid sequences contained in the polypeptides (a) to (c) and contains an amino acid sequence in which one or more amino acids are mutated corresponding to positions selected from the group consisting of 82, 84, 109, 139, 143, and 147 of the amino acid sequence of SEQ ID NO: 1 or 2. The method according to any one of claims 1 and 13 to 15 or the reagent for performing multiplex PCR according to claim 16, comprising a proliferating cell nuclear antigen.

18. A kit for carrying out the method according to any one of claims 1 and 13 to 15 or the multiplex PCR according to claim 16, comprising the reagent according to claim 17.

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