Chimeric ligase

Abstract

To provide a chimeric ligase having improved discrimination ability to a mismatched base pair.SOLUTION: The present invention provides a chimeric ligase, wherein an OB-fold domain or an adenylation domain in a ligase derived from a thermophilic bacterium is substituted with a corresponding domain of a different species derived from a thermophilic bacterium, and the ability to discriminate a mismatched base pair is improved as compared with the ligase before substitution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention broadly relates to chimeric ligases and the like. [Background technology]

[0002] Ligases are essential enzymes in genetic engineering experiments, etc. Some ligases can discriminate between mismatched base pairs near the junction, and such ligases can be used to detect genes with mutations. Specifically, for example, when a nucleic acid sample containing two adjacent probes complementary to a mutant sequence and the mutant sequence is treated with a ligase capable of discriminating mismatched base pairs, the two probes hybridized to the mutant sequence are joined by the ligase because they do not form mismatched base pairs with the mutant sequence. On the other hand, when a sample containing two adjacent probes complementary to a mutant sequence and a nucleic acid sample containing a wild-type sequence is treated with a ligase capable of discriminating mismatched base pairs, the two probes hybridized to the wild-type sequence are joined by the ligase only at a low frequency because they form mismatched base pairs with the wild-type sequence. Therefore, when a nucleic acid sample contains a mutant sequence, a product containing two probes joined by the ligase is obtained with a higher frequency, and the presence or absence of a mutation can be determined by analyzing the product. Therefore, it is possible to detect a gene having a mutation using a ligase capable of discriminating mismatched base pairs.

[0003] Among ligases capable of discriminating between mismatched base pairs, thermostable ligases derived from thermophilic bacteria are extremely useful because they can be applied to gene mutation detection methods, such as the LDR (ligase detection reaction) method and the LCR (ligation chain reaction) method, which involve a heat treatment step. Known examples of ligases derived from thermophilic bacteria capable of discriminating between mismatched base pairs include ligases derived from Thermus thermophilus, Thermus filiformis, Thermus aquaticus, and Thermus species (Non-Patent Documents 1 to 4).

[0004] However, the accuracy of the ability of these thermophilic ligases to discriminate mismatched base pairs varies depending on the bacteria from which they are derived, and the factors that determine the accuracy of the discrimination ability have not yet been elucidated. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Gregory JS Lohman et al. A high-throughput assay for the comprehensive profiling of DNA ligase fidelity. Nucleic Acids Research. 2016, 44(2), e14. [Non-patent document 2] Jae Young Lee et al. Crystal structure of NAD+-dependent DNA ligase: modular architecture and functional implications. The EMBO Journal. 2000, 19, 1119-1129. [Non-patent document 3] Greg Lohman. Substrate specificity and mismatch discrimination in DNA ligases. New England Biolabs, Feature Articles. [Non-patent document 4] Jie Tong. Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D. Nucleic Acids Research. 1999, 27(3), 788-794. Summary of the Invention [Problem to be solved by the invention]

[0006] The problem that the invention aims to solve is to identify the region in the amino acid sequence of a ligase derived from a thermophilic bacterium that is involved in the ability to discriminate between mismatched base pairs, and to provide a novel ligase with improved discrimination ability against mismatched base pairs by modifying this region. [Means for solving the problem]

[0007] As a result of extensive research conducted by the present inventors to solve the above-mentioned problems, they found that replacing the oligomer / oligonucleotide / oligosaccharide-binding domain (OB-fold domain) or adenylation domain of a thermophilic ligase with the corresponding domain from a different species of thermophilic bacterium can sometimes improve the ability to discriminate against mismatched base pairs compared to the ligase before substitution.

[0008] That is, the present application includes the following inventions. [1] A chimeric ligase in which the OB-fold domain or adenylation domain of a thermophilic ligase is replaced with the corresponding domain from a different species of thermophilic bacterium, and the chimeric ligase has improved discrimination ability against mismatched base pairs compared to the ligase before the replacement. [2] A chimeric ligase according to [1], wherein the corresponding domains of a different species derived from a thermophilic bacterium are derived from Thermus species. [3] A chimeric ligase, wherein the OB-fold domain or the adenylation domain of a thermophilic bacterium-derived ligase is: 1) a ligase consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1; 3) a ligase consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, and / or added; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. a chimeric ligase in which the corresponding domain of a ligase from a different species from a thermophilic bacterium is substituted, selected from the group consisting of: [4] The chimeric ligase according to [3], wherein the domain corresponding to the OB-fold domain comprises an amino acid sequence having at least 80% identity with the amino acid sequence from positions 320 to 406 of SEQ ID NO: 1. [5] A chimeric ligase according to [3], wherein the domain corresponding to the adenylation domain comprises an amino acid sequence having at least 80% identity with the amino acid sequence of positions 73 to 319 of SEQ ID NO: 1. [6] A chimeric ligase according to any one of [1] to [5], which has a lower joining rate when a mismatched base pair is present within three bases on either side of the ligation site formed by the ligase, compared to the ligase before substitution. [7] The chimeric ligase according to any one of [1] to [6], wherein the thermophilic ligase before substitution is a ligase derived from Thermus thermophilus, Thermus brockianus, Thermus filiformis, or Thermus oshimai. [8] The thermophilic ligase before substitution was 1) a ligase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 3) a ligase consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added from the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of a base sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. The chimeric ligase according to any one of [1] to [7], which is a ligase selected from the group consisting of: [9] A kit for detecting a mutated target sequence, the kit comprising the chimeric ligase according to any one of [1] to [8].

[10] The kit according to [9], further comprising reagents used in ligase detection reaction (LDR) or ligase chain reaction (LCR).

[11] A method for detecting a mutated target sequence in a sample, comprising the steps of contacting the sample with the chimeric ligase according to any one of [1] to [8]. analyzing the ligase reaction; A method comprising:

[12] The method according to

[11] , wherein the mutation is a single nucleotide polymorphism.

[13] The method according to

[11] or

[12] , wherein the step of analyzing the ligase reaction comprises a ligase detection reaction (LDR) or a ligase chain reaction (LCR).

[14] The method according to

[13] , wherein the presence or absence of a mutated target sequence in a sample is determined based on the presence or absence of an LDR or LCR reaction product. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a novel chimeric ligase derived from a thermophilic bacterium that can discriminate mismatched base pairs with high accuracy, and also to provide a novel method for producing a chimeric ligase derived from a thermophilic bacterium that can discriminate mismatched base pairs with high accuracy. Furthermore, the novel chimeric ligase can also be used to detect mutated target sequences. [Brief explanation of the drawings]

[0010] [Figure 1] The relative positions of the adenylation domain (adenylation), OB-fold domain (OB), zinc finger domain (Zn), helix-hairpin-helix domain (HhH), and BRCT domain (BRCT) in the ligase from Thermus bacteria are shown. [Figure 2] 1 shows the results of evaluating the accuracy of Tth LigA when an ssDNA oligo was used as the bridging nucleic acid. [Figure 3] 1 shows the results of evaluating the accuracy of Tth LigA when a dsDNA amplicon was used as a bridging nucleic acid. [Figure 4] 1 shows the results of evaluating the accuracy of Tsp LigA when an ssDNA oligo was used as the bridging nucleic acid. [Figure 5] 1 shows the results of evaluating the accuracy of Tsp LigA when a dsDNA amplicon was used as a bridging nucleic acid. [Figure 6] The relative conjugation efficiency of Tsp LigA was evaluated when a dsDNA amplicon was used as the bridging nucleic acid, with the relative conjugation efficiency of Tth LigA being set at 100%. [Figure 7] 1 shows the results of evaluating the reaction rates of Tth LigA and Tsp LigA when an ssDNA oligo was used as the bridging nucleic acid. [Figure 8]1 shows the results of evaluating the accuracy of Tth / Tsp chimera LigA, Tth LigA, and Tsp LigA, in which domains 1 to 4 in Tth LigA were each replaced with the corresponding domains in Tsp LigA, when an ssDNA oligo was used as the bridging nucleic acid. [Figure 9] The reaction rates of Tth / Tsp domain 1 chimeric LigA, Tth / Tsp domain 2 chimeric LigA, Tth LigA, and Tsp LigA were evaluated. [Figure 10] 1 shows the results of evaluating the accuracy of Tth / Tsp domain 2 chimeric LigA, Tth LigA, and Tsp LigA when an ssDNA oligo was used as a bridging nucleic acid. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0012] In a first embodiment, a chimeric ligase is provided, in which the OB-fold domain or the adenylation domain of a ligase derived from a thermophilic bacterium is replaced with the corresponding domain from a different species derived from a thermophilic bacterium, and the chimeric ligase has improved discrimination ability against mismatched base pairs compared to the ligase before the replacement.

[0013] As used herein, the term "chimeric ligase" refers to a ligase containing a region derived from a different species, for example, a ligase in which one or more arbitrary regions in the amino acid sequence constituting the ligase, preferably a region (domain) constituting the active site, have been replaced with those from a different ligase, thereby changing a certain property to that derived from the different ligase, or a ligase to which new properties have been imparted by the addition of one or more arbitrary regions in the amino acid sequence constituting the different ligase, preferably a region (domain) constituting the active site. The chimeric ligase provided in this embodiment is a ligase in which the OB-fold domain or adenylation domain has been replaced with the corresponding domain from a different species. The term "corresponding domain" refers to another domain having the same function as the domain before replacement or a highly homologous sequence, and the term "replaced with the corresponding domain" refers to another domain being placed at the same position as the given domain in the ligase before replacement. In this embodiment, another OB-fold domain of a different species is placed at the position of the OB-fold domain in the ligase before replacement, another adenylation domain of a different species is placed at the position of the adenylation domain in the ligase before replacement, or another OB-fold domain and adenylation domain of a different species are placed at the positions of the OB-fold domain and adenylation domain in the ligase before replacement.

[0014] As used herein, the term "thermophilic bacteria" generally refers to microorganisms that can grow in high-temperature environments. Examples of such microorganisms include bacteria, fungi, archaea, and viruses. While there is no particular lower limit to the temperature as long as the bacteria can grow, the optimal growth temperature for thermophilic bacteria is, for example, 40°C or higher.

[0015] Examples of thermophilic bacteria include the genus Thermus, Thermotoga, Thermoactinomyces, Thermobacillus, and Thermobif. Examples of suitable bacteria include those of the genus Thermobifida, Aquifex, Geobacillus, and Bacillus. Of these, the genus Thermus is preferred.

[0016] In one embodiment, the bacterium of the genus Thermus is Thermus thermophilus (Tth), Thermus brockianus (Tbr), Thermus species (Tsp), Thermus oshimai (Tos), or Thermus filiformis (Tfi).

[0017] Examples of thermophilic fungi include those of the genus Rasamsonia, Thermomyces, Byssochlamys, Eupenicillium, and Neosartorya.

[0018] Examples of thermophilic archaea include the genera Thermococcus, Pyrococcus, Thermoproteus, Thermoplasma, Methanothermus, Archaeoglobus, Sulfolobus, Aeropyrum, Pyrolobus, Pyrodictium, and Pyrobaculum. Among these, the genus Thermococcus is preferred.

[0019] Examples of thermophilic viruses include bacteriophages that infect prokaryotes.

[0020] As used herein, "ligase" refers to an enzyme that catalyzes the formation of a phosphodiester bond between nucleic acids. Examples of "ligase" include DNA ligase. Depending on the cofactor used, ligase can be classified into ATP-dependent ligase and NAD-dependent ligase. + They can be classified into ligases that are dependent on the cleavage site.

[0021] As used herein, an "OB-fold domain" is an oligomer / oligonucleotide / oligosaccharide binding domain, generally referring to a domain involved in binding to single-stranded DNA.

[0022] As used herein, "adenylation domain" generally refers to a domain involved in the reaction of covalently attaching adenosine monophosphate (AMP), which in a ligation reaction is involved in forming an adenylated reaction intermediate. Figure 1 shows the relative positions of each domain using a ligase derived from Thermus bacteria as an example.

[0023] As used herein, the term "mismatched base pair" refers to a base pair that is not a Watson-Crick base pair, in which base pairs such as adenine and thymine, adenine and uracil, or guanine and cytosine pair together through hydrogen bonding, but is not a Watson-Crick base pair, such as adenine and cytosine or thymine and guanine.

[0024] As used herein, "discrimination against mismatched base pairs" refers to the ability to distinguish between the presence or absence of mismatched base pairs in the sequences to be joined and to preferentially join sequences that do not have mismatched base pairs. For example, when a ligase joins sequences that do not have mismatched base pairs more frequently than sequences that have mismatched base pairs, the ligase can be said to have discrimination against mismatched base pairs. If the ability to discriminate against mismatched base pairs is improved, it becomes possible to more accurately distinguish between the presence and absence of mismatched base pairs, i.e., it becomes possible to more accurately preferentially join sequences that do not have mismatched base pairs. Furthermore, "improved discrimination ability against mismatched base pairs" may mean improved discrimination ability against a specific mismatched base pair, or improved discrimination ability against multiple types of mismatched base pairs.

[0025] When the chimeric ligase of this embodiment discriminates between the presence or absence of a mismatched base pair in a sequence to be joined, the position of the mismatched base pair in the sequence may be arbitrary, but in order to join sequences without mismatched base pairs with higher accuracy, the mismatched base pair is present preferably within 5 bases, more preferably within 3 bases, 2 bases, or 1 base on both sides of the junction joined by the ligase. When a mismatched base is present, preferably within 5 bases, more preferably within 3 bases on both sides of the junction, the joining rate by the ligase is low. "Within three bases on either side of the junction" refers to the position three bases adjacent, two bases adjacent, or one base adjacent (position adjacent to the junction) from the junction. In addition, the mismatch base pair may be present only on the 5' side of the junction, only on the 3' side of the junction, or on both the 5' and 3' sides of the junction. The chimeric ligase of this embodiment preferably has a lower joining rate when a mismatched base pair is present within three bases on either side of the ligation site formed by the ligase, compared to before the OB-fold domain or the adenylation domain was replaced.

[0026] As used herein, the term "junction" refers to a portion joined by a ligase or a portion that can be a target of a ligase, specifically a phosphodiester bond formed between nucleic acids by a ligase, a portion containing the phosphodiester bond, or a portion between adjacent nucleic acids before the phosphodiester bond is formed. In addition, a portion where a phosphodiester bond has been cleaved, such as a nick in a double-stranded nucleic acid, is also included in the term "junction" in this specification.

[0027] Examples of cases where the joining rate by ligase is low are shown below. For example, when the 5' end of a double-stranded nucleic acid A having a blunt end is joined to the 3' end of a double-stranded nucleic acid B having a blunt end using the chimeric ligase of this embodiment, if a mismatch base pair is present at a position 1 to 3 bases from the 5' end of double-stranded nucleic acid A and / or at a position 1 to 3 bases from the 3' end of double-stranded nucleic acid B, the frequency with which the 5' end of double-stranded nucleic acid A and the 3' end of double-stranded nucleic acid B are joined will be lower than when joining nucleic acids that do not have a mismatch base pair.

[0028] Furthermore, for example, when a double-stranded nucleic acid A having a 5'-overhanging end and a double-stranded nucleic acid B having a 3'-overhanging end are joined by the chimeric ligase of this embodiment, if the overhanging ends form a duplex and a mismatch base occurs within three bases on either side of the junction, the frequency with which the two double-stranded nucleic acids A and B are joined is lower than when nucleic acids without a mismatched base pair are joined.

[0029] Furthermore, for example, when a nick in a double-stranded nucleic acid is joined using the chimeric ligase of this embodiment, if a mismatched base pair is present within three bases on either side of the nick (junction), the frequency with which the nick is joined will be lower than when a nick in a nucleic acid in which no mismatched base pair is present is joined.

[0030] When the chimeric ligase of this embodiment distinguishes between the presence and absence of mismatched base pairs in the sequences to be joined, the number of mismatched base pairs in the sequences varies depending on the sequences to be joined and is not limited to 1. For example, if the chimeric ligase of this embodiment can accurately distinguish between the presence and absence of mismatched base pairs when a mismatched base pair exists within three bases on either side of the junction, and the chimeric ligase does not join certain sequences, there is a high probability that a mismatched base pair existed within three bases on either side of the junction. In this case, a maximum of six mismatched base pairs will be present within three bases on either side of the junction.

[0031] In the case of a ligase with low discrimination ability for mismatched base pairs, two double-stranded nucleic acids will be joined together regardless of whether they have mismatched base pairs or whether mismatched base pairs exist within three bases on either side of the junction between them, but the chimeric ligase of this embodiment accurately discriminates whether two double-stranded nucleic acids have mismatched base pairs, and when they do not have mismatched base pairs, they join nucleic acids more frequently than when they do have mismatched base pairs.When a mismatched base pair exists within three bases on either side of the junction between them, the chimeric ligase can more accurately discriminate whether the two double-stranded nucleic acids have mismatched base pairs, thereby more frequently joining sequences that do not have mismatched base pairs. The chimeric ligase of this embodiment, due to the substitution of the OB-fold domain or the adenylation domain, can more accurately discriminate mismatched base pairs compared to before the substitution. The chimeric ligase of this embodiment preferentially joins nucleic acids that do not have mismatched base pairs, preferably nucleic acids that do not have mismatched base pairs near the junction.

[0032] Since the OB-fold domain or adenylation domain of a ligase is involved in the ability of the ligase to discriminate against mismatched base pairs, replacing the domain makes it possible to adjust the ability of the ligase to discriminate against mismatched base pairs. That is, by replacing the OB-fold domain or adenylation domain of a ligase with an OB-fold domain or adenylation domain derived from a ligase that has a higher ability to discriminate against mismatched base pairs than the ligase before substitution, it is possible to provide a ligase with improved ability to discriminate against mismatched base pairs compared to the ligase before substitution. Furthermore, the ligase before substitution may be a ligase that does not have the ability to discriminate against mismatched base pairs, and such a ligase may be conferred the ability to discriminate against mismatched base pairs by replacing the OB-fold domain or adenylation domain with one that has the discriminating ability. The chimeric ligase of this embodiment may be a ligase in which only the OB-fold domain is replaced, a ligase in which only the adenylation domain is replaced, or a ligase in which both the OB-fold domain and the adenylation domain are replaced, but is preferably a ligase in which only the OB-fold domain or only the adenylation domain is replaced.

[0033] Herein, if the ligase derived from a thermophile before substitution is referred to as ligase I and the ligase derived from a different species of thermophile is referred to as ligase II, the chimeric ligase of this embodiment is a chimeric ligase in which the region other than the OB-fold domain or adenylation domain is derived from ligase I, and the OB-fold domain or adenylation domain is derived from ligase II. Alternatively, the chimeric ligase of this embodiment is a chimeric ligase in which the region other than the OB-fold domain and adenylation domain is derived from ligase I, and the OB-fold domain and adenylation domain are derived from ligase II.

[0034] It is preferable that ligase I and ligase II use the same cofactor, and NAD + Preferably, the ligases are ATP-dependent ligases or ATP-dependent ligases.

[0035] The thermophilic bacterium from which ligase I is derived and the thermophilic bacterium from which ligase II is derived may be any thermophilic bacterium of different genus and species, as long as the ligase after substitution has improved ability to discriminate mismatched base pairs compared to before substitution. However, they are preferably from the same genus but different species, and more preferably from Thermus bacteria but different species.

[0036] In an example of different species of bacteria from the genus Thermus, ligase I may be a ligase from Thermus thermophilus, Thermus brochianus, Thermus filiformis, or Thermus oshimai, and ligase II may be a ligase from Thermus species. Ligases from Thermus species may discriminate mismatched base pairs with higher accuracy than ligases from Thermus thermophilus, Thermus brochianus, Thermus filiformis, or Thermus oshimai. Ligases from Thermus species may discriminate mismatched base pair combinations with higher accuracy than ligases from Thermus thermophilus, Thermus brochianus, Thermus filiformis, or Thermus oshimai. Therefore, chimeric ligases in which the OB-fold domain or adenylation domain of the ligase derived from Thermus thermophilus, Thermus brochianus, Thermus filiformis, or Thermus oshimai was replaced with the OB-fold domain or adenylation domain of the ligase derived from Thermus species showed improved discrimination ability against mismatched base pairs compared to the unsubstituted chimeric ligases.

[0037] In one embodiment, the chimeric ligase of this embodiment is a chimeric ligase in which the OB-fold domain or adenylation domain of a ligase derived from Thermus thermophilus is replaced with the OB-fold domain or adenylation domain of a ligase derived from Thermus species. Thermus species ligase has higher discrimination ability against mismatched base pairs compared to the ligase derived from Thermus thermophilus, but a slower reaction rate. Therefore, a chimeric ligase in which ligase I is derived from Thermus thermophilus and ligase II is derived from Thermus species can be a chimeric ligase with improved discrimination ability against mismatched base pairs and a faster reaction rate.

[0038] If Ligase I has an advantage over Ligase II in that it does not involve an OB-fold domain or an adenylation domain, and if Ligase II has a higher ability to discriminate mismatched base pairs than Ligase I, it will be possible to provide a chimeric ligase derived from Ligase I and Ligase II that retains the advantages of Ligase I while improving its ability to discriminate mismatched base pairs compared to before the substitution.

[0039] In this embodiment, Ligase I and Ligase II may be modified ligases in which one or more amino acids may be added, substituted, and / or deleted. The number of added, substituted, and / or deleted amino acids may be any number as long as the ligase activity is maintained, and can be appropriately determined by those skilled in the art.

[0040] When the ligase I of this embodiment is derived from, for example, Thermus thermophilus, Thermus brochianus, Thermus oshimai, or Thermus filiformis, these ligases are, in one embodiment, enzymes in which 1 to 67, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids have been added, substituted, and / or deleted in any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 5. Here, the sequences of SEQ ID NOs: 2 to 5 are examples of the sequences of ligases derived from Thermus thermophilus, Thermus brochianus, Thermus filiformis, and Thermus oshimai, respectively. Furthermore, when the ligase II of this embodiment is, for example, a ligase derived from Thermus species, in one embodiment, the ligase is an enzyme in which 1 to 67, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids have been added, substituted, and / or deleted in the amino acid sequence shown in SEQ ID NO: 1. Here, the sequence of SEQ ID NO: 1 is an example of the sequence of a ligase derived from Thermus species.

[0041] Furthermore, in this embodiment, ligase I or ligase II may each be a ligase consisting of any one of the amino acid sequences shown in SEQ ID NOs: 2 to 5, or an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 1.

[0042] As used herein, "identity" refers to the percentage (%) of amino acids that are the same at the same position or in the same row when two amino acid sequences are aligned. When two amino acid sequences are aligned, the percentage (%) of amino acids that are the same or similar at the same position or in the same row is called homology. As used herein, sequence homology also includes the case of "identical."

[0043] If an enzyme has 80% identity with a specific enzyme consisting of 100 amino acids, for example, when aligned with the amino acid sequence of the specific enzyme consisting of the 100 amino acids, there will be 80 positions where identical amino acids are lined up in the same position or row.

[0044] Furthermore, as used herein, "similar amino acids" refers to two or more amino acids that have similar chemical or physical properties. General classification of amino acids can be used as a reference for determining such similarity. For example, if two amino acids are classified into the same amino acid group, such as acidic amino acids, basic amino acids, aromatic amino acids, aliphatic amino acids, amino acids with hydroxyl groups, hydrophilic amino acids, or hydrophobic amino acids, the two amino acids are considered to have similar chemical or physical properties.

[0045] In one embodiment, ligase I or ligase II in this embodiment is an enzyme having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 5, or the amino acid sequence set forth in SEQ ID NO: 1, respectively. Furthermore, in one embodiment, ligase I or ligase II in this embodiment is an enzyme having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homology to any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 5, or the amino acid sequence set forth in SEQ ID NO: 1, respectively.

[0046] Furthermore, Ligase I or Ligase II in this embodiment may each be a ligase encoded by a base sequence that hybridizes under stringent conditions with any one of the amino acid sequences shown in SEQ ID NOs: 2 to 5, or with a complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1.

[0047] As used herein, the term "a base sequence that hybridizes under stringent conditions" refers to a DNA base sequence obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a complementary sequence of a wild-type gene encoding the amino acid sequence shown in SEQ ID NOs: 1 to 5 as a probe.

[0048] As used herein, "stringent conditions" refer to conditions under which signals from specific hybrids are clearly distinguished from signals from nonspecific hybrids, and vary depending on the hybridization system used and the type, sequence, and length of the probe. Such conditions can be determined by changing the hybridization temperature, washing temperature, and salt concentration.

[0049] For example, if a strong signal from a nonspecific hybrid is detected, specificity can be improved by increasing the hybridization and washing temperature and, if necessary, decreasing the salt concentration in the washing. If a signal from a specific hybrid is not detected, hybrids can be stabilized by decreasing the hybridization and washing temperature and, if necessary, increasing the salt concentration in the washing.

[0050] In some embodiments, specific examples of stringent conditions include the following: For example, a DNA probe is used as the probe, and hybridization is performed overnight (approximately 8 to 16 hours) using 5x SSC, 1.0% (w / v) blocking reagent for nucleic acid hybridization (Boehringer-Mannheim), 0.1% (w / v) N-lauroyl sarcosine, and 0.02% (w / v) SDS. Washing is performed twice for 15 minutes using 0.1 to 0.5x SSC and 0.1% (w / v) SDS, preferably 0.1x SSC and 0.1% (w / v) SDS. The temperature for hybridization and washing is 65°C or higher, preferably 68°C or higher.

[0051] Examples of base sequences that hybridize under stringent conditions include DNA obtained by hybridizing under the above-mentioned stringent conditions using a filter onto which DNA having a complementary sequence of a wild-type gene derived from a colony or plaque, or a fragment of said DNA, is immobilized; and DNA that can be identified by hybridizing in the presence of 0.5 to 2.0 M NaCl at 40 to 75°C, preferably in the presence of 0.7 to 1.0 M NaCl at 65 to 68°C, and then washing the filter at 55 to 65°C using 0.1 to 1×SSC solution (1×SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Probe preparation and hybridization methods can be carried out in accordance with the methods described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997, etc. Examples of base sequences that hybridize under highly stringent conditions include DNA that can be identified by hybridizing at 65°C in the presence of 1.0 M NaCl, followed by washing the filter at 65°C using 0.5x SSC solution (1x SSC solution is 150 mM sodium chloride, 15 mM sodium citrate).

[0052] In addition, a person skilled in the art can appropriately set conditions for obtaining a base sequence that hybridizes with the complementary sequence of a wild-type gene under stringent conditions, taking into account not only conditions such as the salt concentration of the buffer and temperature, but also other conditions such as probe concentration, probe length, and reaction time.

[0053] In this embodiment, the ligase encoded by the base sequence that hybridizes under stringent conditions with the complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NOs: 1 to 5 is likely to be a ligase having an amino acid sequence that has one to multiple, preferably several, amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the ligase encoded by the base sequence of the wild-type gene, but has the same ligase activity and other activities as the ligase encoded by the base sequence of the wild-type gene.

[0054] In one embodiment, the ligase I in this embodiment is 1) a ligase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 3) a ligase consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added from the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of a base sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. The ligase is selected from the group consisting of: That is, this embodiment is a chimeric ligase, 1) a ligase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 3) a ligase consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added from the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of a base sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. The present invention also provides a chimeric ligase in which the OB-fold domain or the adenylation domain of a ligase selected from the group consisting of:

[0055] In one embodiment, the ligase II in this embodiment is 1) a ligase consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1; 3) a ligase consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, and / or added; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. The ligase is selected from the group consisting of: That is, this embodiment is a chimeric ligase, The OB-fold domain or adenylation domain of the thermophilic bacterium-derived ligase is 1) a ligase consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1; 3) a ligase consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, and / or added; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. The present invention also provides a chimeric ligase in which the corresponding domain of a ligase from a different species of thermophilic bacterium selected from the group consisting of: is substituted with the corresponding domain of a ligase from a different species of thermophilic bacterium, and the ligase has improved discrimination ability against mismatched base pairs compared to before substitution.

[0056] This embodiment is a chimeric ligase comprising: 1) a ligase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 3) a ligase consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added from the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of a base sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. an OB-fold domain or an adenylation domain in a ligase selected from the group consisting of: 1) a ligase consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1; 3) a ligase consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, and / or added; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. The present invention also provides a chimeric ligase in which the OB-fold domain or adenylation domain of a ligase having an amino acid sequence set forth in any one of SEQ ID NOS: 2 to 5 has been substituted with the corresponding domain of a ligase having the amino acid sequence set forth in SEQ ID NOS: 1. These ligases have improved discrimination ability against mismatched base pairs compared to before the substitution.

[0057] The chimeric ligase in this embodiment is a chimeric ligase in which the OB-fold domain or adenylation domain of ligase I is replaced with the OB-fold domain or adenylation domain of ligase II. The position of the OB-fold domain or adenylation domain in the amino acid sequence of ligase I or ligase II can be appropriately determined by those skilled in the art based on information disclosed in publicly known databases such as NCBI. For example, when ligase I or ligase II has any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 5, or SEQ ID NO: 1, respectively, examples of the position of the OB-fold domain or adenylation domain in each amino acid sequence are shown below.

[0058] When the ligase I in this embodiment is derived from Thermus thermophilus, the position of the OB-fold domain is, for example, from positions 323 to 409 in the amino acid sequence shown in SEQ ID NO: 2, and the position of the adenylation domain is, for example, from positions 73 to 322 in the amino acid sequence shown in SEQ ID NO: 2.

[0059] When the ligase I in this embodiment is derived from Thermus broccianus, the position of the OB-fold domain is, for example, from positions 320 to 406 in the amino acid sequence shown in SEQ ID NO: 3, and the position of the adenylation domain is, for example, from positions 73 to 319 in the amino acid sequence shown in SEQ ID NO: 3.

[0060] When the ligase I in this embodiment is derived from Thermus filiformis, the position of the OB-fold domain is, for example, from positions 320 to 406 in the amino acid sequence shown in SEQ ID NO: 4, and the position of the adenylation domain is, for example, from positions 73 to 319 in the amino acid sequence shown in SEQ ID NO: 4.

[0061] When the ligase I in this embodiment is derived from Thermus oshimai, the position of the OB-fold domain is, for example, from positions 320 to 406 in the amino acid sequence shown in SEQ ID NO: 5, and the position of the adenylation domain is, for example, from positions 73 to 319 in the amino acid sequence shown in SEQ ID NO: 5.

[0062] When the ligase II in this embodiment is derived from Thermus species, the position of the OB-fold domain is, for example, from positions 320 to 406 in the amino acid sequence shown in SEQ ID NO: 1, and the position of the adenylation domain is, for example, from positions 73 to 319 in the amino acid sequence shown in SEQ ID NO: 1. When ligase II is a ligase derived from Thermus species, the chimeric ligase of this embodiment is, in one embodiment, a chimeric ligase in which the OB-fold domain or the adenylation domain of ligase I is replaced with an amino acid sequence having at least 80% identity to the amino acid sequence from positions 320 to 406 of SEQ ID NO: 1, or an amino acid sequence having at least 80% identity to the amino acid sequence from positions 73 to 319 of SEQ ID NO: 1. That is, when the ligase II is derived from Thermus species, the chimeric ligase in this embodiment having a substituted OB-fold domain comprises, in one embodiment, an amino acid sequence having at least 80% identity to the amino acid sequence of positions 320 to 406 of SEQ ID NO: 1; the chimeric ligase in this embodiment having a substituted adenylation domain comprises, in one embodiment, an amino acid sequence having at least 80% identity to the amino acid sequence of positions 73 to 319 of SEQ ID NO: 1; and the chimeric ligase in this embodiment having a substituted OB-fold domain and an adenylation domain comprises, in one embodiment, an amino acid sequence having at least 80% identity to the amino acid sequence of positions 320 to 406 of SEQ ID NO: 1 and an amino acid sequence having at least 80% identity to the amino acid sequence of positions 73 to 319 of SEQ ID NO: 1.

[0063] When ligase I in this embodiment is a ligase derived from Thermus thermophilus and ligase II is a ligase derived from Thermus species, and the OB-fold domain is replaced, in one embodiment, the chimeric ligase in this embodiment is a ligase in which the amino acid sequences of positions 1 to 322 and positions 410 to 676 in the chimeric ligase are derived from Thermus thermophilus, and the amino acid sequence of positions 323 to 409 is derived from Thermus species. In one embodiment, the chimeric ligase of this embodiment is a ligase consisting of the amino acid sequence shown in any one of SEQ ID NOs: 6, 7, and 10-12.

[0064] Furthermore, the OB-fold domain or adenylation domain in Ligase I may be replaced with the entire amino acid sequence constituting the domain, or a part of the domain in Ligase I may be replaced as long as the activity of the replaced domain is lost. Furthermore, the OB-fold domain or adenylation domain in Ligase I may be replaced with the entire amino acid sequence constituting the corresponding domain in Ligase II, or a part of the domain in Ligase II may be replaced as long as the activity is maintained.

[0065] Furthermore, the OB-fold domain or adenylation domain in Ligase II may be substituted for the corresponding domain in Ligase I, with an amino acid linker added to the N-terminal and / or C-terminal end of the amino acid sequence. The linker is an amino acid linker consisting of a peptide of preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids, and even more preferably 1 to 6 amino acids. Furthermore, within the above range of 1 to 15 or a suitable range, the number may be 2 or more, 3 or more, or 4 or more. As the linker, a linker used when linking peptides together can be used, and an example thereof is a GS linker containing glycine and serine. Alternatively, the linker may be an amino acid sequence located at the N- or C-terminus of the OB-fold domain or adenylation domain in the amino acid sequence of the original ligase II in which the OB-fold domain or adenylation domain is present. Preferably, a consensus sequence between ligase I and ligase II is used as the linker. Any linker can be selected as long as it has improved discrimination against mismatched base pairs compared to the ligase before substitution.

[0066] This embodiment also provides a nucleic acid encoding a chimeric ligase. The nucleic acid may be either DNA or RNA, or may be a DNA / RNA chimeric nucleic acid. Furthermore, the nucleic acid is not particularly limited as long as it is a nucleic acid capable of expressing the chimeric ligase of this embodiment, but is preferably a nucleic acid having consecutive codons encoding each amino acid in the amino acid sequence of the chimeric ligase, and may also be a nucleic acid having a nucleotide sequence complementary to that of the nucleic acid encoding the chimeric ligase. A nucleic acid having a nucleotide sequence complementary to that of the nucleic acid encoding the chimeric ligase may produce a nucleic acid encoding the chimeric ligase in a host cell; for example, RNA having a sequence encoding the chimeric ligase may be produced using DNA having a nucleotide sequence complementary to that of the nucleic acid encoding the chimeric ligase as a template.

[0067] The nucleic acid in this embodiment may be contained in a vector. The type of vector is not particularly limited, and any vector known to those skilled in the art may be used, including, for example, a plasmid, a cosmid, an episome, an artificial chromosome, a phage, and a viral vector. Thus, this embodiment also provides a vector containing a nucleic acid encoding a chimeric ligase. When the nucleic acid is contained in a vector, the nucleic acid may be incorporated into the vector together with regulatory elements necessary for transcription and translation, such as a promoter, an enhancer, or a terminator, and the nucleic acid may be present contiguously or discontinuously with these elements in the vector. As used herein, the term "vector" refers to a concept that encompasses cloning vectors and expression vectors, and refers to a nucleic acid that carries a gene of interest so as to transform a host, preferably a cell, and promote the expression (e.g., transcription and translation) of the introduced sequence.

[0068] This embodiment also provides a transformant into which the above vector has been introduced. The transformant in this embodiment is a host, preferably a cell (host cell), that expresses the chimeric ligase. As used herein, "transformation" refers to introducing a nucleic acid or vector into a host so that the host expresses the introduced gene (nucleic acid) and produces the desired peptide. A host containing a nucleic acid, preferably a host containing a nucleic acid in the form of a vector, is also called a transformant.

[0069] The nucleic acid, vector, and transformant of this embodiment may be produced by any method known to those skilled in the art, and the produced nucleic acids may be used to produce the chimeric ligase of this embodiment. Furthermore, as the nucleic acid of this embodiment, a nucleic acid having a base sequence complementary to the nucleic acid encoding the chimeric ligase may also be produced by any method known to those skilled in the art.

[0070] The chimeric ligase of this embodiment may be produced by any method known to those skilled in the art, but is preferably produced by, for example, introducing a vector containing a gene that expresses the chimeric ligase of interest into a host to transform the host and allow the host to express the chimeric ligase. It can also be produced using the nucleic acid, vector, or transformant of this embodiment.

[0071] As used herein, "host" refers to an organism, such as a bacterium, fungus, or plant cell, transformed to express a desired protein. The host bacterium may be Escherichia coli, Bacillus subtilis, or actinomycete, and the host fungus may be a yeast or filamentous fungus.

[0072] In one embodiment, the host is E. coli.

[0073] Transformation of host cells may be carried out by any method known to those skilled in the art, such as electroporation or heat shock.

[0074] To transform a host, an exogenous vector encoding the chimeric ligase of interest is prepared. The vector may encode multiple chimeric ligases of interest. The vector may also encode a selectable marker, such as an antibiotic resistance gene, used to select transformants.

[0075] To increase the transformation efficiency, the host is preferably subjected to heat treatment or electrical treatment before transformation, which increases the efficiency of vector uptake in the resulting competent cells.

[0076] The transformed host is also referred to as a transformant. Selection of the transformant, propagation of the transformant, induction of expression of the desired ligase, and recovery of the host expressing the desired chimeric ligase may be carried out by any method known to those skilled in the art.

[0077] Transformants can be obtained by culturing a mixture of vector and host competent cells in a medium containing an antibiotic corresponding to the selection marker encoded by the vector, and selecting the colonies that form. While the type of medium varies depending on the host cell, LB medium is an example of a medium for E. coli. Plate media are preferred for selecting transformants. Culture conditions are appropriately adjusted, for example, at a temperature of approximately 30°C to 37°C and for a culture time of approximately 12 hours to overnight.

[0078] The selected transformant is grown by culturing. Pre-culturing may be performed before the main culturing. The growth of the transformant can also be carried out under the culture conditions used for selecting the transformant, but the culture medium is preferably a liquid medium. The culture temperature and culture time are appropriately adjusted depending on the desired level of growth of the transformant, and are not intended to be limiting, but are appropriately adjusted between about 16°C and 37°C and about 12 hours to overnight.

[0079] The conditions for inducing expression of the target ligase vary depending on the expression system used. While not intended to be limiting, the pET system is a preferred expression system when using E. coli as a host. The pET system is a protein expression system in which T7 RNA polymerase transcribes the target gene under the control of the lacUV5 promoter and is induced in the presence of allolactose or the lactose analog IPTG (isopropyl-β-thiogalactopyranoside).

[0080] In the case of an expression system using the pET system, expression of the desired chimeric ligase can be induced by adding IPTG to the medium and culturing under specified conditions. The culture temperature and time are adjusted appropriately depending on the desired level of expression, and are not intended to be limiting, but are typically adjusted between 1 hour and overnight, and between 16°C and 37°C.

[0081] To recover the expressed chimeric ligase from the host, the host cells are subjected to a disruption process. As used herein, cell disruption refers to the destruction of the cell wall. The disruption method is not particularly limited, but can be broadly divided into mechanical and non-mechanical methods. Examples of mechanical methods include ultrasonic disruption, disruption using known devices such as homogenizers and blenders. Non-mechanical methods are methods that do not fall under the mechanical method category, such as chemical methods using surfactants or enzymes such as lysozyme. When disrupting host cells using surfactants or enzymes such as lysozyme, protease inhibitors, DNase, etc. may be added as needed in addition to the surfactant or lysozyme. Non-mechanical methods are not limited to chemical methods and may also utilize osmotic pressure differences.

[0082] Various conditions, such as temperature conditions, used in the process of disrupting host cells can be appropriately determined by those skilled in the art. For example, mechanical methods are generally performed on ice because heat is generated during the disruption process. Non-mechanical chemical methods include the lysis reagent method, in which the host cell wall is disrupted by suspending it in a buffer containing a surfactant or lysozyme and treating it at room temperature. However, when a non-mechanical method is used to disrupt host cells in the present invention, it is preferable that the treatment temperature is not room temperature. Room temperature may be, for example, 10 to 30°C.

[0083] The conditions for non-mechanical disruption of host cells at temperatures other than room temperature can be determined appropriately by those skilled in the art. For example, when disrupting host cells using a lysis reagent, among other non-mechanical methods, the treatment temperature is adjusted by mixing the host in a buffer containing necessary reagents, such as a surfactant or lysozyme, on ice. When the mixture of the host and buffer containing the reagent is removed from the ice and suspended using a known device such as a vortex mixer, it is preferable to suspend the mixture quickly, for example, within 10 seconds, so that the temperature of the mixture does not rise to room temperature. The procedure of removing the cells from the ice, suspending them using a vortex mixer, returning them to ice, and then removing them from the ice and suspending them using a vortex mixer may be repeated. When suspending without using a known device such as a vortex mixer, it is preferable to always perform the suspension procedure on ice. Avoiding incubation at room temperature after mixing and suspension allows for rapid transition to the heating step. If the heating step is not to be transitioned to immediately after mixing and suspension, it is preferable to store the mixture on ice.

[0084] After the disruption step, the host cells are subjected to a heating step. The heating step is carried out to denature the host cell-derived proteins, and is carried out at a temperature below 64°C at which the host cell-derived proteins denature. The temperature used in the heating step is also referred to as the "heating temperature" or the "treatment temperature," and these terms are used interchangeably. Denaturation occurs when the helical or sheet structure of the protein is disrupted, and the temperature at which the protein denatures can be determined by known methods for analyzing the secondary structure of proteins, such as circular dichroism spectroscopy or fluorescence spectroscopy.

[0085] As used herein, "a temperature below 64°C at which a host cell-derived protein is denatured" refers to a temperature equal to or higher than a specific temperature at which the structure of the host cell-derived protein changes and the function of the protein is lost, but lower than 64°C. It also refers to a temperature at which the host cell-derived protein is denatured but the thermophilic bacterium-derived protein or its homologue is not denatured.

[0086] The heating temperature can be appropriately determined by those skilled in the art depending on the growth temperature of the thermophilic bacterium.

[0087] In one embodiment, the heating temperature is 50°C or higher.

[0088] The heating temperature is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, or 63°C. The heating temperature is preferably 53°C to 62°C, more preferably 55°C to 60°C. The heating step time varies depending on the heating temperature, but may be any time as long as the host cell-derived protein is denatured.

[0089] In one embodiment, the heating temperature is about 53 to 62°C, preferably about 55 to 60°C.

[0090] In one embodiment, when the heating temperature is within the range of 50 to 60°C, the heating step time is, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, or 19 minutes.

[0091] Heating may be performed using any device known to those skilled in the art, for example, an incubator such as a heat block, or, in the case of industrial production, a heating tank.

[0092] The host and / or the protein produced by the host may be cooled after the heating step. Proteins produced by the host are usually separated from other components by steps such as centrifugation, but by cooling the host and / or the protein produced by the host, the insoluble fraction and the soluble fraction can be separated more clearly than when not cooled, thereby increasing the recovery amount of the target ligase contained in either fraction. Cooling is preferably performed on ice, but may be performed at a temperature lower than room temperature, for example, at 0 to 10°C. The cooling time may be any time as long as the insoluble fraction and the soluble fraction can be clearly separated, for example, 1 to 30 minutes. The cooling step is preferably performed promptly after the heat treatment.

[0093] The chimeric ligase produced from the host is collected after cooling using centrifugation or the like.

[0094] The produced chimeric ligase may be purified by a method known to those skilled in the art, such as affinity chromatography, ion exchange chromatography, or gel filtration chromatography.

[0095] This embodiment also provides a method for producing a chimeric ligase, which includes a step of replacing the OB-fold domain or adenylation domain of a thermophilic ligase with a corresponding domain from a different species of thermophilic bacterium, thereby improving the ability to discriminate against mismatched base pairs compared to the ligase before the replacement. Instead of the replacement step, the desired sequence may be synthesized. For example, when producing a chimeric ligase through a transformant, a vector containing a gene expressing the chimeric ligase may be synthesized.

[0096] The performance of the chimeric ligase of this embodiment is evaluated by comparison with a control, e.g., the ligase before substitution, to determine, for example, improved discrimination of mismatched base pairs.

[0097] Here, the ability to discriminate against mismatched base pairs may be evaluated by any method known to those skilled in the art, but for example, whether a sequence having a mismatched base pair can be discriminated can be evaluated by treating a sample containing a nucleic acid having a mismatched base pair and its ligation partner nucleic acid with a chimeric ligase and then analyzing the ligation product.When evaluating the ability to discriminate against a specific mismatched base pair, the ability to discriminate against the specific mismatched base pair can be evaluated by treating a sample containing the nucleic acid having the mismatched base pair and its ligation partner nucleic acid with a ligase and then analyzing the ligation product. An example of a method for evaluation by analyzing ligation products is a method using the relative joining rate (%) (which may also be the relative joining efficiency (%)). The method for calculating the relative joining rate (%) can be determined appropriately depending on the sequences to be joined and the object to be compared. For example, when the object to be compared is the accuracy of the ligase before domain replacement, the relative joining rate (%) can be determined by dividing the amount of ligation product when a nucleic acid sample is treated with the chimeric ligase after domain replacement by the amount of ligation product when the nucleic acid sample without mismatched base pairs is treated with the ligase before domain replacement, and multiplying this value by 100. Alternatively, the relative joining rate (%) can be determined by dividing the amount of ligation product when a nucleic acid sample is treated with the ligase by the amount when all of the nucleic acids contained in the sample are joined, and multiplying this value by 100. When the amplification product of the ligation product is analyzed to evaluate the ability to discriminate between mismatched base pairs, the relative conjugation rate (%) may be calculated from the PCR efficiency (e) in the amplification reaction, the difference in Ct value from the control (ΔCt), etc., and can be calculated, for example, using the following formula 1. <Expression 1> Relative bonding rate (%) = {1 / (1+e)Δ Ct}×100

[0098] When the relative joining rate (%) is used to evaluate whether the chimeric ligase of this embodiment has improved discrimination ability against mismatched base pairs compared to the ligase before substitution, the evaluation is carried out, for example, as follows. By comparing the relative joining rate of the ligase before substitution for a nucleic acid having a mismatched base pair with the relative joining rate of the chimeric ligase after substitution for the same nucleic acid, the accuracy of the mismatched base pair discrimination ability of the ligase before domain substitution and the chimeric ligase after domain substitution is compared, and whether the accuracy of the discrimination ability has improved by domain substitution is evaluated.

[0099] The relative joining rate (%) when a nucleic acid sample having mismatched base pairs is treated with ligase may be referred to as the misjoining rate (%). The closer the misjoining rate (%) is to 0%, the more likely it is that the ligase did not join nucleic acids with mismatched base pairs. Furthermore, the closer the relative joining rate (%) is to 100% when a nucleic acid sample without mismatched base pairs is treated with the ligase, the more likely it is that the ligase has high joining activity. Therefore, for example, a chimeric ligase that shows a relative joining rate (%) of nearly 100% and a misjoining rate (%) of nearly 0% when a nucleic acid sample without mismatched base pairs is treated with the chimeric ligase can be evaluated as a chimeric ligase that joins sequences without mismatched base pairs with high accuracy. If the chimeric ligase after domain substitution has a lower misjunction rate than the ligase before domain substitution and has the same or higher relative junction rate when a nucleic acid sample without mismatched base pairs is treated, it can be evaluated that the domain substitution has improved the ability to discriminate against mismatched base pairs.

[0100] In a second embodiment, there is provided a method for detecting a mutated target sequence in a sample, the method comprising contacting the sample with a chimeric ligase as provided in the first embodiment, and analyzing the ligase reaction.

[0101] As used herein, "mutation" refers to one or more bases in which a base substitution, deletion, or insertion has occurred, or the state in which the base substitution or insertion has occurred. "Mutation" can be used interchangeably with "polymorphism." The position of the base where the base substitution, deletion, or insertion occurs, the number of bases substituted or deleted, and the number of bases inserted vary depending on the target sequence being analyzed. The type of mutation is not particularly limited, but includes missense mutation, nonsense mutation, frameshift mutation, silent mutation, etc. In this embodiment, the mutation may be a polymorphism, and is preferably a single nucleotide polymorphism.

[0102] In this embodiment, the detection of the mutated target sequence is made possible by utilizing the property of the chimeric ligase to discriminate between mismatched base pairs, i.e., to join sequences that do not have mismatched base pairs with high accuracy.

[0103] Specifically, when a sample requiring mutation detection contains nucleic acids that are joined with high accuracy by a chimeric ligase when a mutated sequence is present, and the sample is treated with the chimeric ligase, a reaction product of the chimeric ligase is obtained when a mutated target sequence is present in the sample, making it possible to detect the mutated target sequence by the chimeric ligase.

[0104] Probes are used for such detection. Specifically, two adjacent probes complementary to a mutated target sequence are added to a sample requiring mutation detection, and the sample is then treated with the ligase. If the sample contains a mutated target sequence, the two probes hybridized to the mutated target sequence will not form mismatched base pairs with the mutated target sequence, and will be joined with high accuracy by the chimeric ligase. On the other hand, if the sample contains a wild-type sequence, the two probes hybridized to the wild-type sequence will form mismatched base pairs with the wild-type sequence, and will be joined only at a low frequency or not at all by the chimeric ligase. Therefore, if a sample treated with the chimeric ligase contains a mutated target sequence, a reaction product consisting of two probes joined by the chimeric ligase is frequently obtained, and the presence or absence of a mutation can be determined by analyzing this sample treated with the chimeric ligase. In other words, a mutated target sequence can be detected.

[0105] In this embodiment, a sample requiring mutation detection may contain a target sequence for which mutation detection is desired and a probe necessary for mutation detection. The target sequence may be contained in the sample in any form, such as part of genomic DNA, a cell sample, a tissue sample, or as an amplification product obtained by amplifying a region containing the target sequence by PCR or other methods. However, it is preferable for the target sequence to be contained in the sample as an amplification product. When the target nucleic acid is contained in the sample as an amplification product, the region to be amplified and its length may be determined appropriately by those skilled in the art. The target nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. The target sequence contained in the sample may be a mixture of a mutated target sequence and a wild-type target sequence, or may consist solely of a mutated target sequence, or may consist solely of a wild-type target sequence. In addition to the target sequence, the sample may also contain any other sequences, buffers, reagents, etc.

[0106] The method of this embodiment includes a step of contacting a sample with a chimeric ligase, which catalyzes a ligation reaction of probes that are joined when a mutation is present in the sample. In this embodiment, the chimeric ligase may be added to the sample at any timing. It may be contained in the sample from the beginning, or may be added after the step of hybridizing the probe to the target sequence. The hybridization step and the ligation reaction using the chimeric ligase may be performed in parallel, or the ligation reaction may be performed after the hybridization step. Furthermore, each reaction condition, such as the time for reacting the chimeric ligase with the sample and the treatment temperature, can be appropriately determined by one skilled in the art.

[0107] The method of this embodiment may include a step of analyzing a ligase reaction. The analysis of a ligase reaction broadly refers to the analysis of a ligation reaction using a chimeric ligase, and specifically refers to the analysis of a ligation reaction for detecting a mutated target sequence. The analysis of a ligation reaction for detecting a mutated target sequence is not particularly limited and may be any method known to those skilled in the art. Known methods for detecting a mutated target sequence using a ligase include, for example, a ligase detection reaction (LDR method) and a ligase chain reaction (LCR method). The step of analyzing a ligase reaction preferably includes the LDR method or the LCR method.

[0108] In this embodiment, the step of analyzing the ligase reaction includes analyzing the ligation reaction products. The ligation reaction products may include reaction products that are frequently produced by joining when a mutated target sequence is present. By analyzing the ligation reaction products, the presence or absence of the mutated target sequence can be determined. The method for analyzing the ligation reaction product is not particularly limited and can be carried out by any method known to those skilled in the art, such as electrophoresis, amplification reactions such as PCR, microarray analysis, analysis using magnetic beads, etc. The reaction product may be labeled by any method, such as with a fluorescent substance or a specific sequence, and then analyzed.

[0109] A ligase that does not have the ability to discriminate between mismatched base pairs will ligate even if the double-stranded sequence formed by the target sequence to be ligated and the probe used for detection contains mismatched base pairs, but a chimeric ligase can discriminate between sequences that do not have mismatched base pairs near the junction during ligation. Utilizing this property, the chimeric ligase can detect mutated target sequences. This embodiment also provides a chimeric ligase for detecting mutated target sequences.

[0110] LDR method In this embodiment, when the step of analyzing the ligase reaction includes the LDR method, i.e., when a mutated target sequence is detected by the LDR method, the sample to be contacted with the chimeric ligase preferably contains three components: the target sequence to be detected for mutation, a nucleic acid containing a sequence complementary to the mutated target sequence (hereinafter referred to as the first nucleic acid), and a nucleic acid containing the 5'- or 3'-adjacent sequence of the target sequence and a complementary sequence (hereinafter referred to as the second nucleic acid).

[0111] The first nucleic acid and the second nucleic acid may be double-stranded nucleic acids, but are preferably single-stranded nucleic acids, and may also be referred to as a labeled primer and a common primer, respectively.

[0112] The length of the first nucleic acid and the second nucleic acid is from several nucleotides to several hundred nucleotides, preferably from about 10 to 200 nucleotides.

[0113] In one embodiment, the length of the first nucleic acid and the second nucleic acid is several nucleotides to several hundred nucleotides, for example, 10 to 200, 10 to 150 nucleotides, and preferably 12 to 100 nucleotides.

[0114] The specific procedures for the LDR method can be appropriately determined by those skilled in the art, but an example of each procedure will be described below.

[0115] First, in a sample to be contacted with the chimeric ligase, the first nucleic acid and the second nucleic acid are hybridized to a single-stranded sequence containing the target sequence. If the target sequence is contained in the sample in a double-stranded state, the sample may be heated to denature the double-stranded target sequence into a single-stranded sequence, and then hybridization may be performed.

[0116] In parallel with hybridization, or after hybridization is complete, the sample is contacted with a chimeric ligase. When a target sequence contains a mutation, the nucleic acids can be joined with high accuracy by a chimeric ligase. For example, if a single-stranded sequence containing the mutated target sequence forms a complementary double-stranded sequence with a first nucleic acid, the first nucleic acid and the second nucleic acid hybridized to the single-stranded sequence containing the mutated target sequence can be joined with high accuracy by a chimeric ligase capable of discriminating between mismatched base pairs. On the other hand, if the target sequence does not contain a mutation, the nucleic acids will be joined only at a low frequency by the chimeric ligase, or not at all. For example, if a single-stranded sequence containing an unmutated wild-type target sequence forms a double-stranded sequence with a mismatched base pair with a first nucleic acid, the first nucleic acid and the second nucleic acid hybridized to the single-stranded sequence containing the mutated target sequence will be joined only at a low frequency or not at all by the chimeric ligase capable of discriminating between mismatched base pairs.

[0117] When contacting a sample with a chimeric ligase, it is preferable that a mismatched base pair be present within three bases on either side of the junction to enable the chimeric ligase to more accurately discriminate between mismatched base pairs. That is, it is preferable to design the base sequence of the first nucleic acid so that, when hybridized to a single-stranded sequence containing a target sequence without a mutation, a mismatched base pair is present within three bases on either side of the junction. For example, the following describes an example in which, when the first nucleic acid and the second nucleic acid hybridize to a single-stranded sequence containing a target sequence, the second nucleic acid is present at the 3'-end of the first nucleic acid. In this case, it is preferable that the base complementary to the mutated base in the target sequence be located at the 3'-end of the first nucleic acid, one base adjacent to the 3'-end, and / or two bases adjacent to the 3'-end. In this arrangement, when a base complementary to the mutated base in the target sequence is present, a mismatched base pair will occur within 1, 2, and / or 3 bases on either side of the junction when the first nucleic acid hybridizes to a single-stranded sequence containing the target sequence without the mutation, allowing the chimeric ligase to recognize the mismatched base pair more accurately. Thus, when a mismatched base is present within 3 bases on either side of the junction, the joining rate by the chimeric ligase will be low.

[0118] Here, the first nucleic acid and the second nucleic acid may each contain an arbitrary sequence, as long as they each contain a sequence complementary to the first nucleic acid and the second nucleic acid as a continuous sequence. The position and length of the arbitrary sequence in each nucleic acid may be determined appropriately by those skilled in the art, but it is preferable that the arbitrary sequence be contained in a position other than within 3 bases on either side of the junction when hybridized to a single-stranded sequence containing a target sequence. Furthermore, the arbitrary sequence may be a sequence that serves as a label when analyzing a sample.

[0119] After the ligase reaction, if a mutated target sequence is present in the sample, a reaction product containing the first nucleic acid and the second nucleic acid ligated by the chimeric ligase will be generated, but if the mutated target sequence is not present, such a reaction product will be generated only at a low frequency or not at all. Therefore, the presence or absence of a mutated target sequence in a sample can be determined based on the presence or absence of the reaction product of the LDR method.

[0120] As described above, the chimeric ligase can be applied to the LDR method, which requires a ligase with highly accurate mismatch discrimination ability, and therefore, the present embodiment also provides a method for detecting a target sequence mutated by the LDR method.The present embodiment also provides a chimeric ligase for detecting a target sequence mutated by the LDR method.

[0121] Furthermore, the number of mutated target sequences detected by the LDR method of this embodiment is not limited to one type. Multiple types of mutated target sequences can be detected by adding first nucleic acids corresponding to multiple mutations to a sample.

[0122] LCR Method In this embodiment, when the step of analyzing the ligase reaction includes the LCR method, i.e., when a mutated target sequence is detected by the LCR method, the sample to be contacted with the chimeric ligase preferably contains five elements: the target sequence to be detected for mutation, a nucleic acid (first nucleic acid) containing a sequence complementary to the mutated target sequence, a nucleic acid (second nucleic acid) containing a sequence complementary to the 5'- or 3'-flanking sequence of the target sequence, a nucleic acid containing the mutated target sequence (hereinafter referred to as the third nucleic acid), and a nucleic acid (hereinafter referred to as the fourth nucleic acid) containing the 5'- or 3'-flanking sequence of the target sequence.

[0123] The first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid may be double-stranded nucleic acids, but are preferably single-stranded nucleic acids.

[0124] The length of the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid is from several nucleotides to several hundred nucleotides, preferably from about 10 to 200 nucleotides.

[0125] In one embodiment, the length of the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid is several nucleotides to several hundred nucleotides, for example, 10 to 200, 10 to 150 nucleotides, and preferably 12 to 100 nucleotides.

[0126] The specific reaction steps of the LCR method can be appropriately determined by those skilled in the art, but an example of each step will be described below.

[0127] In a sample to be contacted with a ligase, the first and second nucleic acids are hybridized to a single-stranded sequence containing the target sequence, and the third and fourth nucleic acids are hybridized to complementary strands of the single-stranded sequence containing the mutated target sequence. If the target sequence is contained in the sample in a double-stranded state, the sample may be heated to denature the double-stranded target sequence into single strands, and then hybridization may be performed.

[0128] After hybridization is complete, the sample is contacted with a chimeric ligase. When a target sequence contains a mutation, nucleic acids can be joined with high accuracy by a chimeric ligase. For example, when a single-stranded sequence containing a mutated target sequence forms a complementary double-stranded sequence with a first nucleic acid, a first nucleic acid and a second nucleic acid hybridized to the single-stranded sequence containing the mutated target sequence are joined with high accuracy by a chimeric ligase capable of discriminating between mismatched base pairs. Alternatively, when a complementary strand of a single-stranded sequence containing a mutated target sequence forms a complementary double-stranded sequence with a third nucleic acid, a third nucleic acid and a fourth nucleic acid hybridized to the complementary strand of the single-stranded sequence containing the mutated target sequence are joined with high accuracy by a chimeric ligase capable of discriminating between mismatched base pairs. On the other hand, if the target sequence does not contain a mutation, the nucleic acids are joined only at a low frequency or not at all by the chimeric ligase. For example, if a single-stranded sequence containing a target sequence without a mutation forms a double-stranded sequence with a mismatched base pair with a first nucleic acid, the first nucleic acid and the second nucleic acid hybridized to the single-stranded sequence containing the mutated target sequence are joined only at a low frequency or not at all by the chimeric ligase capable of discriminating between mismatched base pairs. Alternatively, if the complementary strand of the single-stranded sequence containing a target sequence without a mutation forms a double-stranded sequence with a mismatched base pair with a third nucleic acid, the third nucleic acid and the fourth nucleic acid hybridized to the complementary strand of the single-stranded sequence containing the mutated target sequence are joined only at a low frequency or not at all by the chimeric ligase capable of discriminating between mismatched base pairs.

[0129] When contacting a sample with a chimeric ligase, it is preferable that a mismatched base pair be present within three bases on either side of the junction to enable the chimeric ligase to more accurately discriminate between mismatched base pairs. That is, it is preferable to design the base sequence of the first nucleic acid or the third nucleic acid so that a mismatched base pair is present within three bases on either side of the junction when hybridized to a single-stranded sequence containing a target sequence without a mutation or a complementary strand of a single-stranded sequence containing a target sequence without a mutation. For example, when the first nucleic acid and the second nucleic acid hybridize to a single-stranded sequence containing a target sequence, if the second nucleic acid is present on the 3' end side of the first nucleic acid, it is preferable that the base complementary to the mutated base in the target sequence is located at the 3' end of the first nucleic acid, one base adjacent to the 3' end, and / or two bases adjacent to the 3' end. Furthermore, for example, when the third nucleic acid and the fourth nucleic acid hybridize to the complementary strand of a single-stranded sequence containing a target sequence, if the fourth nucleic acid is present on the 3'-end side of the third nucleic acid, it is preferable that the mutated base in the target sequence is located at the 3'-end of the third nucleic acid, one base adjacent to the 3'-end, and / or two bases adjacent to the 3'-end. In this arrangement, if a base complementary to a mutated base in the target sequence or a mutated base in the target sequence is present, when the first nucleic acid or the third nucleic acid hybridizes to a single-stranded sequence containing the target sequence without the mutation or the complementary strand of the single-stranded sequence containing the target sequence without the mutation, respectively, a mismatched base pair will be generated within 1, 2, and / or 3 bases on either side of the junction, allowing the chimeric ligase to recognize the mismatched base pair more accurately. Thus, when a mismatched base is present within 3 bases on either side of the junction, the joining rate by the ligase will be low.

[0130] Here, the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid may each contain an arbitrary sequence, as long as the nucleic acid contains each sequence contained therein as a continuous sequence. The position and length of the arbitrary sequence in each nucleic acid may be determined appropriately by one skilled in the art, but it is preferable that the arbitrary sequence be contained in a position other than within 3 bases on either side of the junction when hybridized to a single-stranded sequence containing the target sequence or its complementary strand. Furthermore, the arbitrary sequence contained in the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid may be a sequence that serves as a label when analyzing a sample.

[0131] After the ligase reaction, if a mutated target sequence is present in the sample, a reaction product containing the first nucleic acid and the second nucleic acid, and a reaction product containing the third nucleic acid and the fourth nucleic acid, which are ligated by the chimeric ligase, are generated. These two reaction products may form a double strand.

[0132] These two reaction products and the target sequence contained in the sample can act as single-stranded sequences containing the mutated target sequence or complementary strands of the single-stranded sequences containing the mutated target sequence, and can react with the first, second, third, and fourth nucleic acids in the sample. Therefore, by further repeating the hybridization and ligation steps described above, if the sample contains a mutated target sequence, the sequence containing the mutated target sequence and its complementary strand can be exponentially increased. If the mutated target sequence is not present in the sample, such reaction products will be produced only at a low frequency or not at all. Therefore, the presence or absence of the mutated target sequence in the sample can be determined from the presence or absence of the LCR reaction products.

[0133] As described above, the chimeric ligase can be applied to the LCR method, which requires a ligase with highly accurate mismatch discrimination ability, and therefore, this embodiment also provides a method for detecting a mutated target sequence by the LCR method. This embodiment also provides a chimeric ligase provided as a first embodiment for detecting a mutated target sequence by the LCR method.

[0134] Furthermore, the number of mutated target sequences detected by the LCR method of this embodiment is not limited to one type. Multiple types of mutated target sequences can be detected by adding first nucleic acids and third nucleic acids corresponding to multiple mutations to the sample.

[0135] The applicability of a chimeric ligase to the LDR or LCR method can be evaluated by any method using a means for confirming the ability to discriminate between mismatched base pairs, for example, by subjecting the chimeric ligase to an experiment that reproduces the steps of the LDR or LCR method, or by subjecting the chimeric ligase to an experiment that performs steps similar to those of the LDR or LCR method.

[0136] Furthermore, whether a chimeric ligase is applicable to the LDR or LCR method can be evaluated, for example, by reacting a sample containing a mutated target sequence, a sequence complementary to the mutated target sequence, a sequence complementary to the unmutated target sequence, and a sequence complementary to the adjacent sequence of the target sequence with the chimeric ligase, and comparing the ligation products containing the sequence complementary to the mutated target sequence and the sequence complementary to the adjacent sequence of the target sequence with the ligation products containing the sequence complementary to the unmutated target sequence and the sequence complementary to the adjacent sequence of the target sequence. That is, whether a chimeric ligase is applicable to the LDR or LCR method can be evaluated based on the amount or proportion of ligation products containing the sequence complementary to the unmutated target sequence and the sequence complementary to the adjacent sequence of the target sequence, which are generated by ligation despite the presence of a mismatched base pair near the junction. For example, evaluation may be based on the misligation rate (%).

[0137] The applicability of a chimeric ligase to the LDR or LCR method can also be evaluated by reacting the ligase with a sample containing, for example, a mutated target sequence, an unmutated target sequence, and a single-stranded circular sequence containing a sequence complementary to the mutated target sequence, the single-stranded circular sequence being opened by a nick near the mutation site. Preferably, the mutated target sequence and the unmutated target sequence in the sample are contained as amplification products. In this case, ligation of the nick generates a circular sequence containing a sequence complementary to the mutated target sequence as a ligation product. Therefore, whether a chimeric ligase can discriminate between mismatched base pairs can be evaluated by comparing the ligation product containing the circular sequence when the mutated target sequence is added with the ligation product containing the circular sequence when the non-mutated target sequence is added. The applicability of a chimeric ligase to the LDR or LCR method can also be evaluated by analyzing the amplification product, which can be obtained by ligation to circularize the nick-containing region and amplify it using PCR or other methods. That is, the applicability of a chimeric ligase to the LDR or LCR method can be evaluated based on the amount or proportion of ligation products containing the circular sequence or amplification products of the nick-containing region, which are generated by joining the mismatched base pair despite being located near the junction. For example, evaluation may be based on the misjunction rate (%).

[0138] (Kit for detecting mutated target sequences) In a third embodiment, there is provided a kit for detecting a mutated target sequence, the kit comprising the chimeric ligase provided as the first embodiment.

[0139] The chimeric ligase provided as a kit of this embodiment may be provided together with any buffer, nucleic acid, other necessary reagents, etc. The chimeric ligase is preferably provided together with reagents used in ligase detection reaction (LDR) or ligase chain reaction (LCR), and may also be provided together with other reagents, such as dithiothreitol, water, potassium chloride, EDTA, Tris-HCl, BSA, DMSO, glycerol, DNA or RNA enzyme inhibitors, surfactants, etc. The reagents used in LDR may include a nucleic acid comprising a sequence complementary to the mutated target sequence and a nucleic acid comprising a sequence complementary to the 5'- or 3'-flanking sequence of the target sequence. The reagents used in LCR may include a nucleic acid comprising a sequence complementary to the mutated target sequence, a nucleic acid comprising the mutated target sequence, a nucleic acid comprising a sequence complementary to the 5'- or 3'-flanking sequence of the target sequence, and a nucleic acid comprising the 5'- or 3'-flanking sequence of the target sequence.

[0140] The kit of this embodiment may be provided in such a manner that the chimeric ligase, any buffer, nucleic acid, and other necessary reagents are contained in the same container, or the reagents may be provided in separate containers. When the reagents are provided in separate containers, the containers may be provided together in a single box or the like.

[0141] The kit of this embodiment makes it possible to detect a mutated target sequence by reacting a sample requiring mutation detection with the chimeric ligase included in the kit and, if necessary, reagents and the like provided therewith. Detection of a mutated target sequence is preferably achieved by treating a sample requiring mutation detection with the kit of this embodiment containing the chimeric ligase and then analyzing the sample. Detection of a mutated target sequence is more preferably achieved by treating a sample requiring mutation detection with the kit of this embodiment containing the chimeric ligase and then analyzing the ligation product that is generated when a mutation is present.

[0142] The kit of this embodiment may be a kit for detecting a target sequence mutated by LDR, or may be a kit for detecting a target sequence mutated by LCR.

[0143] The present embodiment also provides a method for detecting a mutated target sequence using a kit comprising the chimeric ligase.

[0144] The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples. [Example]

[0145] <Design of chimeric protein recombination site> Amino acid homology analysis was performed on LigA derived from Thermus species (Tsp) (sequence number 1), LigA derived from Thermus thermofilus (Tth) (sequence number 2), LigA derived from Thermus brockianus (Tbr) (sequence number 3), LigA derived from Thermus thermophilus (Tfi) (sequence number 4), and LigA derived from Thermus Oshimai (Tos) (sequence number 5), and it was found that the amino acid sequences of amino acids 316 to 329 (AYKFPAEEKETRLL = sequence X), amino acids 421 to 443 (KEGKVHRCPNPLCPAKRFEAIRH = sequence Y), and amino acids 602 to 616 (TGELSRPREEVKALL = sequence Z) are conserved between species. An adenylation domain (domain 1) is located upstream of sequence X, an oligomer-binding domain (OB-fold domain) and a zinc finger domain (domain 2 together) are located between sequences X and Y, a helix-hairpin-helix domain (domain 3) is located between sequences Y and Z, and a BRCT domain (domain 4) is located downstream of sequence Z. Figure 1 shows an example of the relative positions of the adenylation domain (adenylation), OB-fold domain (OB), zinc finger domain (Zn), helix-hairpin-helix domain (HhH), and BRCT domain (BRCT) in a ligase derived from Thermus bacteria. Using sequences X to Z, which are conserved across species, we generated chimeric LigA expression constructs by recombining domains 1 to 4 of Tth LigA with those of Tsp LigA using the Gibson Assembly recombination method (SEQ ID NOS: 6 to 9). The regions containing sequences X to Z at their termini were recombined. Furthermore, chimeric LigA expression constructs were prepared by recombining domain 2 of Tbr LigA, Tfi LigA, or Tos LigA with the same domain of Tsp LigA (SEQ ID NOs: 10 to 12). All constructs had a 6xHis sequence and a TEV protease recognition sequence added to their N-termini (SEQ ID NO: 13).

[0146] [Table 1] JPEG2026007482000002.jpg245170JPEG2026007482000003.jpg245170JPEG2026007482000004.jpg245170JPEG20260074820 00005.jpg245170JPEG2026007482000006.jpg245170JPEG2026007482000007.jpg245170JPEG2026007482000008.jpg106170

[0147] <Transformation> The pET vectors containing the Tsp LigA construct, Tth LigA construct, Tbr LigA construct, Tfi LigA construct, Tos LigA construct, Tth / Tsp (domain 1) LigA construct, Tth / Tsp (domain 2) LigA construct, Tth / Tsp (domain 3) LigA construct, Tth / Tsp (domain 4) LigA construct, Tbr / Tsp (domain 2) LigA construct, Tfi / Tsp (domain 2) LigA construct, and Tos / Tsp (domain 2) LigA construct were transformed into BL21(DE3) competent cells (Thermo Fisher, Cat: EC0114). The transformed E. coli cells were plated on ampicillin-containing LB plates and cultured overnight at 37°C to allow colony formation.

[0148] <Induction of enzyme protein expression> A single colony was inoculated into a small volume of carbenicillin-containing liquid LB medium and cultured overnight at 37°C with shaking (225 rpm, preculture). The next day, 1 / 40 of the preculture was added to fresh carbenicillin-containing liquid LB medium and cultured for 4 hours with shaking at 37°C (225 rpm, main culture). After confirming that the OD600 of the main culture was between 0.4 and 0.6, IPTG (Fujifilm Wako, Cat: 099-05013) was added to a final concentration of 0.1 mM and cultured for 3 hours with shaking at 30°C (225 rpm, IPTG induction). The IPTG-induced E. coli solution was collected and centrifuged (10,000 x g, 10 minutes, 4°C) to obtain a pellet of E. coli. The wet weight was measured and the pellet was frozen overnight or longer in a -80°C freezer.

[0149] <Lysis of E. coli and collection of soluble fraction> Transfer the frozen Escherichia coli onto ice, add 5 mL of BugBuster (Novagen, Cat: 70921-4) containing 1,000 U / mL rLysozyme (Novagen, Cat: 71110-4) + 1 / 200 Volume Protease inhibitor cocktail (Millipore, Cat: 539134-1MLCN) per 1 g of wet weight of Escherichia coli, and immediately vortex to completely suspend it without warming. Pipette the Escherichia coli suspension into 1.5 mL microtubes, transfer them to each heat block set at 60 °C, and perform a shaking reaction for 15 minutes (600 rpm). Immediately after the heat treatment, transfer it onto an aluminum block placed on ice, perform a cooling treatment for 15 minutes, and age the precipitate. After centrifugation (16,000 xg, 20 minutes, 4 °C), collect the soluble fraction and transfer it to a new 1.5 mL microtube.

[0150] <His Affinity Purification> Transfer 250 μL of His-Affinity Gel (included in His-Spin Protein Miniprep kit (ZymoResearch, Cat: P2002)) into a His-Spin column (included in His-Spin Protein Miniprep kit), and remove the gel dispersion solution by centrifugation (15,000 xg, 20 seconds, 4 °C). Add 300 μL of the soluble fraction, and perform a shaking treatment at 25 °C for 5 minutes (1,500 rpm). Centrifuge (same as above), and discard the flow-through solution by decantation. Add 250 μL of washing buffer (10 mM imidazole (Nacalai Tesque, Cat: 08787-22), 0.03% Triton X-100 (FUJIFILM Wako Pure Chemical Corporation, Cat: 160-24751)), gently vortex to mix, and centrifuge (same as above). Discard the flow-through solution, and repeat the same washing operation two more times. Set the column in a new 1.5 mL microtube, add 250 μL of His-Elution Buffer (included in His-Spin Protein Miniprep kit), mix by vortex for about 10 seconds, and let it stand on ice for 1 minute or more. Recover the eluate by centrifugation (same as above). Accurately record the volume of the eluate.

[0151] <Measurement of protein concentration in eluate> Protein concentration was measured using an absorption spectrometer by measuring the absorbance at 280 nm, which is derived from the side chains of the aromatic amino acids (tyrosine, tryptophan) that make up the protein. His-Elution Buffer was used as a blank solution. The yield (µg protein / µg wet weight of E. coli) was calculated by multiplying the eluate volume by the measured value and dividing the result by the wet weight of the E. coli used.

[0152] <Ligase activity evaluation - ssDNA oligo cross-linking ligase reaction> Each eluate was diluted with LigA diluent (10 mM Tris-HCl, pH 7.4, 100 mM potassium chloride (Nacalai Tesque, Cat. No. 28538-75), 0.1 mM EDTA (Thermo Fisher Scientific, Cat. No. 15575-020), 1 mM dithiothreitol, 0.1% Triton X®-100, 50% glycerol) to a protein concentration of 3.4 ng / μL. The diluted LigA solution was added to a reaction solution containing 20 nM template DNA oligo (any of SEQ ID NOs: 14 to 20), 20 nM bridge ssDNA oligo (any of SEQ ID NOs: 21 to 28), and 1× HiFi Taq Ligase Buffer (New England Biolabs, Cat. No. M0647S, included buffer) at a concentration of 10 v / v% and mixed thoroughly. Heat denaturation was performed at 95°C for 5 minutes, and unless otherwise specified, the ligase reaction was carried out for 1 hour at 50°C. After the reaction was completed, the mixture was quickly cooled, and a reaction stop solution (80% formamide (Fujifilm Wako, Cat: 066-02301), 100 mM EDTA) was added in an amount 1.5 times the reaction solution, mixed, and then allowed to stand on ice.

[0153] [Table 2] JPEG2026007482000010.jpg87170

[0154] <Ligase activity evaluation - dsDNA amplicon cross-linking ligase reaction> Each eluate was diluted with LigA diluent (10 mM Tris-HCl, pH 7.4, 100 mM potassium chloride (Nacalai Tesque, Cat. No. 28538-75), 0.1 mM EDTA (Thermo Fisher Scientific, Cat. No. 15575-020), 1 mM dithiothreitol, 0.1% Triton X®-100, 50% glycerol) to a protein concentration of 3.4 ng / μL. The diluted LigA solution was added to a reaction solution containing 10 nM template DNA oligo (a DNA oligo of any one of SEQ ID NOs: 14 to 20), 10 nM bridged dsDNA amplicon (a DNA oligo of any one of SEQ ID NOs: 29 to 36), and 1× HiFi Taq Ligase Buffer (New England Biolabs, Cat. No. M0647S, included buffer), at a concentration of 10 v / v%. The diluted LigA solution was then mixed thoroughly. Heat denaturation was carried out at 95°C for 5 minutes, and the ligase reaction was carried out for 15 cycles, each cycle consisting of 50°C for 3 minutes and 80°C for 15 seconds.

[0155] [Table 3]

[0156] <Ligase activity evaluation - quantitative real-time PCR reaction> The quenched reaction mixture was diluted 40,000-fold with UltraPure DNase / RNase-Free Distilled Water and added in an amount of 2 μL to a quantitative real-time PCR reaction mixture consisting of 1X Prime Time PCR Assay (Integrated DNA Technologies, consisting of 20 μM DNA oligos of SEQ ID NOs: 37 and 38 and 10 μM DNA oligos of SEQ ID NO: 39, which are modified with the fluorescent molecule FAM at the 5' end and the quencher molecule Iowa Black® FQ at the 3' end) and 1X Prime Time Gene Expression Master Mix (Integrated DNA Technologies, Cat. 1055771). After 3 minutes of heat denaturation at 95°C, a total of 45 PCR cycles were performed, each cycle consisting of 15 seconds of heat denaturation at 95°C and 30 seconds of extension at 60°C. Fluorescent signals were detected once after each cycle. The Ct value was calculated as the position at which the second derivative (a curve differentiated twice) of the measured fluorescence signal amplification curve was maximized.

[0157] [Table 4]

[0158] Example 1 The efficiency (%) of circularization of the detection template DNA oligos through nicking was evaluated in the presence of either a bridged ssDNA oligo containing sequences perfectly complementary to the 13 bases on the 5' and 3' ends of the detection template DNA oligos used to detect each BRAF gene mutation and forming a nick when annealed to the 5' and 3' ends of the detection template DNA oligos (conditions corresponding to the mutation variants of the detection template DNA oligo and the bridged ssDNA oligo). Alternatively, a BRAF bridged ssDNA oligo similarly forms a nick when annealed to the detection template DNA oligo, but is not perfectly complementary to the detection template DNA oligo and forms a mismatch with the detection template DNA oligo on the 3' end (mismatch condition).

[0159] Figure 2 shows the relative conjugation rates for each mismatch condition using Tth LigA, where the conjugation rate for the corresponding mutant variant of the detection template DNA oligo and bridging ssDNA oligo is set to 100%. While the relative conjugation rate for all mismatch combinations was generally less than 1%, showing good accuracy, eight combinations exceeded 1%, resulting in misconjugation rates of up to 3.65%. Note that Tth LigA has been reported to have high accuracy in a previous study (Nucleic Acids Research, 1996, Vol. 24, No. 14, 3071-3078).

[0160] <Example 2> In the test of Example 1, accuracy was evaluated when amplicons amplified and purified from plasmid DNA containing each BRAF gene mutation were used as the bridging nucleic acid (bridged dsDNA amplicon) instead of ssDNA oligos. Figure 3 shows the relative junction rates under each mismatch condition, assuming that the junction amount under conditions in which the detection template DNA oligo and the bridged dsDNA amplicon matched the mutation variants, using Tth LigA. When bridged dsDNA amplicons were used, 13 combinations with accuracy exceeding 5% were observed. A total of 19 combinations had accuracy exceeding 1%.

[0161] Example 3 Previous literature2 (Nucleic Acids Research, 1999, Vol. 27, No. 3) has reported that Tsp LigA has higher accuracy than Tth LigA. Therefore, we evaluated the accuracy of Tsp LigA instead of the Tth LigA used in Example 1 (Figure 4). Compared with Tth LigA (Figure 2), the accuracy was improved, exceeding 1% in only one combination.

[0162] Example 4 Similarly, accuracy was evaluated using dsDNA amplicons as bridging nucleic acids, substituting Tsp LigA for Tth LigA used in the test in Example 2 (FIG. 5). Compared to Tth LigA (FIG. 3), accuracy was improved, with three combinations with accuracy exceeding 5% and a total of eight combinations with accuracy exceeding 1%.

[0163] On the other hand, compared to Tth LigA, the circularization efficiency of Tsp LigA under fully complementary conditions using dsDNA amplicons as bridging nucleic acids was lower, especially when the detection template DNA oligos E1, D, and G were used, which was less than half that of Tth LigA (Figure 6). This suggests that the circularization efficiency is lower when long dsDNA is used as bridging nucleic acids.

[0164] <Example 5> The low ligation activity of Tsp LigA when dsDNA amplicons were used as bridging nucleic acids was believed to be due to the reaction rate, and the reaction rate was evaluated when ssDNA oligos were used as bridging nucleic acids. Among the test conditions in Examples 1 and 3, the ligase reaction time was set to 5, 10, 20, 30, 45, and 60 minutes, and BRAF E1 was used as the detection template DNA oligo and bridging ssDNA oligo. While the ligation reaction with Tth LigA nearly reached a plateau within 10 minutes of reaction, Tsp LigA required approximately 45 minutes to reach a plateau. As expected, the slow reaction rate was believed to be the cause of the low reaction rate when dsDNA was used as the bridging nucleic acid.

[0165] Example 6 To create an enzyme that combines the high fidelity of Tsp LigA with the rapid reactivity of Tth LigA, Tth / Tsp chimeric LigA was synthesized by replacing domains 1-4 of Tth LigA with the corresponding domains of Tsp LigA. Under the test conditions of Example 1, BRAF E1 was used as the detection template DNA oligo, a BRAF E1 ssDNA oligo was used as the fully complementary bridging nucleic acid, and a BRAF G ssDNA oligo was used as the mismatch bridging nucleic acid. As shown in Figure 8, under the condition using the fully complementary bridging nucleic acid (Perfect Match), both chimeric LigAs exceeded the reaction efficiency of Tsp LigA. Under the condition using the mismatch bridging nucleic acid (Mismatch), the chimeric LigAs (chimera1, chimera2) incorporating domains 1 and 2 of Tsp LigA showed improved fidelity compared to Tth LigA.

[0166] Example 7 The reaction rates of the Tth / Tsp domain 1 chimeric LigA (Fig. 9, Chimera 1) and Tth / Tsp domain 2 chimeric LigA (Fig. 9, Chimera 2), which showed improved reactivity and accuracy compared to Tth LigA in Example 6, were evaluated in the same manner as in Example 5. The results showed that both chimeric LigAs increased the amount of conjugated product compared to both Tsp LigA and Tth LigA, and also significantly improved the reaction rate compared to Tsp LigA. This effect was even more pronounced in the Tth / Tsp domain 2 chimeric LigA, in which domain 2 of Tth LigA was replaced with domain 2 of Tsp LigA.

[0167] Example 8 In Examples 6 and 7, the reactivity and accuracy of the Tth / Tsp domain 2 chimeric LigA, which combines the fidelity of Tsp LigA with the reactivity of Tth LigA, was evaluated when a dsDNA amplicon was used as the bridging nucleic acid. BRAF E1 was used as the detection template DNA oligo, and the BRAF E1 dsDNA amplicon was used as the fully complementary bridging nucleic acid. The BRAF G dsDNA amplicon or BRAF K dsDNA amplicon was used as the mismatch bridging nucleic acid. As shown in Figure 10, the relative conjugation efficiency of the Tth / Tsp domain 2 chimeric LigA (Figure 9, Chimera2), when used with Tsp LigA, was reduced to half, but recovered to approximately 80%. On the other hand, when the BRAF G dsDNA amplicon was used as the bridging nucleic acid, Tth LigA resulted in approximately 20% misjunctions, whereas the chimeric LigA conferred the same accuracy as Tsp LigA.

[0168] Example 9 To confirm that the chimeric LigA incorporating domain 2 of Tsp LigA universally confers high fidelity, we constructed chimeric LigAs in which domain 2 of Tbr LigA, Tfi LigA, and Tos LigA was replaced with domain 2 of Tsp LigA, and evaluated their effect on improving fidelity. The detection template DNA oligos used in the ligase reaction were any of SEQ ID NOs: 40, 43, 46, 49, 14, and 52, the bridging ssDNA oligos that were completely complementary to each detection template DNA oligo were any of SEQ ID NOs: 41, 44, 47, 50, 21, and 53, and the mismatch bridging ssDNA oligos were any of SEQ ID NOs: 42, 45, 48, 51, 28, and 24.

[0169] [Table 5] JPEG2026007482000014.jpg62170

[0170] Furthermore, during the quantitative real-time PCR reaction, three types of primer-probe sets, from SEQ ID NO: 54 to SEQ ID NO: 62, that matched the gene variant species to be analyzed were used.

[0171] [Table 6]

[0172] As shown in Table 7, among the 16 combinations of detection template DNA oligos and mismatch bridge ssDNA oligos, the use of chimeric LigA improved accuracy in 14 of them. In particular, the improvement in accuracy due to the Tsp LigA domain 2 recombinant was more pronounced in combinations where accuracy was low when wild-type LigA was used.

[0173] [Table 7]

Claims

1. A chimeric ligase in which the OB-fold domain or adenylation domain of a ligase derived from a thermophilic bacterium is replaced with the corresponding domain of a different species derived from a thermophilic bacterium, and the chimeric ligase has improved discrimination ability against mismatched base pairs compared to the ligase before the replacement.

2. 2. The chimeric ligase of claim 1, wherein the corresponding domains of different species from thermophilic bacteria are derived from Thermus species.

3. A chimeric ligase, wherein the OB-fold domain or the adenylation domain of a ligase derived from a thermophilic bacterium is 1) a ligase consisting of the amino acid sequence shown in SEQ ID NO: 1; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1; 3) a ligase consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 have been deleted, substituted, and / or added; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of the base sequence encoding the amino acid sequence shown in SEQ ID NO:

1. a chimeric ligase in which the corresponding domain of a ligase from a different species from a thermophilic bacterium is substituted, selected from the group consisting of:

4. The chimeric ligase of claim 3, wherein the domain corresponding to the OB-fold domain comprises an amino acid sequence having at least 80% identity with the amino acid sequence from positions 320 to 406 of SEQ ID NO:

1.

5. The chimeric ligase of claim 3, wherein the domain corresponding to the adenylation domain comprises an amino acid sequence having at least 80% identity with the amino acid sequence from positions 73 to 319 of SEQ ID NO:

1.

6. The chimeric ligase according to any one of claims 1 to 5, which has a lower joining rate when a mismatched base pair is present within three bases on either side of the ligation site formed by the ligase, compared to the ligase before substitution.

7. The chimeric ligase according to any one of claims 1 to 5, wherein the thermophilic bacterium-derived ligase before substitution is a ligase derived from Thermus thermophilus, Thermus brockianus, Thermus filiformis, or Thermus oshimai.

8. The thermophilic ligase before substitution was 1) a ligase consisting of an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 2) a ligase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5; 3) a ligase consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5 have been deleted, substituted, and / or added; and 4) A ligase encoded by a base sequence that hybridizes under stringent conditions with a complementary sequence of a base sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 2 to 5. The chimeric ligase according to any one of claims 1 to 5, which is a ligase selected from the group consisting of:

9. A kit for detecting a mutated target sequence, comprising the chimeric ligase of any one of claims 1 to 5.

10. 10. The kit of claim 9, further comprising reagents used in a ligase detection reaction (LDR) or a ligase chain reaction (LCR).

11. A method for detecting a mutated target sequence in a sample, comprising the steps of contacting the sample with a chimeric ligase according to any one of claims 1 to 5; analyzing the ligase reaction; A method comprising:

12. 12. The method of claim 11, wherein the mutation is a single nucleotide polymorphism.

13. 12. The method of claim 11, wherein the step of analyzing the ligase reaction comprises a ligase detection reaction (LDR) or a ligase chain reaction (LCR).

14. 14. The method of claim 13, wherein the presence or absence of a mutated target sequence in a sample is determined based on the presence or absence of an LDR or LCR reaction product.

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