Design method for primer set, primer set, base sequence determination method for total length of mitochondrial DNA, design device for primer set, computer program, and storage medium

Abstract

To simplify a process of determining the total base sequence of mitochondrial DNA by using a highly common primer capable of using a fragmented sample.SOLUTION: According to the present invention, total length sequences of known mitochondrial DNA of a plurality of biological species which belong to a specific biological classification are acquired; conserved regions are extracted by aligning the acquired total length sequences of the mitochondrial DNA; primer candidate sequences are set from base sequences of each of the extracted conserved regions; combinations of the primer candidate sequences with which 3 to 6 types of fragments that cover the total length of the mitochondrial DNA can be obtained, and a desired length can be obtained as the length of an overlapping region with a neighboring fragment, are identified as primer set candidates; and at least one of the primer set candidates is selected as the primer set.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present disclosure relates to a method for designing a primer set for determining the full-length nucleotide sequence of mitochondrial DNA, a primer set, a method for determining the full-length nucleotide sequence of mitochondrial DNA, a device for designing a primer set, a computer program, and a storage medium.

Background Art

[0002] As a method for investigating or monitoring species present in the environment, there is a method of recovering nucleic acids derived from organisms from the environment, analyzing the recovered nucleic acids, and identifying the species from which the nucleic acids are derived. Nucleic acids derived from organisms are nucleic acids that contain genetic information of organisms and are released from organisms into the environment, and are included in nucleic acids present in the environment such as environmental DNA. As a method for analyzing such environmental DNA, a method of identifying the species from which mitochondrial DNA collected from the environment is derived using the nucleotide sequence of known mitochondrial DNA as a reference sequence is known. And in order to analyze environmental DNA using the nucleotide sequence of mitochondrial DNA as a reference sequence, it is necessary that sequence information of mitochondrial DNA of various organisms be registered in a database. At this time, in order to improve the accuracy of analysis, it is desirable that the full nucleotide sequences of mitochondrial DNA of more species be accumulated in the database and the database be enriched.

[0003] In order to enrich the database and efficiently obtain information associating the entire nucleotide sequence of mitochondrial DNA with biological species for a wide range of biological species, for example, it is desirable to satisfy the following conditions. That is, as a first condition, as a primer for obtaining a mitochondrial DNA sequence, it is desirable to reduce the load required for primer design by using a primer that can be commonly applied to a certain range of biological species. As a second condition, even when the quality of environmental DNA containing mitochondrial DNA is relatively low and mitochondrial DNA that is not circular but fragmented to a certain extent is used as an analysis target, it is desirable that the nucleotide sequence can be analyzed. As a third condition, it is desirable that the construction of the entire nucleotide sequence of mitochondrial DNA can be easily carried out with as few man-hours as possible.

[0004] As a method for obtaining the entire nucleotide sequence of mitochondrial DNA, for example, in Patent Document 1, a configuration is disclosed in which 24 known primer pairs designed for humans are used to amplify a relatively short human mitochondrial genome fragment and determine the full length of mitochondrial DNA. In Non-Patent Document 1, a method of amplifying the entire length of mitochondrial DNA as a single fragment using a single primer is disclosed, and in Non-Patent Documents 2 and 3, methods of amplifying the entire length of mitochondrial DNA as two fragments using two primer pairs are disclosed. In addition, Patent Document 2 discloses a method of amplifying all DNA fragments of the entire genome including mitochondrial DNA.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, in the method described in Patent Document 1, since it is necessary to design a relatively large number of primer sequences according to the number of mitochondrial genome fragments to be amplified, it is difficult to design primer sequences applicable to a wide range of species, and it is considered difficult to satisfy the above first condition. Also, in the methods described in Non-Patent Documents 1 to 3, since it is necessary to use an unfragmented circular mitochondrial DNA sample to amplify a relatively long DNA fragment, it is considered difficult to satisfy the above second condition. Further, in the method described in Patent Document 2, mitochondrial DNA is amplified by amplifying all DNA fragments of the entire genome, but since DNA other than mitochondrial DNA is also amplified, the overall operation becomes complicated and the cost and the load for data processing increase, and it is considered difficult to satisfy the above third condition. Therefore, there has been a demand for a technique using primers with high commonality that can be used in a relatively wide range of species, that can be used even with a sample in which mitochondrial DNA is fragmented to some extent, and that can simplify the process of determining the entire nucleotide sequence of mitochondrial DNA.

Means for Solving the Problems

[0008] The present disclosure can be realized in the following forms. (1) According to one embodiment of the present disclosure, a method for designing a primer set for determining the full-length nucleotide sequence of mitochondrial DNA is provided. This method for designing a primer set includes obtaining the full-length sequences of known mitochondrial DNA of a plurality of species belonging to a specific biological classification, aligning the obtained full-length sequences of the mitochondrial DNA, and extracting conserved regions from the full-length sequences of the mitochondrial DNA, which are regions that meet a predetermined criterion as a criterion indicating a high degree of nucleotide conservation. Using the nucleotide sequences of the extracted conserved regions, primer candidate sequences are set. When the primer candidate sequences are combined and used as a primer set to amplify mitochondrial DNA, three or more and six or fewer fragments that cover the full length of the mitochondrial DNA are obtained, and a combination of primer candidate sequences that gives a desired length as the length of the overlapping region with an adjacent fragment is specified as a primer set candidate, and at least one of the primer set candidates is selected as the primer set. According to the method for designing a primer set of this embodiment, a primer set having a nucleotide sequence with a high degree of conservation in species belonging to a "specific biological classification" is obtained, and a primer set that gives three or more and six or fewer fragments that cover the full length of the mitochondrial DNA is obtained. Therefore, it is possible to obtain a highly common primer that can be used in a relatively wide range of species. In addition, since mitochondrial DNA can be amplified as three or more and six or fewer fragments, even if the mitochondrial DNA used as a template for PCR is not circular but partially degraded, it is possible to amplify a desired DNA fragment and obtain a DNA fragment having a length suitable for nucleotide sequence analysis by a long-read sequencer. (2) In the method for designing a primer set of the above embodiment, the specific biological classification may be a class of the biological classification hierarchy or a classification hierarchy lower than the class. With such a configuration, it becomes possible to determine the full-length sequence of mitochondrial DNA by a simple process using a common primer set for various organisms belonging to a class or a classification hierarchy lower than the class. (3) In the method for designing a primer set of the above-described form, the specific biological classification may be the Mammalia or Aves. With such a configuration, it becomes possible to determine the full-length mitochondrial DNA sequence by a simple process using a common primer set for various organisms belonging to the Mammalia or Aves. (4) In the method for designing a primer set of the above-described form, the primer set candidates may be specified such that the length of the overlapping region is 500 bases or more and 4000 bases or less. With such a configuration, it is possible to increase the likelihood of selecting all available primer sequences without omission and specifying appropriate primer set candidates. (5) In the method for designing a primer set of the above-described form, the primer set candidates may be specified such that the length of the overlapping region is 500 bases or more and 3000 bases or less. With such a configuration, even when a sample containing mitochondrial DNA from a plurality of different types of organisms belonging to a common specific biological classification is used as a template for amplification, based on the sequence differences in the overlapping region, the amplified DNA fragments can be distinguished for each biological species, and the sequences of the amplified DNA fragments can be joined together for each biological species. (6) In the method for designing a primer set of the above-described form, the primer set may be specified such that when mitochondrial DNA is amplified using the primer set, four fragments that cover the full length of the mitochondrial DNA are obtained. With such a configuration, it is possible to improve the accuracy of determining the full-length mitochondrial DNA sequence by amplifying the desired DNA fragments. (7) In the method for designing a primer set according to the above aspect, using each of the primer pairs composed of a pair of primers constituting the primer set candidate, a mitochondrial DNA fragment is experimentally amplified using DNA derived from an organism belonging to the specific biological classification as a template, and based on the amplification result, the primer set is selected from the primer set candidate so as to be composed of a primer pair determined to have obtained a desired mitochondrial DNA fragment. With such a configuration, the accuracy of amplifying a desired DNA fragment to determine the full-length mitochondrial DNA sequence can be increased. (8) In the method for designing a primer set according to the above aspect, the setting of the primer candidate sequence is to identify a highly conserved sequence, which is a base sequence of a site with a relatively high degree of base conservation among each of the conserved regions, and a first parameter representing the high degree of sequence specificity of the highly conserved sequence with respect to the sequence of mitochondrial DNA of a species belonging to the specific biological classification is equal to or higher than a predetermined first reference value, and a second parameter representing the high degree of sequence specificity of the highly conserved sequence with respect to the sequence of mitochondrial DNA of a species not belonging to the specific biological classification is equal to or lower than a second reference value predetermined as a value smaller than the first reference value. The sequence may be used as the primer candidate sequence. With such a configuration, the specificity of the primer set for a species belonging to a specific biological classification can be further increased. (9) In the method for designing the primer set of the above-described form, the first parameter is the ratio of the number of species in which the number of mismatched bases is 1 or less when the highly conserved sequence is aligned with the full-length sequences of mitochondrial DNAs of the plurality of species belonging to the specific biological classification, to the total number of the plurality of species belonging to the specific biological classification. The second parameter is the ratio of the number of species in which the number of mismatched bases is 1 or less when the highly conserved sequence is aligned with the full-length sequences of mitochondrial DNAs of the plurality of species not belonging to the specific biological classification, to the total number of the plurality of species not belonging to the specific biological classification. The first reference value may be 85%, and the second reference value may be 15%. With such a configuration, the specificity of the primer set for the species belonging to the specific biological classification can be further enhanced. (10) According to another form of the present disclosure, a primer set for determining the base sequence of the entire length of mitochondrial DNA is provided. This primer set includes a first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing on the 3'-terminal side, a second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing on the 3'-terminal side, a third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing on the 3'-terminal side, and a fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing on the 3'-terminal side. Each of the primers has a length of 100 bases or less. According to the primer set of this form, the base sequences of the entire lengths of mitochondrial DNAs of various species belonging to the class Mammalia can be determined by using a common primer set. (11) According to still another aspect of the present disclosure, a primer set for determining the full-length nucleotide sequence of mitochondrial DNA is provided. This primer set comprises a first primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing at the 3'-end side, a second primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 12 in the sequence listing at the 3'-end side, a third primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing at the 3'-end side, and a fourth primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 16 in the sequence listing at the 3'-end side, and each of the said primers has a length of 100 bases or less. According to the primer set of this aspect, the full-length nucleotide sequence of mitochondrial DNA of various species belonging to the avian class can be determined by using a common primer set. (12) According to still another aspect of the present disclosure, a primer set for determining the full-length nucleotide sequence of mitochondrial DNA is provided. This primer set comprises a first primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 130 and SEQ ID NO: 131 in the sequence listing at the 3'-end side, a second primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 132 and SEQ ID NO: 133 in the sequence listing at the 3'-end side, a third primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 134 and SEQ ID NO: 135 in the sequence listing at the 3'-end side, and a fourth primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 136 and SEQ ID NO: 137 in the sequence listing at the 3'-end side, and each of the said primers has a length of 100 bases or less. According to the primer set of this aspect, the full-length nucleotide sequence of mitochondrial DNA of various species belonging to the mammalian class can be determined by using a common primer set. (13) According to still another aspect of the present disclosure, a primer set for determining the full-length nucleotide sequence of mitochondrial DNA is provided. This primer set comprises a first primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 138 and SEQ ID NO: 139 in the sequence listing at the 3'-terminal side, a second primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 140 and SEQ ID NO: 141 in the sequence listing at the 3'-terminal side, a third primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 142 and SEQ ID NO: 143 in the sequence listing at the 3'-terminal side, and a fourth primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 144 and SEQ ID NO: 145 in the sequence listing at the 3'-terminal side, and each of the primers has a length of 100 bases or less. According to the primer set of this aspect, the full-length nucleotide sequence of mitochondrial DNA of various species belonging to the avian class can be determined by using a common primer set. (14) According to still another aspect of the present disclosure, a method for determining the full-length nucleotide sequence of mitochondrial DNA is provided. This method for determining the full-length nucleotide sequence of mitochondrial DNA comprises preparing a DNA sample containing mitochondrial DNA, using the DNA sample as a template, and using a primer set designed by the primer set design method according to any one of (1) to (9) to amplify DNA fragments, analyzing the nucleotide sequence of each of the amplified DNA fragments, and constructing the full-length sequence of mitochondrial DNA using the nucleotide sequence of each of the DNA fragments. According to the method for determining the full-length nucleotide sequence of mitochondrial DNA of this aspect, it becomes possible to efficiently and easily decode the full-length sequence of mitochondrial DNA in various species in which the full-length sequence of mitochondrial DNA has not been decoded. (15) According to still another aspect of the present disclosure, a method for determining the full-length nucleotide sequence of mitochondrial DNA is provided. This method for determining the full-length nucleotide sequence of mitochondrial DNA prepares a DNA sample containing mitochondrial DNA, uses the DNA sample as a template, and performs amplification of DNA fragments using the primer set according to any one of (10) to (13). Then, the nucleotide sequence of each of the amplified DNA fragments is analyzed, and the full-length sequence of mitochondrial DNA is constructed using the nucleotide sequences of the respective DNA fragments. According to this method for determining the full-length nucleotide sequence of mitochondrial DNA in this form, it becomes possible to efficiently and easily decode the full-length sequences of mitochondrial DNA in various species in which the full-length sequences of mitochondrial DNA are unread. (16) In the method for determining the full-length nucleotide sequence of mitochondrial DNA of the above form, as the DNA sample, it may be possible to prepare a sample containing mitochondrial DNA derived from a plurality of types of organisms. With such a configuration, the operation of determining the full-length sequences of mitochondrial DNA of a plurality of types of organisms can be simplified. (17) In the method for determining the full-length nucleotide sequence of mitochondrial DNA of the above form, as the DNA sample, in addition to mitochondrial DNA, it may be possible to prepare a sample containing genomic DNA different from mitochondrial DNA. With such a configuration, since a more diverse sample can be used, the degree of freedom in selecting a sample containing DNA that serves as a template for determining the full-length sequence of mitochondrial DNA can be increased. The present disclosure can be realized in various forms other than the above. For example, it can be realized in forms such as a device for designing a primer set for determining the full-length nucleotide sequence of mitochondrial DNA, a computer program that causes a computer to execute a method for designing a primer set for determining the full-length nucleotide sequence of mitochondrial DNA, a storage medium storing the program, a database in which the full nucleotide sequence of mitochondrial DNA is registered, and a method for analyzing environmental DNA using the database.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] A. Primer set design device: FIG. 1 is a block diagram functionally showing the configuration of a primer set design apparatus 10 (hereinafter also referred to as “design apparatus 10”) for determining a mitochondrial DNA sequence as an embodiment of the present disclosure. The design apparatus 10 includes a CPU 110, a storage unit 120, a RAM 130, an input interface 140, an output interface 150, and a communication interface 160. These components are interconnected by a bus. The CPU 110 controls the overall operation of the design apparatus 10 by expanding a program 122 stored in the storage unit 120 into the RAM 130. An operation unit 170 such as a keyboard or a mouse is connected to the input interface 140, and a display unit 180 such as a liquid crystal display is connected to the output interface 150.

[0011] The CPU 110 realizes the functions of an acquisition unit 111, an extraction unit 112, a setting unit 113, a specifying unit 114, and a selection unit 115 by executing a program 122 stored in the storage unit 120.

[0012] The acquisition unit 111 acquires the full-length sequences of known mitochondrial DNAs of a plurality of species belonging to a specific biological classification. The extraction unit 112 aligns the full-length sequences of the mitochondrial DNAs acquired by the acquisition unit 111, and extracts a conserved region, which is a region satisfying a criterion predetermined as a criterion indicating the degree of conservation of bases, from the full-length sequences of the mitochondrial DNAs. The setting unit 113 sets a plurality of primer candidate sequences using the base sequences of the respective conserved regions extracted by the extraction unit 112. The specifying unit 114 specifies a primer set candidate by combining the primer candidate sequences set by the setting unit 113. The selection unit 115 selects at least one of the primer set candidates specified by the specifying unit 114 as a primer set. The operations related to the design of the primer set will be described in detail later.

[0013] As the storage unit 120, for example, a hard disk, a storage medium, a nonvolatile memory, a storage device (SSD) composed of a nonvolatile memory, or the like can be used. A program 122 is stored in the storage unit 120.

[0014] B. Primer set design method: FIG. 2 is a flowchart showing a method for designing a primer set for determining a mitochondrial DNA sequence as an embodiment of the present disclosure. Hereinafter, the method for designing the primer set shown in FIG. 2 will be described based on an embodiment of being executed using the design apparatus 10 shown in FIG. 1. However, the method for designing the primer set of the present embodiment may be executed using an apparatus other than the design apparatus 10. The primer set designed by the design method shown in FIG. 2 is a primer set for determining the base sequence of the entire length of mitochondrial DNA (hereinafter, also referred to as the "mitochondrial DNA full-length sequence"), and is used to determine the mitochondrial DNA full-length sequence of a biological species belonging to a specific biological classification.

[0015] When designing a primer set, first, the acquisition unit 111 acquires known mitochondrial DNA full-length sequences of a plurality of biological species belonging to a specific biological classification (step T100). Here, the "specific biological classification" represents a relatively closely related group of organisms for determining the mitochondrial DNA full-length sequence by commonly using the primer set designed by the method for designing the primer set of the present embodiment as a universal primer. Specifically, for example, it can be the "class" of the biological classification hierarchy, or a classification hierarchy lower than the class (for example, "order", "family", "genus", etc.). For example, in the class Mammalia or Aves, since the conservation of the mitochondrial DNA full-length sequence among species is relatively high, by setting the "specific biological classification" as the "class", a primer set as a universal primer that can be commonly used in a wider range of biological species can be designed. Also, for example, in the class Actinopterygii, since the conservation of the mitochondrial DNA full-length sequence among species is relatively low, by setting the "specific biological classification" as a classification hierarchy lower than the class, the accuracy when amplifying a mitochondrial DNA fragment using the designed primer set can be increased. By setting the "specific biological classification" as a classification hierarchy higher than the "species", such as "class", "order", "family", "genus", etc., it becomes possible to design a primer set that can be commonly used among different species.

[0016] From the viewpoint of improving the accuracy when amplifying mitochondrial DNA fragments using the designed primer sets, the number of species from which the full-length mitochondrial DNA sequences are obtained in Project T100 is preferably, for example, 800 or more species, more preferably 1000 or more species, and even more preferably 1200 or more species. Also, within the range where the data processing load is acceptable, it can be, for example, 3000 or fewer species, or it may be 2000 or fewer species. As long as it is within the available range, the full-length mitochondrial DNA sequences of more species may be obtained. Also, for obtaining the full-length mitochondrial DNA sequences, a publicly available database containing information on the full-length mitochondrial DNA sequences of various species may be appropriately used. For example, the acquisition unit 111 can access the external database 190 via the communication interface 160 and the Internet to perform the acquisition operation of Project T100. Alternatively, when the database to be used is stored in the storage unit 120, the acquisition unit 111 can perform the acquisition operation of Project T100 by accessing the storage unit 120.

[0017] Next, the extraction unit 112 aligns the full-length mitochondrial DNA sequences of a plurality of species belonging to the same specific biological classification obtained in Project T110, and extracts a "conserved region", which is a region that satisfies a criterion predetermined as a criterion indicating the degree of conservation of bases, from the aligned full-length mitochondrial DNA sequences (Project T110). The alignment of the full-length mitochondrial DNA sequences in Project T110 can be performed using conventionally known well-known sequence alignment algorithms, such as MAFFT, ClustalW, MUSCLE, ClustalOmega, Kalign, etc. The degree of conservation at each position (each base) of the aligned mitochondrial DNA can be evaluated, for example, by scoring the Shannon information content as the degree of conservation. The formula for expressing the base conservation degree using the Shannon information content is shown below as formula (1).

[0018]

Number

[0019] However, in formula (1), I represents the base conservation degree, and pk represents the probability of occurrence of each base A, T, G, and C at each base position. The second term on the right side is the Shannon information amount at the base position being focused on. That is, it indicates the bias in the occurrence frequency of each base, with a minimum value of 0 (when only one type of base appears) and a maximum value of 2 (when the four types of bases appear with equal probability). For example, if only A appears at the base position being focused on, that is, when the bases are completely conserved among the sequences, the second term on the right side becomes 0. In the entire right side, by subtracting the value of this second term on the right side from the maximum value of 2, the higher the value indicates that the bases are biased towards being conserved as one type of base. That is, the value on the right side serves as an index indicating the base conservation degree.

[0020] The "conserved region" extracted in step T110 can be, for example, in a group of aligned mitochondrial DNA sequences, when the operation of calculating the average value of the base conservation degree at each position of a "specific-length sequence" having a specific length is continuously performed while shifting the position of the "specific-length sequence" by one base at a time, the range where the average value of the conservation degree of the "specific-length sequence" continuously exceeds a preset threshold. That is, the above-mentioned "region that satisfies the criterion predetermined as a criterion indicating the high degree of base conservation" can be the "region where the average value of the conservation degree of a sequence having a specific length continuously exceeds the threshold". For example, when the "specific-length sequence" is a sequence of 20 consecutive bases, and the average value of the conservation degree of each base constituting the "specific-length sequence" is sequentially calculated, after a "specific-length sequence" whose average value of the conservation degree exceeds the threshold is discovered, assume that the operation of calculating the average value of the conservation degree of the "specific-length sequence" is performed 10 times while shifting by one base at a time and the state where the average value of the conservation degree exceeds the threshold is maintained, and the average value of the conservation degree of the "specific-length sequence" becomes below the threshold at the 11th time of the operation of shifting by one base. In this case, a 30-base range where the average value of the conservation degree of the "specific-length sequence" continuously exceeds the threshold is specified as the "conserved region".

[0021] In step T110, by appropriately setting the "predetermined criterion indicating the high degree of preservation of bases", that is, the "threshold value of the average degree of preservation in a specific-length sequence", it is possible to identify a considerable number of "preserved regions" where bases with a sufficiently high degree of preservation are continuous. For example, the above-mentioned criterion (threshold value) may be set so that "preserved regions" of about 30 to 70 are identified.

[0022] Note that the extraction of the "preserved region" in step T110 may be performed by a method different from the above. For example, instead of the above-mentioned Shannon information amount, the ratio of the most frequent base may be used as an index of the degree of preservation. The "ratio of the most frequent base" is a value indicating what percentage of the most frequently occurring base occupies the whole at each position (each base) of the aligned mitochondrial DNA. For example, when the same base appears in all of the aligned mitochondrial DNA at a specific position, the "ratio of the most frequent base" at that position is 100%. In this case, when the operation of calculating the average value of the ratios of the most frequent bases of each base in the "specific-length sequence" is continuously performed while shifting the position of the "specific-length sequence" by one base at a time, the range where the average value of the ratios of the most frequent bases in the "specific-length sequence" continuously exceeds a preset threshold value may be defined as the "preserved region". Note that the method using the Shannon information amount as an index of the degree of preservation is desirable from the viewpoint that it can evaluate the degree of preservation considering bases other than the most frequent base, which is different from the method using the ratio of the most frequent base as an index of the degree of preservation.

[0023] After extracting a plurality of storage regions in Project T110, the setting unit 113 sets a "primer candidate sequence" using the base sequences of each "storage region" (Project T120). The setting of the "primer candidate sequence" in Project T120 is performed by identifying a "high conservation sequence", which is the base sequence of a site with a relatively high degree of base conservation among each "storage region". The "high conservation sequence" can be, for example, the "specific length sequence" (hereinafter referred to as the "highest conservation sequence") with the highest average value of conservation degrees within each "storage region". For example, when a "storage region" in the range of 30 bases is extracted as described above, this "storage region" contains 11 consecutive "specific length sequences" of 20 bases in length, each shifted by 1 base, whose average value of conservation degrees exceeds the threshold. In such a case, the "highest conservation sequence", which is the sequence with the largest average value of conservation degrees among these 11 "specific length sequences", can be set as the "high conservation sequence". Then, the identified "high conservation sequence" can be set as the "primer candidate sequence".

[0024] The identification of the "highly conserved sequence" for setting the "primer candidate sequence" in step T120 may be performed by a method different from the above. For example, as in the case where the GC content in the "highest conserved sequence" within the above-mentioned "conserved region" is excessively high or excessively low, it may not be appropriate to directly use the "highest conserved sequence" as the "primer candidate sequence". In such a case, for the "highest conserved sequence", about 2 to 5 bases adjacent to at least one of the 5'-side and 3'-side of the "highest conserved sequence" within the conserved region can be added to obtain a "highly conserved sequence". Alternatively, the sequences at least at one end of the 5'-side and 3'-side of the "highest conserved sequence" can be deleted by about 2 to 5 bases to obtain a "highly conserved sequence". For example, when there are regions with relatively high GC content on both the 5'-side and 3'-side of the "highest conserved sequence", adjacent sequences of several bases included in the "conserved region" can be added to the 5'-side of the "highest conserved sequence", and the sequences of several bases at the 3'-end of the "highest conserved sequence" can be deleted. When adding bases at one end of the "highest conserved sequence" and deleting bases at the other end, the length of the added bases and the length of the deleted bases may be the same or different.

[0025] The "primer candidate sequence" set in step T120 is a candidate for a primer used when amplifying mitochondrial DNA fragments using the mitochondrial DNA of an organism belonging to the above-mentioned "specific biological classification" as a template. Therefore, it is required to bind (anneal) sufficiently to the mitochondrial DNA of the above-mentioned "organism belonging to the specific biological classification". From this perspective, it is desirable that the "primer candidate sequence" can bind with no more than 1 mismatched base to the mitochondrial DNA of a species of organism belonging to the above-mentioned "specific biological classification".

[0026] As described above, in order to ensure that the "primer candidate sequence" sufficiently binds to the mitochondrial DNA of a species belonging to the "specific biological classification", rather than using the "highly conserved sequence" as the "primer candidate sequence" as it is, further selection may be performed on the "highly conserved sequence" to set the "primer candidate sequence". Specifically, for example, the "primer candidate sequence" may be set by narrowing down to the "highly conserved sequences" for which a "first parameter" representing the degree of sequence specificity with respect to the sequence of the mitochondrial DNA of species belonging to the "specific biological classification" is equal to or higher than a predetermined "first reference value". Further, the "primer candidate sequence" may be set by narrowing down to the "highly conserved sequences" for which a "second parameter" representing the degree of sequence specificity with respect to the sequence of the mitochondrial DNA of species not belonging to the "specific biological classification" is equal to or lower than a "second reference value" predetermined as a value smaller than the above "first reference value".

[0027] The above "first parameter" can be, for example, the ratio of the number of species with 1 or fewer mismatched bases when the "highly conserved sequence" is aligned with the full-length sequences of the mitochondrial DNA of a plurality of species belonging to the "specific biological classification" to the total number of the plurality of species belonging to the above "specific biological classification". Also, the above "second parameter" can be the ratio of the number of species with 1 or fewer mismatched bases when the "highly conserved sequence" is aligned with the full-length sequences of the mitochondrial DNA of a plurality of species not belonging to the "specific biological classification" to the total number of the plurality of species not belonging to the above "specific biological classification". The above "first reference value" can be, for example, 85%. Also, the above "second reference value" can be, for example, 15%.

[0028] In the above, the "first parameter" and the "second parameter" were set based on the number of mismatches when aligning the "highly conserved sequence" with the full-length sequences of mitochondrial DNA of species belonging or not belonging to a "specific biological classification", but different indicators may be used. For example, based on the number of gaps when aligning the "highly conserved sequence" with the full-length sequences of mitochondrial DNA of species belonging or not belonging to a "specific biological classification", or the length of the longest continuous base sequence that is aligned without mismatches and gaps, etc., the "first parameter" and the "second parameter" may be set.

[0029] Note that the length of the "primer candidate sequence" is not limited to 20 bases, and can be, for example, any length in the range of 17 to 25 bases. However, from the perspective of ensuring the accuracy of amplifying the desired fragment by PCR, it is necessary to ensure a sufficient primer length, and from the perspective of making the primer set to be designed a universal primer that can be commonly used in species belonging to the "specific biological classification" described above, it is desirable to suppress the primer length. Therefore, 20 bases can be cited as a particularly desirable length of the "primer candidate sequence".

[0030] When the "primer candidate sequence" is set in step T120, next, the specific part 114 combines these "primer candidate sequences" to specify a "primer set candidate" (step T130). The specification of the "primer set candidate" is performed by combining a plurality of "primer candidate sequences" so that when mitochondrial DNA is amplified using the specified "primer set candidate" as a primer set, three or more and six or less fragments that cover the entire length of the mitochondrial DNA are obtained, and a desired length is obtained as the length of the overlapping region with adjacent fragments.

[0031] FIG. 3 is an explanatory diagram schematically showing an example of the configuration of a "primer set candidate" specified in step T130. FIG. 3 shows an example of a "primer set candidate" from which four fragments covering the entire length of mitochondrial DNA (mt) can be obtained. The primer set candidate shown in FIG. 3 is composed of eight primers, primer α1, α2, β1, β2, γ1, γ2, δ1, and δ2, which are selected from a plurality of "primer candidate sequences" set in step T120. That is, the primer set candidate shown in FIG. 3 includes a first primer pair composed of forward primer α1 and reverse primer α2, a second primer pair composed of forward primer β1 and reverse primer β2, a third primer pair composed of forward primer γ1 and reverse primer γ2, and a fourth primer pair composed of forward primer δ1 and reverse primer δ2. By performing PCR using the first primer pair composed of primers α1 and α2, "fragment A" is obtained. By performing PCR using the second primer pair composed of primers β1 and β2, "fragment B" is obtained. By performing PCR using the third primer pair composed of primers γ1 and γ2, "fragment C" is obtained. By performing PCR using the fourth primer pair composed of primers δ1 and δ2, "fragment D" is obtained. In FIG. 3, fragments A to D are schematically shown as being arranged at equal intervals with the same length, but FIG. 3 does not accurately represent the ratio of the dimensions of each part.

[0032] In the fragments obtained when amplifying mitochondrial DNA using the primer set candidate, there are overlapping regions between adjacent fragments. In FIG. 3, "fragment A" and "fragment B" form an "overlapping region AB", "fragment B" and "fragment C" form an "overlapping region BC", "fragment C" and "fragment D" form an "overlapping region CD", and "fragment D" and "fragment A" form an "overlapping region DA".

[0033] When identifying "primer set candidates" in Project T130, as described above, "primer candidate sequences" are combined so that a desired length can be obtained as the length of the overlapping region. The sequence of each overlapping region is used to accurately join the sequences of each DNA fragment in the correct order in order to accurately join the sequences of each DNA fragment in the correct order when amplifying mitochondrial DNA using a primer set to obtain each fragment and sequencing each DNA fragment, and then connecting the sequences of each DNA fragment to obtain the full-length mitochondrial DNA sequence. That is, by overlapping the corresponding overlapping regions, it becomes possible to easily construct the full-length mitochondrial DNA sequence.

[0034] Here, in each overlapping region, "primer candidate sequences" with high conservation are arranged at both ends, but in the intermediate region sandwiched between these "primer candidate sequences", there are regions with lower conservation than the "primer candidate sequences" (hereinafter also referred to as "non-conserved regions"). That is, as described above, since the "primer candidate sequences" are set from each of the conserved regions, which are regions where highly conserved bases are continuous, there are regions with relatively low conservation where the state of continuous highly conserved bases is interrupted between the "primer candidate sequences" existing at a distance. Such "non-conserved regions" can be said to be regions where the base sequences differ to some extent between mitochondrial DNAs derived from organisms of different species even if they belong to "specific biological classifications".

[0035] Therefore, even when amplifying mitochondrial DNA fragments using the same primer set, by comparing the sequences of "non-conserved regions" within the overlapping regions of homologous fragments such as "Fragment A" shown in FIG. 3, for example, homologous fragments can be distinguished for each biological species. Therefore, for example, even when using a sample containing mitochondrial DNA from multiple different types of organisms belonging to a common specific biological classification as a template for amplification, homologous fragments can be distinguished for each biological species as described above, and fragments derived from homologous organisms having overlapping regions of the same sequence can be joined together. As a result, it becomes possible to appropriately construct the full-length mitochondrial DNA sequences of multiple types of organisms for each biological species. In order to distinguish the biological species from which the template DNA is derived for each fragment based on the differences in the sequences in the overlapping regions, it is necessary to ensure a certain length of the entire overlapping region so that a non-conserved region of sufficient length is included in the overlapping region. From this perspective, it is desirable to specify "primer set candidates" such that the length of each overlapping region is 500 bases or more.

[0036] Also, if the overlapping region is made excessively long, as a result, the length of each fragment obtained by PCR becomes longer. The longer the length of each fragment, the more likely the accuracy of sequencing each fragment will decrease, and the sequencing operation will become more complicated. Furthermore, the longer the length of each fragment, the more difficult it is to obtain read data including the entire fragment length, and the data processing load increases. Therefore, from the perspective of ensuring an opportunity to select all available primer sequences without omission and specifying appropriate primer set candidates, the length of each overlapping region is preferably 4000 bases or less. Also, from the perspective of reducing the PCR fragment length, increasing the sequencing accuracy, and enhancing the effect of facilitating the acquisition of read data, it is preferably 3000 bases or less.

[0037] As described above, the longer the length of the fragment to be amplified, the more likely the amplification accuracy is to decrease. Further, when the mitochondrial DNA contained in the DNA sample used as the amplification template includes fragmented rather than complete circular DNA, the longer the length of the fragment to be amplified, the higher the possibility that amplification will not be sufficiently performed. Therefore, from the viewpoint of suppressing the fragment length obtained by PCR and enhancing the amplification accuracy and amplification efficiency, in the present embodiment, the number of types of fragments obtained by the "primer set candidate" is set to 3 or more, and preferably 4 or more.

[0038] On the other hand, the more fragments obtained by amplification, the shorter the length of each fragment and the higher the accuracy of amplification and sequencing. However, if the length of each fragment is excessively shortened, it becomes difficult to sufficiently secure the length of the overlapping region including the region with relatively low conservation, and the operation of constructing the mitochondrial DNA full-length sequence from the amplified fragments may become difficult. Further, in the mitochondrial DNA full-length sequence, regions with relatively low conservation are contained continuously to some extent. In such regions, it becomes difficult to set an overlapping region having primer candidate sequences at both ends, and it becomes difficult to suppress the maximum value of the fragment length, which may cause the variation in the fragment length to become undesirably large. In addition, when the fragment length is shortened to increase the number of types of fragments, the man-hours of experimental operations for obtaining the mitochondrial DNA full-length sequence increase. Therefore, in the present embodiment, the number of types of fragments obtained by the "primer set candidate" is set to 6 or less, and preferably 5 or less. From the viewpoint of ensuring the amplification accuracy and amplification efficiency and suppressing the man-hours of experimental operations for obtaining the mitochondrial DNA full-length sequence, the number of types of fragments obtained by the "primer set candidate" is preferably 4.

[0039] Note that the length of each fragment obtained by amplification using the "primer set candidates" needs to be somewhat longer than the total length of the overlapping regions at both ends of the fragment. Therefore, even when the length of the overlapping region is, for example, about 500 bases, it is desirable that the length of the above fragment be 2000 bases or more, and from the viewpoint of allowing a longer overlapping region, it is more desirable that the length be 4000 bases or more. Also, from the viewpoint of suppressing a decrease in the accuracy of sequencing due to the increase in the length of each fragment, the complication of the sequencing operation, or the increase in the data processing load, it is desirable that the length of each of the above fragments be 9000 bases or less, and more desirable that the length be 8000 bases or less.

[0040] In step T130, by fully searching through combinations of a plurality of primer candidate sequences set in step T120, three or more and six or less fragments covering the entire length of mitochondrial DNA are obtained, and a combination of primers (in the example of FIG. 3, eight primers: α1, α2, β1, β2, γ1, γ2, δ1, δ2) arranged such that a desired length is obtained as the length of the overlapping region with an adjacent fragment is extracted and specified as a primer set candidate. The number of primers required to obtain three or more and six or less fragments is 6, 8, 10, or 12. On the other hand, as described above, a relatively large number of sequences are set as primer candidate sequences. Therefore, by fully searching through combinations of a plurality of primer candidate sequences, a combination of primer candidate sequences of the above-described desired number, in which an overlapping region of a desired length is formed, can be extracted relatively easily.

[0041] After identifying primer set candidates in Project T130, the selection unit 115 selects at least one of these primer set candidates as the primer set (Project T140). If there is one set of primer set candidates identified in Project T130, the said primer set candidate may be selected as the primer set. If there are multiple primer set candidates identified in Project T130, all of these multiple primer set candidates may be selected as the primer set, or alternatively, a part of the multiple primer set candidates may be selected as the primer set. The selection operation in Project T140 is executed according to a predetermined criterion, such as excluding primer set candidates with particularly large variation in fragment length in combinations of 3 or more and 6 or fewer fragments obtained by amplification from among the multiple primer set candidates, and then selecting the primer set. The selection unit 115 can output and display the selected primer set to the display unit 180 via the output interface 150.

[0042] Note that in the CPU 110, without realizing the function of the selection unit 115, the primer set candidates identified by the identification unit 114 may be output from the design device 10 to the display unit 180 or the like, and the operation of selecting the primer set from the primer set candidates may be executed outside the design device 10. As a method of selecting a part of the multiple primer set candidates as the primer set outside the design device 10, for example, a preliminary experiment of performing PCR using the mitochondrial DNA derived from an organism belonging to the above-mentioned "specific biological classification" as a template with each primer set candidate can be cited, and a combination that gives better results can be selected as the primer set. Specifically, for example, for each primer set candidate, PCR can be performed using each primer pair constituting the primer set candidate, and the primer set candidate constituted by the primer pair that can obtain a fragment of the desired length can be selected as the primer set.

[0043] Each primer constituting the primer set selected in step T140 is set based on the "highly conserved sequence" set within each "storage region" described in step T120, that is, the "primer candidate sequence". In addition to the sequence that anneals to mitochondrial DNA serving as a template when performing amplification by PCR, it may further have a different sequence. Specifically, on the 5' side of the aforementioned "primer candidate sequence", a sequence of about 80 bases at most, preferably 60 bases or less, and different from the adjacent sequence within the above-mentioned "storage region" may be added as a "5'-side additional sequence". When used as a primer in PCR, it is considered that the above "5'-side additional sequence" does not anneal to mitochondrial DNA, but addition of such a sequence is allowed at the 5' end, which is different from the 3' side where the DNA elongation reaction proceeds. As described above, since the "primer candidate sequence" is about 20 bases (for example, about 16 to 27 bases, preferably about 18 to 25 bases), the length of each primer may be, for example, 100 bases or less, and preferably 80 bases or less.

[0044] In the embodiment related to the above-described method for designing a primer set, a part of the configuration realized by hardware may be replaced with software, or conversely, a part of the configuration realized by software may be replaced with hardware. Further, when a part or all of the functions of the present disclosure are realized by software, the software (computer program) can be provided in a form stored in a computer-readable storage medium. The "computer-readable storage medium" includes not only portable storage media such as flexible disks and CD-ROMs, but also various internal storage devices in a computer such as various RAMs and ROMs, and external storage devices fixed to a computer such as hard disks. That is, the "computer-readable storage medium" has a broad meaning including any storage medium that can fixedly store data rather than temporarily.

[0045] C. Primer set: FIG. 4 is an explanatory diagram showing an example of a primer set designed by the above-described primer set design method as an example of the primer set of the present embodiment, which can be used to determine the full-length sequence of mitochondrial DNA of mammals. FIG. 5 is an explanatory diagram showing an example of another primer set of the embodiment, which is a primer set designed by the above-described primer set design method and can be used to determine the full-length sequence of mitochondrial DNA of birds. FIG. 6 is an explanatory diagram showing an example of still another primer set of the present embodiment, which is a primer set designed by the above-described primer set design method and can be used to determine the full-length sequence of mitochondrial DNA of mammals. FIG. 7 is an explanatory diagram showing an example of still another primer set of the embodiment, which is a primer set designed by the above-described primer set design method and can be used to determine the full-length sequence of mitochondrial DNA of birds.

[0046] Figures 4 to 7 show primer sets composed of four sets of primer pairs (first to fourth primer pairs) that cover the full length of mitochondrial DNA. In Figures 4 to 7, the forward primer of each primer pair is indicated by attaching "for" to the name of the primer, and the reverse primer is indicated by attaching "rev" to the name of the primer. The conditions for designing each primer set will be described in detail later. In addition, in the four fragments obtained by amplifying mammalian mitochondrial DNA using the primer set shown in Figure 4, the lengths of each overlapping region (overlapping regions AB to DA in Figure 3) are approximately 1300 bases, approximately 3000 bases, approximately 1100 bases, and approximately 1200 bases, respectively. Also, in the four fragments obtained by amplifying avian mitochondrial DNA using the primer set shown in Figure 5, the lengths of each overlapping region are approximately 1600 bases, approximately 1700 bases, approximately 3000 bases, and approximately 2000 bases, respectively. Further, in the four fragments obtained by amplifying mammalian mitochondrial DNA using the primer set shown in Figure 6, the lengths of each overlapping region (overlapping regions AB to DA in Figure 3) are approximately 3100 bases, approximately 3000 bases, approximately 1300 bases, and approximately 600 bases, respectively. Also, in the four fragments obtained by amplifying avian mitochondrial DNA using the primer set shown in Figure 7, the lengths of each overlapping region are approximately 600 bases, approximately 3000 bases, approximately 2500 bases, and approximately 1100 bases, respectively.

[0047] In the primer sets shown in Figures 4 to 7, at least one of the primers constituting each primer set may further have an additional sequence with a length of about 60 bases or less at the 5'-end.

[0048] D. Method for sequencing mitochondrial DNA: FIG. 8 is a flowchart showing a method for determining the nucleotide sequence of the entire mitochondrial DNA of the present embodiment. Such a method for determining the full-length sequence of mitochondrial DNA is executed, for example, to determine the full-length sequence of mitochondrial DNA of a species for which the full-length mitochondrial DNA sequence is not registered in order to enrich a database in which sequence information of nucleic acids possessed by various species is registered.

[0049] When determining the nucleotide sequence of the entire mitochondrial DNA, first, a DNA sample containing mitochondrial DNA is prepared (step T200). The DNA sample prepared in step T200 may be a sample containing only mitochondrial DNA derived from one type of organism, or a sample containing mitochondrial DNA derived from multiple types of organisms. Further, the DNA sample prepared in step T200 may be a sample containing only mitochondrial DNA as DNA, or a sample containing genomic DNA different from mitochondrial DNA in addition to mitochondrial DNA. Further, the mitochondrial DNA contained in the DNA sample prepared in step T200 may exist as circular DNA, or at least a part thereof may be fragmented.

[0050] After preparing the DNA sample in step T200, using the prepared DNA sample as a template and using a primer set designed by the primer set design method described with reference to FIG. 2, amplification of a DNA fragment by PCR is performed (step T210). That is, amplification of a fragment of mitochondrial DNA contained in the above DNA sample is performed. As a result, three or more and six or less fragments that cover the entire length of mitochondrial DNA of a species belonging to a specific biological classification for which a known full-length mitochondrial DNA sequence was obtained in step T100 of FIG. 2 are amplified. In step T210, from the viewpoint of efficiently amplifying a desired type of fragment, the PCR reaction for amplification may be performed separately for each primer pair constituting the primer set. The number of cycles in the PCR performed in step T210 can be, for example, 25 or more and 35 or less. Further, the annealing temperature in PCR can be, for example, 50°C or more and 60°C or less.

[0051] A primer set used in step T210. As an example of a primer set designed by the primer set design method shown in FIG. 2, the primer sets shown in FIGS. 4 to 7 can be cited. By using the primer set shown in FIG. 4 or FIG. 6, four fragments that are fragments of mammalian mitochondrial DNA present in a DNA sample and cover the entire length of the mitochondrial DNA are amplified. By using the primer set shown in FIG. 5 or FIG. 7, four fragments that are fragments of avian mitochondrial DNA present in a DNA sample and cover the entire length of the mitochondrial DNA are amplified.

[0052] After amplifying the mitochondrial DNA fragment in step T210, the base sequence of each of the amplified DNA fragments is analyzed (step T220). As described above, the primer set of the present embodiment can be designed such that the length of the mitochondrial DNA fragment obtained by amplification is about 2000 to 8000 bases. In the present embodiment, the analysis of the base sequence of each such fragment is performed using a long-read sequencer (for example, MinION of Oxford Nanopore Technologies, Sequel of PacBio, etc. MinION and Sequel are registered trademarks). While a general short-read sequencer that has been widely used in the past reads a sequence of about 200 bases at a time, a long-read sequencer is a device that can read a continuous DNA sequence of several thousand bases or more or 10,000 bases or more at a time. Therefore, by using a long-read sequencer, the base sequence of each fragment amplified in step T210 can be analyzed by a longer read instead of analyzing it as short and fragmented fragments of about 300 bases, and the man-hours of experimental operations required for the base sequence analysis can be greatly reduced. Furthermore, by using a long-read sequencer, it is also possible to analyze complex structures that are difficult to analyze with short-read sequencers, such as long repetitive sequences.

[0053] After analyzing the nucleotide sequences of each amplified DNA fragment in Project T220, the full-length sequence of mitochondrial DNA is constructed using the nucleotide sequences of each DNA fragment (Project T230). Specifically, first, the nucleotide sequence of each fragment is determined from the shared sequences between the reads obtained by the long-read sequencer. Then, the full-length sequence of circular mitochondrial DNA is constructed by overlapping the corresponding overlapping regions present at both ends of each fragment.

[0054] According to the method for designing the primer set of the present embodiment configured as described above, a primer set having a nucleotide sequence with a high degree of conservation in a biological species belonging to a "specific biological classification" and capable of obtaining three or more and six or less fragments covering the full length of mitochondrial DNA can be prepared. Therefore, primers with high commonality that can be used in a relatively wide range of biological species can be obtained. In addition, since mitochondrial DNA can be amplified as three or more and six or less fragments, even if the mitochondrial DNA used as a template for PCR is not circular but partially degraded, it is possible to amplify the desired DNA fragment and obtain a DNA fragment with a length suitable for nucleotide sequence analysis by a long-read sequencer. Then, by analyzing the nucleotide sequences of each fragment thus obtained, the full-length sequences of mitochondrial DNA of various biological species belonging to a "specific biological classification" can be determined by a simple process.

[0055] In the present embodiment, when determining the full-length sequence of mitochondrial DNA from the DNA fragments obtained by amplification using the primer set, the full-length sequence of mitochondrial DNA can be easily constructed by overlapping the overlapping regions at both ends of the DNA fragments. At this time, even when the sample used as a template for amplification contains mitochondrial DNA derived from multiple types of organisms, as described above, by comparing the sequences of non-conserved regions in the overlapping regions, the biological species from which the DNA fragments are derived can be distinguished and the full-length sequences of mitochondrial DNA can be constructed separately for each biological species, thereby determining the full-length sequences of mitochondrial DNA for each biological species.

[0056] Also, according to the method for designing the primer set of the present embodiment, the number of types of fragments obtained can be adjusted within the range of 3 or more and 6 or less according to the length distribution state of DNA in the sample used as the template for PCR. For example, when the proportion of relatively short fragments is high as the DNA distribution state in the sample, the number of types of fragments obtained may be set to be larger. Thereby, it becomes possible to optimize the balance among the amplification efficiency in PCR, the amplification accuracy, and the simplification of the steps of the operations related to sequencing.

[0057] Furthermore, by using a primer set that can obtain 3 or more and 6 or less fragments that cover the entire length of mitochondrial DNA, the length of each DNA fragment obtained by amplification can be suppressed to, for example, about 8000 bases or less. Therefore, even when using a long-read sequencer, it becomes possible to use a DNA polymerase with higher reading accuracy that can also be used in a general short-read sequencer, and the accuracy can be improved when determining the full-length sequence of the mitochondrial DNA of the target biological species.

[0058] The primer set designed by the primer set design method as described above can be commonly used when determining the full-length sequences of mitochondrial DNAs of various biological species belonging to the same "specific biological classification" as described above. Therefore, when examining the full-length sequences of mitochondrial DNAs of various biological species, the complicated process of preparing different primer sets for each biological species can be suppressed. In addition, since the number of DNA fragments amplified by the primer set is 3 or more and 6 or less, the data processing load for connecting the base sequences of each fragment can be suppressed. As a result, it becomes possible to efficiently and easily decode the full-length sequences of mitochondrial DNAs in various biological species whose full-length sequences of mitochondrial DNAs are un-decoded, and the database can be efficiently enriched. When amplifying a fragment of mitochondrial DNA using the primer set designed by the primer set design method of the present embodiment to derive the full-length sequence of mitochondrial DNA, the method for amplifying the DNA fragment may be other than the PCR method. For example, the MDA (Multiple Displacement Amplification) method may be used.

[0059] Also, in the present embodiment, when setting the "primer candidate sequence" from the "highly conserved sequence", as described above, the first parameter representing the high sequence specificity of the "highly conserved sequence" with respect to the sequence of the mitochondrial DNA of a biological species belonging to the "specific biological classification" is equal to or higher than a predetermined first reference value, and the second parameter representing the high sequence specificity of the "highly conserved sequence" with respect to the sequence of the mitochondrial DNA of a biological species not belonging to the "specific biological classification" is equal to or lower than a second reference value, which is a value smaller than the first reference value. It is desirable to narrow down the sequence as the "primer candidate sequence". Thereby, the specificity of the primer set for biological species belonging to the "specific biological classification" can be further enhanced.

Example

[0060] <Design of the First Primer Set> For each of the Mammalia and Aves, which are "specific biological classifications", primer sets for determining the full-length mitochondrial DNA sequence were designed by the primer set design method shown in FIG. 2.

[0061] (Setting of primer candidate sequences) For each of the Mammalia and Aves, the full-length mitochondrial DNA sequences of multiple species were obtained (step T100). Specifically, information on the full-length mitochondrial DNA sequences of 1,379 species belonging to Mammalia and 990 species belonging to Aves was obtained from the nucleotide sequence database RefSeq of the National Center for Biotechnology Information in the United States. Thereafter, for each of mammals and birds, alignment was performed using MAFFT (Katoh et al, Mol Biol Evol, 30(4):772-780, 2013), which is a sequence alignment tool. For each base in the alignment, the Shannon information content was calculated and scored as the conservation degree, and a conserved region that satisfies the criterion indicating the high conservation degree of the bases was extracted (step T110).

[0062] In step T110, for the sequence of a 20-base region (specific-length sequence) from one end of the aligned sequence group, the average conservation degree was calculated, and the calculated average value was defined as the "conservation degree of the region". Similar calculations were performed to calculate the conservation degree of the 20-base region while shifting one base at a time from one end to the other end, and the result was compared with a predetermined threshold value. Then, the range in which the conservation degree of the 20-base region continuously exceeds the threshold value was specified as the "region that satisfies the criterion predetermined as the criterion indicating the high conservation degree of the bases", that is, the "conserved region".

[0063] Then, a "primer candidate sequence" was set from each of the identified "storage regions" (step T120). Here, within each "storage region", a "highly conserved sequence" was identified using the consensus sequence (highest conservation sequence) of the 20 bases with the highest conservation degree, and a "primer candidate sequence" was set. Forty "primer candidate sequences" were obtained for mammals and 55 for birds. When setting the "primer candidate sequence", as described above, when the GC content of the 20-base "highest conservation sequence" was excessively high, bases were added or deleted at least at one of the 5'-end and 3'-end of the "highest conservation sequence" to identify the "highly conserved sequence". Therefore, the length of the set "primer candidate sequence" is in the range of 18 to 25 bases. Also, when setting the "primer candidate sequence", based on thermodynamic parameters, those that did not meet the conditions generally considered desirable as primers among the "highly conserved sequences" were excluded. As a specific example, using Primer3, which is a tool for designing PCR primers, those that did not meet the condition of "Tm value being 55°C or higher and 65°C or lower" were excluded.

[0064] FIG. 9 is an explanatory diagram showing the "primer candidate sequences" obtained for mammals, and FIG. 10 is an explanatory diagram showing the "primer candidate sequences" obtained for birds. In FIG. 9, the positions of the mitochondrial DNA of each "primer candidate sequence" are shown by representing them as coordinates on the nucleotide sequence of the mitochondrial DNA of Cavia porcellus (guinea pig) (RefSeq ID: NC_000884.1), with the two ends of the sequence being "Start" and "End", respectively. Also, in FIG. 10, the positions of the mitochondrial DNA of each "primer candidate sequence" are shown by representing them as coordinates on the nucleotide sequence of the mitochondrial DNA of Prodotiscus insignis (Japanese firefly) (RefSeq ID: NC_039892.1), with the two ends of the sequence being "Start" and "End", respectively. In order to examine whether these "primer candidate sequences" can anneal to the mitochondrial DNA sequences of how many species belonging to the same "specific biological classification", analysis was performed using PrimerProspector (Walters et al, Bioinformatics, 27(8), 2011).

[0065] FIG. 11 is an explanatory diagram showing, in a bar graph, the results of examining the theoretical sensitivity of matching when each of the 40 "primer candidate sequences" set in step T120 is annealed to each of the mitochondrial DNA sequences of 1,379 organisms belonging to the class Mammalia obtained in step T100. FIG. 12 is an explanatory diagram showing, in a bar graph, the results of examining the theoretical sensitivity of matching when each of the 55 "primer candidate sequences" set in step T120 is annealed to each of the mitochondrial DNA sequences of 990 organisms belonging to the class Aves obtained in step T100. In FIGS. 11 and 12, the horizontal axis indicates the "primer candidate sequences", and the vertical axis indicates the proportion of each species belonging to each of the class Mammalia and the class Aves in which the "primer candidate sequences" bind at a specific matching rate. In FIGS. 11 and 12, the black columns indicate the proportion of species having mitochondrial DNA sequences to which each "primer candidate sequence" binds with 0 mismatches, and the gray columns indicate the proportion of species having mitochondrial DNA sequences to which each "primer candidate sequence" binds with 1 or fewer mismatches. In FIGS. 11 and 12, the position where the proportion of the species shown on the vertical axis is 90% is indicated by a broken line.

[0066] As shown in FIG. 11, it was confirmed that 35 out of the 40 "primer candidate sequences" set for the class Mammalia bind to mitochondrial DNA with 1 or fewer mismatches for 90% or more of the species belonging to the class Mammalia. Also, as shown in FIG. 12, it was confirmed that 49 out of the 55 "primer candidate sequences" set for the class Aves bind to mitochondrial DNA with 1 or fewer mismatches for 90% or more of the species belonging to the class Aves. As described above, it was shown that the "primer candidate sequences" set in step T120 have high sensitivity to the base sequences of the mitochondrial DNA of the species belonging to the "specific biological classification" targeted.

[0067] (Design of primer set) For the "primer candidate sequences" obtained above, all combinations of the number of "primer candidate sequences" corresponding to the number of fragments obtained when amplifying mitochondrial DNA using a primer set were listed according to the set number of fragments. Then, for each combination, the length of the fragment generated when using the "primer candidate sequence" and the length of the overlapping region with the adjacent fragment were calculated, and combinations of "primer candidate sequences" that could obtain the desired fragment length and overlapping region length were acquired and specified as "primer set candidates" (step T130). Specifically, the number of fragments to be obtained was set to 4, and "primer set candidates" were specified such that the length of the overlapping region was 500 bases or more and 3000 bases or less. Then, from these "primer set candidates", a "primer set" was selected based on the conservation degree of the primers constituting the "primer set candidates" (step T140). When selecting such a "primer set", for "primer set candidates" with a high conservation degree of the constituent primers, an experiment was further conducted to actually amplify mitochondrial DNA fragments using the "primer set candidates", and those that gave good results were selected as the "primer set". The sequences of each primer constituting the "primer set" for mammals selected in this way are shown in FIG. 4 described above. Also, the sequences of each primer constituting the "primer set" for birds selected in this way are shown in FIG. 5 described above. Note that the experimental results of actually amplifying mitochondrial DNA fragments using the "primer set candidates" will be described later.

[0068] <Evaluation of Primer Sets> (Evaluation Targeting Cattle, Pigs, Sheep, and Chickens) For cattle, pigs, sheep belonging to the class Mammalia, and chickens belonging to the class Aves, DNA was extracted from the meat sold at a general butcher shop, and DNA fragments were amplified using the primer sets shown in FIGS. 4 and 5. Note that the primer sets shown in FIGS. 4 and 5, the primer sets shown in FIGS. 6, 7, 18, 20, and 21 described later, and the primer pairs set based on FIG. 28 described later include primers in which locations indicated by W, R, Y, M, N, etc., which indicate correspondence to multiple types of bases, are present in the sequence. When amplifying DNA fragments, such primers used were primers in which all possible sequences were mixed in equal amounts.

[0069] Specifically, for about 25 mg of meat of cattle, pigs, sheep, and chickens, genomic DNA was extracted using a NucleoSpin Tissue (Macherey-Nagel) kit (step T200). To 1 ng of the obtained genomic DNA, 2× KAPA Hifi HS Ready Mix (Genetics Japan) and 0.4 μM of primer DNA were added, and a thermal cycle reaction was performed (step T210). The conditions of the thermal cycle reaction were 1 cycle of 98°C for 3 minutes, 30 cycles of 3 steps of 98°C for 30 seconds, 55°C for 30 seconds, and 72°C for 3 minutes, and 1 cycle of 72°C for 5 minutes.

[0070] FIG. 13 is an explanatory diagram showing the result of confirming, by electrophoresis, the reaction product obtained by performing an amplification reaction using the primer set for mammals shown in FIG. 4. Further, FIG. 14 is an explanatory diagram showing the result of confirming, by electrophoresis, the reaction product obtained by performing an amplification reaction using the primer set for avians shown in FIG. 5. As shown in FIGS. 13 and 14, when the primer set for mammals was used, amplification of fragments of the expected sizes was confirmed for each of the four primer pairs for three species, namely bovine, porcine, and ovine. When the primer set for avians was used, amplification of fragments of the expected size was confirmed for chicken. Further, it was confirmed that fragments of the expected sizes were not amplified for species belonging to a "specific biological classification" different from the target of the primer set.

[0071] (Evaluation by sequencing) Among the samples shown in FIGS. 13 and 14, for the samples in which amplification of fragments of the expected sizes was confirmed (samples in which "specific biological classification" corresponds between the species from which the extracted DNA is derived and the target of the primer set), 25 fmol each of the four fragments were mixed and sequenced using MinION (Oxford Nanopore Technologies). A sequencing library was constructed using the Ligation Sequencing kit (SQK-LSK109, Oxford Nanopore Technologies) and the Native Barcoding Expansion 1-12 (EXP-NBD104, Oxford Nanopore Technologies), and sequencing was performed for 72 hours using an R9.4.1 flow cell (Oxford Nanopore Technologies) (step T220).

[0072] Since the adapters used during sequencing are added to the reads obtained by sequencing, the adapter sequences are removed from the obtained DNA sequences using Porechop (Wick et al., Microb. Genom. 2017; 3(10): e000132), filtering based on the quality scores of the reads is performed using Chopper (De Coster & Rademakers, Bioinformatics, 2023; 39(5): btad311), and the reads are sorted by fragment type using Amplicon_sorter (Vierstraete & Braeckman, Ecol Evol, 2022). Then, for each sorted fragment, the sequence of the entire fragment was determined using the common sequence between the reads. After that, the sequences of each fragment were combined at the overlapping regions using Minimus2 (Sommer et al., BMC Bioinformatics, 2007; 8 64), and error correction was performed using Medaka (https: / / github.com / nanoporetech / medaka) to construct the sequence of the full-length mitochondrial DNA (step T230). To examine whether the full-length sequences of the mitochondrial DNA obtained for each of cattle, pigs, sheep, and chickens were correctly constructed, a search was performed against the nucleotide sequence database of the National Center for Biotechnology Information in the United States using the search program Blastn (Altschul et al, J Mol Biol, 215(3), 1990).

[0073] FIG. 15 is an explanatory diagram showing the results of searching each of the mitochondrial DNA full-length sequences constructed as described above against a database. In FIG. 15, the species with the highest sequence identity with the constructed full-length sequence of mitochondrial DNA is shown as the "Blastn top hit species". Also, in FIG. 15, the length of the constructed full-length sequence of mitochondrial DNA is shown as the "Contig Length". As shown in FIG. 152, in any of the samples, the full-length sequence of mitochondrial DNA of the same species as the species from which the analyzed DNA sample was derived was hit, and a match rate of 99% or more was observed. From this, it was shown that by using the primer sets shown in FIGS. 4 and 5, an error rate comparable to that of conventionally known methods for determining nucleotide sequences (for example, when using a sequencer manufactured by Illumina or the error rate when determining nucleotide sequences by the Sanger method, which is generally about 0.1%) can be achieved. Also, since it was possible to construct even complex regions that are generally difficult to handle when using a short-read sequencer, the method using the above primer sets was shown to be an excellent method capable of constructing the full-length sequence of mitochondrial DNA even when it contains complex regions.

[0074] (Further evaluation targeting other organisms) To confirm that the primer set designed by the primer set design method according to the present disclosure can be widely applied to various biological species belonging to the target "specific biological classification (here, Mammalia or Aves)", DNA extracted from other biological species was used as a target to evaluate the primer set. Specifically, for Sika deer, Brown bear, Steller sea lion, Racoon, Japanese badger, Wild boar, European rabbit belonging to Mammalia, and Japanese Green Pigeon, Common kestrel, Wild Duck, Green pheasant, Japanese grossbeak belonging to Aves, the construction of the full-length sequence of mitochondrial DNA of each biological species was carried out using tissues obtained from carnivorous or carcass samples. Extraction of genomic DNA from tissues, amplification of DNA fragments using the primer sets shown in FIGS. 4 and 5, sequencing, and construction of the full-length mitochondrial DNA sequence were performed in the same manner as the evaluation for the previously described cattle, pigs, sheep, and chickens. In addition, to examine whether the full-length sequence of the obtained mitochondrial DNA was correctly constructed, a search was performed using the Blastn search program (Altschul et al, J Mol Biol, 215(3), 1990) against the nucleotide sequence database of the National Center for Biotechnology Information in the United States.

[0075] Figures 16 and 17 are explanatory diagrams showing the results of searching each of the full-length mitochondrial DNA sequences constructed as described above against a database. Figure 16 shows the results for the above-mentioned species belonging to the class Mammalia, and Figure 17 shows the results for the above-mentioned species belonging to the class Aves. In Figures 16 and 17, the species with the highest sequence identity with the constructed full-length mitochondrial DNA sequence is shown as the "Blastn top hit species". Also, in Figures 16 and 17, the length of the constructed full-length mitochondrial DNA sequence is shown as the "Contig Length". As shown in Figures 16 and 17, for most samples, the full-length mitochondrial DNA sequence of the same species as the species from which the analyzed DNA sample was derived was hit, and a match rate of 99% or more was observed. Even for the Japanese Green Pigeon, which had a 97% match rate with the database, there was no major structural difference, and it was an error caused by fluctuations in the copy number of repetitive sequences. From the above analysis, it was shown that by using the primer sets shown in Figures 4 and 5, the full-length mitochondrial DNA sequence can be constructed with extremely high accuracy and without major structural errors for a wide range of species belonging to the "specific biological classification (here, Mammalia or Aves)" targeted.

[0076] <Selection of Primer Sets from Primer Set Candidates> The experimental results of actually amplifying mitochondrial DNA fragments using the "primer set candidates" identified in step T130 will be described below.

[0077] FIG. 18 is an explanatory diagram showing the "primer set candidate" identified in step T130, which is the "primer set candidate" identified by combining the "primer candidate sequences" related to the Aves shown in FIG. 10, and having the highest conservation degree (average of base conservation degrees calculated using the Shannon information amount). Note that the "primer set" shown in FIG. 5 selected from the "primer set candidates" in step T140 had the second highest conservation degree (average of base conservation degrees calculated using the Shannon information amount) among the "primer set candidates".

[0078] FIG. 19 is an explanatory diagram showing the results of PCR using, as a template, genomic DNA extracted from each of chicken, pigeon, and ostrich, by synthesizing each primer constituting the primer set candidate shown in FIG. 18. As shown in FIG. 19, when the primer pairs of primer pair numbers 3 and 4 were used, fragments of the desired length were amplified, but when the primer pairs of primer pair numbers 1 and 2 were used, little or no amplification was observed. From the above, it was shown that the usefulness as a "primer set" may be relatively greatly affected by factors that cannot be predicted only by the conservation degree of the constituent primers, depending on, for example, "specific biological classification". Therefore, when selecting a "primer set" from the "primer set candidates" in step T140, it is considered desirable to perform a preliminary experiment of actually synthesizing the primer set and amplifying it using mitochondrial DNA as a template. Note that the "primer set" for Mammalia shown in FIG. 4 had the highest conservation degree of the constituent primers among the "primer set candidates" obtained by combining the "primer candidate sequences" shown in FIG. 9.

[0079] <Regarding the number of fragments obtained by the primer set> In the above-described embodiment, an example was shown in which mitochondrial DNA was amplified using a primer set that can obtain four types of fragments covering the entire length of mitochondrial DNA. Hereinafter, an example using a primer set that can obtain less than four types (two or three types) of fragments covering the entire length of mitochondrial DNA will be described.

[0080] FIG. 20 is an explanatory diagram showing an example of a primer set for mammals designed to obtain two types of fragments covering the entire length of mitochondrial DNA. FIG. 21 is an explanatory diagram showing an example of a primer set for mammals designed to obtain three types of fragments covering the entire length of mitochondrial DNA. Here, the primer set for the two types of fragments shown in FIG. 20 was selected from the four pairs of eight primers included in the primer set for the four types of fragments shown in FIG. 4, and two pairs of primer pairs with high conservation were used. Both of these two pairs of primer pairs are designed to obtain fragments with a length of 10,000 bases or more. In addition, the primer set for the three types of fragments shown in FIG. 21 was designed so that the length of the overlapping region is 500 bases or more and 3000 bases or less, and three types of fragments with a fragment length of 4000 bases or more and 9000 bases or less can be obtained from the "primer candidate sequences" for mammals shown in FIG. 9. Using these primer sets, an amplification reaction of mitochondrial DNA fragments was performed under the same conditions as the primer sets shown in FIGS. 4 and 5 described above.

[0081] FIG. 22 is an explanatory diagram showing the result of amplification using the primer set shown in FIG. 20 for two types of fragments, and FIG. 23 is an explanatory diagram showing the result of amplification using the primer set shown in FIG. 21 for three types of fragments. FIGS. 22 and 23 show the results of confirming the amplification products obtained by amplification by electrophoresis for each primer pair constituting each primer set.

[0082] As shown in Fig. 22, when using primer sets for two types of fragments, DNA fragment amplification was observed in cows among mammalian species, but no amplification was observed in pigs and sheep. Thus, when using primer sets for two types of fragments, it was confirmed that the success rate of replicating the desired DNA fragment is low.

[0083] In contrast, as shown in Fig. 23, when using primer sets for three types of fragments, amplification was observed in all primer pairs of all samples. However, when using sheep-derived DNA as a template and the primer pair with primer pair number 3 shown in Fig. 21, fragments shorter than the fragment length set during the design of the primer set were mainly amplified, resulting in a relatively low proportion of fragments of the desired length. Thus, when using primer sets for three types of fragments, although the desired DNA fragment can be amplified, depending on the combination of the biological species from which the DNA used as a template is derived and the primer pair, the possibility that unwanted fragments are co-amplified was recognized. Therefore, from the perspective of suppressing the amplification of unwanted fragments and increasing the accuracy of constructing the full-length mitochondrial DNA sequence, it was confirmed that it is desirable to design primer sets so that four or more types of fragments covering the full length of mitochondrial DNA can be obtained.

[0084] <Design of the Second Primer Set> Separate from the above-described "Design of the First Primer Set", in the same manner as the "Design of the First Primer Set", for each of the mammalian and avian classes, which are "specific biological classifications", primer sets for determining the full-length mitochondrial DNA sequence were designed by the primer set design method shown in Fig. 2.

[0085] (Setting of Primer Candidate Sequences) The "Design of the Second Primer Set" was carried out using the same database as the "Design of the First Primer Set", but since it was carried out after the "Design of the First Primer Set", the number of data registered in the database had increased compared to when the "Design of the First Primer Set" was carried out. Therefore, in step T100, the number of species from which the full-length mitochondrial DNA sequences were obtained from the database was large for both mammals and birds. Information on the full-length mitochondrial DNA sequences of 1,620 species belonging to mammals and 1,081 species belonging to birds was obtained.

[0086] In step T110, as in the "Design of the First Primer Set", for each of mammals and birds, the full-length mitochondrial DNA sequences were aligned and conserved regions were extracted. Then, in step T120, using the "sequences with the highest conservation degree" in each "conserved region", 38 "sequences with high conservation degree" for mammals and 55 "sequences with high conservation degree" for birds were identified. Furthermore, based on thermodynamic parameters, those that did not meet the conditions generally considered desirable as primers among the "sequences with high conservation degree" were excluded from the target. As a specific example, using Primer3, which is a tool for designing PCR primers, those that did not meet the conditions of "Tm value is 55°C or higher and 65°C or lower", "self-complementary score is 50 or lower", "self-complementary score in the 3' end region is 50 or lower", "hairpin formation score is 50 or lower", and "3' end structure stability is 4 or lower" were excluded. As a result, it was narrowed down to 28 "sequences with high conservation degree" for mammals and 34 "sequences with high conservation degree" for birds.

[0087] In Project T120, the "highly conserved sequences" were further narrowed down using the high sequence specificity of the mitochondrial DNA sequences of species belonging to "specific biological classifications" as an index. That is, a "first parameter" was calculated to represent how well each "highly conserved sequence" could bind to the mitochondrial DNA of species belonging to the corresponding "specific biological classification", and a screening was performed to narrow down the "highly conserved sequences" whose obtained "first parameter" value was equal to or higher than a predetermined "first reference value". Also, the "highly conserved sequences" were narrowed down using the high sequence specificity of the mitochondrial DNA sequences of species not belonging to "specific biological classifications" as an index. That is, a "second parameter" was calculated to represent how well each "highly conserved sequence" would bind to the mitochondrial DNA of species not belonging to the corresponding "specific biological classification", and a screening was performed to narrow down the "highly conserved sequences" whose obtained "second parameter" value was equal to or lower than a "second reference value" predetermined as a value smaller than the above "first reference value".

[0088] Such analysis regarding sequence specificity was performed using PrimerProspector. Specifically, first, the full-length mitochondrial DNA sequences that could be obtained from the database were divided into sequences derived from organisms belonging to "specific biological classifications" which are "target organisms" and sequences derived from organisms belonging to "organisms not belonging to specific biological classifications" which are "non-target organisms". That is, they were divided into sequences derived from mammals and non-mammals, or sequences derived from birds and non-birds. Then, PrimerProspector analysis was performed regarding the sequence specificity with the above-mentioned "highly conserved sequences".

[0089] The "first parameter" is defined as the ratio of the number of species within the biological species (all target organisms) belonging to the "specific biological classification" for which the "highly conserved sequence" and the mitochondrial DNA sequence can be aligned with no more than 1 mismatch, and the "first reference value" is set at 85%. The "second parameter" is defined as the ratio of the number of species among the biological species (all non-target organisms) that do not belong to the "specific biological classification" for which the "highly conserved sequence" and the mitochondrial DNA sequence can be aligned with no more than 1 mismatch, and the "second reference value" is set at 15%.

[0090] Figures 24 and 25 are explanatory diagrams showing the results of examining the "first parameter" and the "second parameter" for each "highly conserved sequence" plotted two-dimensionally. Figure 24 shows the results for mammals, and Figure 25 shows the results for birds. In Figures 24 and 25, the horizontal axis represents the "first parameter", and the vertical axis represents the "second parameter". In Figures 24 and 25, the "first reference value" of 85% for the "first parameter" and the "second reference value" of 15% for the "second parameter" are indicated by dashed lines respectively. In Figures 24 and 25, the "highly conserved sequences" for which the "first parameter" is equal to or greater than the "first reference value" and the "second parameter" is equal to or less than the "second reference value" are indicated by black circles, and the "highly conserved sequences" that do not satisfy the above two conditions are indicated by black triangles. In step T120, the "highly conserved sequences" that satisfy the above two conditions were set as "primer candidate sequences". As a specific example, in mammals, 13 out of 28 "highly conserved sequences" were set as "primer candidate sequences", and in birds, 20 out of 34 "highly conserved sequences" were set as "primer candidate sequences".

[0091] Figure 26 is an explanatory diagram showing the "primer candidate sequences" set for mammals. Also, Figure 27 is an explanatory diagram showing the "primer candidate sequences" set for birds.

[0092] (Design of primer sets) For each of the Mammalia and Aves, combinations were listed in which fragments with a length of 4,000 to 8,000 bases were obtained by using the "primer candidate sequences" set as described above as primers for PCR, and PCR amplification experiments were conducted for all of them. For each combination of primers, the length of the amplification product was confirmed by electrophoresis, and primer combinations (primer pairs) that gave amplification products of the expected length were identified. For these primer pairs, primer sets that covered the entire length of mitochondrial DNA in four fragments and had overlapping regions (overlapping regions AB to DA in Fig. 3) with lengths of 500 to 4,000 bases were listed and identified as "primer set candidates" (step T130). Then, among these "primer set candidates", the one with the smallest average length of the four amplified fragments was selected as the "primer set" (step T140). The "primer set" selected in this way is the primer set shown in Figs. 6 and 7 described above.

[0093] <Confirmation of the effect of narrowing down based on high sequence specificity> In the "design of the second primer set", as described above, when setting the "primer candidate sequence" from the "highly conserved sequence" in step T120, narrowing down was performed based on sequence specificity for the "target organism" and "non-target organisms". The effect of performing such narrowing down was verified as follows.

[0094] First, from among the 28 "highly conserved sequences" for Mammalia shown in Fig. 24, for primers (Pr1) that satisfy the "sequence specificity condition" of "satisfying both the condition that the 'first parameter' is equal to or greater than the 'first reference value' and the condition that the'second parameter' is equal to or less than the'second reference value'", five primer pairs were set up by combining primers (Pr2) that similarly satisfy the "sequence specificity condition" and primers (Pr3 to Pr6) that do not satisfy the "sequence specificity condition", respectively.

[0095] FIG. 28 is an explanatory diagram showing the sequences of each of the primers (Pr1 to Pr6). Also, in FIG. 24, each of the primers (Pr1 to Pr6) is indicated by an arrow. Using these 5 sets of primer pairs, PCR was performed on mammals (target organisms) and birds (non-target organisms).

[0096] For cattle, pig, sheep, grizzly bear belonging to mammals, and chicken, green pigeon, common kestrel, duck belonging to birds, DNA was extracted using tissues obtained from meat or carcass samples, and DNA fragment amplification was performed using the above-mentioned 5 sets of primer pairs. The conditions for DNA extraction and PCR were the same as those adopted in "Evaluation of Primer Sets" in "Design of the First Primer Set".

[0097] FIG. 29 is an explanatory diagram showing the results of confirming by electrophoresis the reactants obtained by performing an amplification reaction using the above-mentioned 5 sets of primer pairs with DNA derived from the above-mentioned 4 mammalian animals and 4 avian animals as templates. In FIG. 29, for each primer, 4 lanes are shown as the results of performing PCR on mammalian animals, and these lanes show the results for cattle, pig, sheep, and grizzly bear from left to right. Also, in FIG. 29, for each primer, 4 lanes are shown as the results of performing PCR on avian animals, and these lanes show the results for chicken, green pigeon, common kestrel, and duck from left to right.

[0098] Figure 30 is an explanatory diagram summarizing the breakdown of the five sets of primer pairs used and the results of performing amplification reactions using each primer pair. In Figure 30, for each primer, the "first parameter", that is, the percentage (%) of the number of mammalian species belonging to the class Mammalia whose mitochondrial DNA sequence can be aligned with each primer with no more than one mismatch with respect to the total number of mammalian species from which the sequences were obtained, is shown as "Mammal specificity". Also, the percentage (%) related to the "second parameter" is shown as "Non-mammal specificity".

[0099] As shown in FIGS. 29 and 30, when using a primer pair (Pr1-Pr2) in which both primers constituting the primer pair satisfy the "sequence specificity condition", amplification was observed when using genomic DNA of any mammal as a template, but no amplification was observed in any species when using genomic DNA of the class Aves as a template. In contrast, in a primer pair combining primer (Pr1) and a primer that does not satisfy the "sequence specificity condition", cases where amplification was observed for genomic DNA of the class Aves (Pr1-Pr3, Pr1-Pr4, Pr1-Pr5) or cases where amplification was weak for genomic DNA of mammals in the first place (Pr1-Pr3, Pr1-Pr6) were seen. From these results, it is considered that by narrowing down highly conserved sequences through analysis of sequence specificity, the effect of suppressing amplification using DNA derived from organisms other than the target organism as a template and specifically amplifying mitochondrial DNA fragments derived from the target organism can be enhanced. That is, it can be said that narrowing down highly conserved sequences through analysis of sequence specificity is useful for designing more suitable primer pairs as universal primers. Although a sample containing mitochondrial DNA, which is adjusted when determining the full-length mitochondrial DNA sequence, is generally considered to contain almost no DNA of non-target biological classifications, by performing narrowing down of primers based on sequence specificity for "target organisms" and "non-target organisms", it was confirmed that the effect of enhancing the performance as a primer for determining the full-length sequence of mitochondrial DNA can be obtained.

[0100] (Evaluation by Sequencing) Using the primer set shown in Fig. 6, which is the primer set selected in "Design of the Second Primer Set", the full-length sequences of mitochondrial DNA for various mammalian animals were determined. That is, PCR was performed using DNA derived from cattle, pig, sheep, Hokkaido shika deer, grizzly bear, Steller sealion, racoon, Japanese badger, wild boar, and rabbit as templates, and the full-length sequences of mitochondrial DNA for each animal were determined. Also, using the primer set shown in Fig. 7, the full-length sequences of mitochondrial DNA for various avian species were determined. That is, PCR was performed using DNA derived from chicken, green pigeon, common kestrel, duck, green pheasant, and Japanese grossbeak as templates, and the full-length sequences of mitochondrial DNA for each animal were determined.

[0101] The determination of the full-length sequences of mitochondrial DNA was performed by performing PCR using DNA derived from mammals or birds as templates with each primer pair constituting each primer set, and recovering the obtained fragments. The specific method for determining the full-length sequences of mitochondrial DNA was the same as the method described in "Evaluation by Sequencing" of "Design of the First Primer Set". To examine whether the correct sequences could be constructed for the full-length sequences of mitochondrial DNA obtained for each animal, a search was performed using the Blastn search program (Altschul et al, J Mol Biol, 215(3), 1990) against the nucleotide sequence database of the National Center for Biotechnology Information in the United States.

[0102] Figures 31 and 32 are explanatory diagrams showing the results of searching each of the full-length mitochondrial DNA sequences constructed as described above against a database. Figure 31 shows the results for the above-described species belonging to the class Mammalia, and Figure 17 shows the results for the above-described species belonging to the class Aves. In Figures 31 and 32, the species with the highest sequence identity with the constructed full-length mitochondrial DNA sequence is shown as the "Blastn top hit species". Also, in Figures 31 and 32, the length of the constructed full-length mitochondrial DNA sequence is shown as the "Contig Length". As shown in Figures 31 and 32, for all samples, the full-length mitochondrial DNA sequence of the same species as the species from which the analyzed DNA sample was derived was hit, and in most samples, a match rate of 99% or more was observed. Even for the Green Pigeon, which had a 97% match rate with the database, there was no major structural difference, and it was an error caused by fluctuations in the copy number of the repetitive sequence. From the above analysis, it was shown that by using the primer sets shown in Figures 6 and 7, the full-length mitochondrial DNA sequence can be constructed with extremely high accuracy and without major structural errors for a wide range of species belonging to the "specific biological classification (here, Mammalia or Aves)" targeted.

[0103] The present disclosure is not limited to the above-described embodiments and the like, and can be realized in various configurations without departing from the gist thereof. For example, the technical features in the embodiments corresponding to the technical features in each form described in the summary of the invention can be appropriately replaced or combined in order to solve some or all of the above-described problems or to achieve some or all of the above-described effects. Also, if the technical feature is not described as essential in this specification, it can be appropriately deleted.

[0104] The present disclosure can also be realized in the following forms. [Application Example 1] A method for designing a primer set for determining the nucleotide sequence of the entire length of mitochondrial DNA, comprising: obtaining the full-length sequences of mitochondrial DNA of a plurality of species belonging to a specific biological classification; aligning the obtained full-length sequences of mitochondrial DNA, and extracting conserved regions, which are regions satisfying a criterion predetermined as a criterion indicating a high degree of nucleotide conservation, from the full-length sequences of mitochondrial DNA; using the nucleotide sequences of the extracted conserved regions to set primer candidate sequences; when amplifying mitochondrial DNA using a combination of the primer candidate sequences as a primer set, identifying combinations of primer candidate sequences that can obtain three or more and six or fewer fragments covering the entire length of mitochondrial DNA and can obtain a desired length as the length of the overlapping region with adjacent fragments, as primer set candidates; selecting at least one of the primer set candidates as the primer set; A method for designing a primer set. [Application Example 2] A method for designing a primer set according to Application Example 1, wherein the specific biological classification is a class in the taxonomic hierarchy of organisms or a taxonomic class lower than the class; A method for designing a primer set. [Application Example 3] A method for designing a primer set according to Application Example 2, wherein the specific biological classification is the class Mammalia or the class Aves; A method for designing a primer set. [Application Example 4] A method for designing a primer set according to any one of Application Examples 1 to 3, wherein the primer set candidates are identified such that the length of the overlapping region is 500 bases or more and 4000 bases or less; A method for designing a primer set. [Application Example 5] A method for designing a primer set according to any one of Application Examples 1 to 4, wherein Specify the primer set candidate so that the length of the overlapping region is 500 bases or more and 3000 bases or less. Method for designing a primer set. [Application Example 6] A method for designing a primer set according to any one of Application Examples 1 to 5, When amplifying mitochondrial DNA using the primer set, specify the primer set so that four fragments covering the entire length of mitochondrial DNA are obtained. Method for designing a primer set. [Application Example 7] A method for designing a primer set according to any one of Application Examples 1 to 6, Using each of the primer pairs consisting of a pair of primers constituting the primer set candidate, experimentally amplify a mitochondrial DNA fragment using DNA derived from an organism belonging to the specific biological classification as a template, Based on the amplification result, select the primer set from the primer set candidates so that it is composed of a primer pair determined to have obtained a desired mitochondrial DNA fragment. Method for designing a primer set. [Application Example 8] A method for designing a primer set according to any one of Application Examples 1 to 7, The setting of the primer candidate sequence is Among each of the conserved regions, identify a highly conserved sequence that is the nucleotide sequence of a site with a relatively high degree of nucleotide conservation, A sequence in which a first parameter representing the degree of sequence specificity of the highly conserved sequence with respect to the sequence of mitochondrial DNA of a species belonging to the specific biological classification is equal to or greater than a predetermined first reference value, and a second parameter representing the degree of sequence specificity of the highly conserved sequence with respect to the sequence of mitochondrial DNA of a species not belonging to the specific biological classification is equal to or less than a second reference value determined in advance as a value smaller than the first reference value is used as the primer candidate sequence. Method for designing a primer set. [Application Example 9] The primer set design method according to Application Example 8, wherein the first parameter is the ratio of the number of species with 1 or fewer mismatched bases when the highly conserved sequence is aligned with the full-length sequences of mitochondrial DNA of the plurality of species belonging to the specific biological classification, to the total number of the plurality of species belonging to the specific biological classification; the second parameter is the ratio of the number of species with 1 or fewer mismatched bases when the highly conserved sequence is aligned with the full-length sequences of mitochondrial DNA of the plurality of species not belonging to the specific biological classification, to the total number of the plurality of species not belonging to the specific biological classification; the first reference value is 85%; the second reference value is 15% A primer set design method. [Application Example 10] A primer set for determining the base sequence of the entire length of mitochondrial DNA, comprising a first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 1 and 2 in the Sequence Listing at the 3'-terminal side; a second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 3 and 4 in the Sequence Listing at the 3'-terminal side; a third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 5 and 6 in the Sequence Listing at the 3'-terminal side; a fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 7 and 8 in the Sequence Listing at the 3'-terminal side; and each of the primers has a length of 100 bases or less A primer set. [Application Example 11] A primer set for determining the base sequence of the entire length of mitochondrial DNA, comprising a first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 9 and 10 in the Sequence Listing at the 3'-terminal side; A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 11 and 12 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 13 and 14 in the Sequence Listing on the 3'-terminal side, A fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 15 and 16 in the Sequence Listing on the 3'-terminal side, comprising, each of said primers having a length of 100 bases or less primer set. [Application Example 12] A primer set for determining the base sequence of the full length of mitochondrial DNA, A first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 130 and 131 in the Sequence Listing on the 3'-terminal side, A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 132 and 133 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 134 and 135 in the Sequence Listing on the 3'-terminal side, A fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 136 and 137 in the Sequence Listing on the 3'-terminal side, comprising, each of said primers having a length of 100 bases or less primer set. [Application Example 13] A primer set for determining the base sequence of the full length of mitochondrial DNA, A first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 138 and 139 in the Sequence Listing on the 3'-terminal side, A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 140 and 141 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 142 and 143 in the Sequence Listing on the 3'-terminal side, A fourth primer pair consisting of a pair of primers each containing the base sequences of SEQ ID NOs: 144 and 145 of the Sequence Listing at the 3'-end side, comprising, each of said primers having a length of 100 bases or less primer set. [Application Example 14] A method for determining the full-length base sequence of mitochondrial DNA, comprising: preparing a DNA sample containing mitochondrial DNA, using said DNA sample as a template and amplifying DNA fragments using a primer set designed by the primer set design method according to any one of Application Examples 1 to 9, analyzing the base sequence of each of the amplified DNA fragments, constructing the full-length sequence of mitochondrial DNA using the base sequences of each of said DNA fragments A method for determining the full-length base sequence of mitochondrial DNA. [Application Example 15] A method for determining the full-length base sequence of mitochondrial DNA, comprising: preparing a DNA sample containing mitochondrial DNA, using said DNA sample as a template and amplifying DNA fragments using the primer set according to any one of Application Examples 10 to 13, analyzing the base sequence of each of the amplified DNA fragments, constructing the full-length sequence of mitochondrial DNA using the base sequences of each of said DNA fragments A method for determining the full-length base sequence of mitochondrial DNA. [Application Example 16] A method for determining the full-length base sequence of mitochondrial DNA according to Application Example 14 or 15, comprising: preparing, as said DNA sample, a sample containing mitochondrial DNA derived from a plurality of types of organisms A method for determining the full-length base sequence of mitochondrial DNA. [Application Example 17] The method for determining the nucleotide sequence of the entire length of mitochondrial DNA according to any one of Application Examples 14 to 16, As the DNA sample, in addition to mitochondrial DNA, a sample containing genomic DNA different from mitochondrial DNA is prepared Method for determining the nucleotide sequence of the entire length of mitochondrial DNA. [Application Example 18] A primer set design device for determining the nucleotide sequence of the entire length of mitochondrial DNA, An acquisition unit that acquires the full-length sequences of known mitochondrial DNAs of a plurality of species belonging to a specific biological classification, An extraction unit that aligns the full-length sequences of the mitochondrial DNA acquired by the acquisition unit and extracts a conserved region that is a region satisfying a criterion predetermined as a criterion indicating a high degree of nucleotide conservation from the full-length sequence of the mitochondrial DNA, A setting unit that sets primer candidate sequences using the nucleotide sequences of the respective conserved regions extracted by the extraction unit, When the mitochondrial DNA is amplified using a combination of the primer candidate sequences as a primer set, three or more and six or less fragments that cover the entire length of the mitochondrial DNA are obtained, and a combination of primer candidate sequences that gives a desired length as the length of the overlapping region with an adjacent fragment is specified as a primer set candidate, A primer set design device comprising: [Application Example 19] A computer program for designing a primer set for determining the nucleotide sequence of the entire length of mitochondrial DNA, An acquisition function for acquiring the full-length sequences of known mitochondrial DNAs of a plurality of species belonging to a specific biological classification, An extraction function for aligning the full-length sequences of the mitochondrial DNA acquired by the execution of the acquisition function and extracting a conserved region that is a region satisfying a criterion predetermined as a criterion indicating a high degree of nucleotide conservation from the full-length sequence of the mitochondrial DNA, A function of setting primer candidate sequences using the base sequences of each of the storage areas extracted by the execution of the extraction function; A function of specifying, as primer set candidates, combinations of the primer candidate sequences that, when used as primer sets to amplify mitochondrial DNA, result in three or more and six or fewer fragments that cover the entire length of the mitochondrial DNA and a desired length as the length of the overlapping region with an adjacent fragment; A computer program that causes a computer to execute the above. [Application Example 20] A storage medium that stores the computer program according to Application Example 19. Storage medium.

Explanation of Signs

[0105] 10…Design device 110…CPU 111…Acquisition unit 112…Extraction unit 113…Setting unit 114…Specification unit 115…Selection unit 120…Storage unit 122…Program 130…RAM 140…Input interface 150…Output interface 160…Communication interface 170…Operation unit 180…Display unit 190…External database

Claims

1. A method for designing a primer set for determining the nucleotide sequence of the entire length of mitochondrial DNA, comprising: obtaining the full-length sequences of mitochondrial DNA of a plurality of species belonging to a specific biological classification; aligning the obtained full-length sequences of mitochondrial DNA, and extracting conserved regions, which are regions that satisfy a criterion predetermined as a criterion indicating a high degree of nucleotide conservation, from the full-length sequences of mitochondrial DNA; setting primer candidate sequences using the nucleotide sequences of the extracted conserved regions; identifying combinations of primer candidate sequences as primer set candidates, such that when mitochondrial DNA is amplified using the combinations of primer candidate sequences as a primer set, three or more and six or fewer fragments that cover the entire length of mitochondrial DNA are obtained, and a desired length is obtained as the length of the overlapping region with an adjacent fragment; selecting at least one of the primer set candidates as the primer set A method for designing a primer set.

2. The method for designing a primer set according to claim 1, wherein the specific biological classification is a class in the taxonomic hierarchy of organisms or a taxonomic class lower than a class A method for designing a primer set.

3. The method for designing a primer set according to claim 2, wherein the specific biological classification is Mammalia or Aves A method for designing a primer set.

4. The method for designing a primer set according to claim 1, wherein the primer set candidates are identified such that the length of the overlapping region is 500 bases or more and 4000 bases or less A method for designing a primer set.

5. The method for designing a primer set according to claim 4, wherein the primer set candidates are identified such that the length of the overlapping region is 500 bases or more and 3000 bases or less A method for designing a primer set.

6. The method for designing a primer set according to claim 1, wherein the primer set is identified such that when mitochondrial DNA is amplified using the primer set, four fragments that cover the entire length of mitochondrial DNA are obtained A method for designing a primer set.

7. The method for designing a primer set according to claim 1, wherein Using each of the primer pairs consisting of a pair of primers that make up the primer set candidate, a mitochondrial DNA fragment is experimentally amplified using DNA derived from an organism belonging to the specific biological classification as a template, Based on the results of the amplification, the primer set is selected from the primer set candidates so as to be composed of a primer pair that is determined to have obtained the desired mitochondrial DNA fragment A method for designing a primer set.

8. A method for designing a primer set according to claim 1, The setting of the primer candidate sequence is Among each of the conserved regions, a highly conserved sequence that is the nucleotide sequence of a site with a relatively high degree of nucleotide conservation is identified, A first parameter representing the degree of sequence specificity of the highly conserved sequence with respect to the sequence of mitochondrial DNA of a species belonging to the specific biological classification is equal to or greater than a predetermined first reference value, and a second parameter representing the degree of sequence specificity of the highly conserved sequence with respect to the sequence of mitochondrial DNA of a species not belonging to the specific biological classification is less than the first reference value and equal to or less than a predetermined second reference value. This is done by using the sequence as the primer candidate sequence. A method for designing a primer set.

9. A method for designing a primer set according to claim 8, The first parameter is the ratio of the number of species with 1 or fewer mismatched bases when the highly conserved sequence is aligned with the full-length sequences of mitochondrial DNA of the plurality of species belonging to the specific biological classification, to the total number of the plurality of species belonging to the specific biological classification, The second parameter is the ratio of the number of species with 1 or fewer mismatched bases when the highly conserved sequence is aligned with the full-length sequences of mitochondrial DNA of the plurality of species not belonging to the specific biological classification, to the total number of the plurality of species not belonging to the specific biological classification, The first reference value is 85%, The second reference value is 15% A method for designing a primer set.

10. A primer set for determining the nucleotide sequence of the entire length of mitochondrial DNA, A first primer pair consisting of a pair of primers each containing the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing on the 3'-end side, A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 3 and 4 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 5 and 6 in the Sequence Listing on the 3'-terminal side, A fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 7 and 8 in the Sequence Listing on the 3'-terminal side, comprising, each of said primers having a length of 100 bases or less primer set.

11. A primer set for determining the base sequence of the full length of mitochondrial DNA, A first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 9 and 10 in the Sequence Listing on the 3'-terminal side, A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 11 and 12 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 13 and 14 in the Sequence Listing on the 3'-terminal side, A fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 15 and 16 in the Sequence Listing on the 3'-terminal side, comprising, each of said primers having a length of 100 bases or less primer set.

12. A primer set for determining the base sequence of the full length of mitochondrial DNA, A first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 130 and 131 in the Sequence Listing on the 3'-terminal side, A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 132 and 133 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 134 and 135 in the Sequence Listing on the 3'-terminal side, A fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 136 and 137 in the Sequence Listing on the 3'-terminal side, comprising, each of said primers having a length of 100 bases or less primer set.

13. A primer set for determining the base sequence of the full length of mitochondrial DNA, A first primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 138 and 139 in the Sequence Listing on the 3'-terminal side, A second primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NOs: 140 and 141 in the Sequence Listing on the 3'-terminal side, A third primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 142 and 143 in the Sequence Listing on the 3'-end side, a fourth primer pair consisting of a pair of primers each containing the base sequence of SEQ ID NO: 144 and 145 in the Sequence Listing on the 3'-end side, comprising: each of the primers has a length of 100 bases or less primer set.

14. A method for determining the full-length base sequence of mitochondrial DNA, comprising: preparing a DNA sample containing mitochondrial DNA, using the DNA sample as a template and using a primer set designed by the primer set design method according to any one of Claims 1 to 9 to amplify DNA fragments, analyzing the base sequence of each of the amplified DNA fragments, constructing the full-length sequence of mitochondrial DNA using the base sequence of each of the DNA fragments A method for determining the full-length base sequence of mitochondrial DNA.

15. A method for determining the full-length base sequence of mitochondrial DNA, comprising: preparing a DNA sample containing mitochondrial DNA, using the DNA sample as a template and using the primer set according to any one of Claims 10 to 13 to amplify DNA fragments, analyzing the base sequence of each of the amplified DNA fragments, constructing the full-length sequence of mitochondrial DNA using the base sequence of each of the DNA fragments A method for determining the full-length base sequence of mitochondrial DNA.

16. The method for determining the full-length base sequence of mitochondrial DNA according to Claim 14, comprising: preparing, as the DNA sample, a sample containing mitochondrial DNA derived from a plurality of types of organisms A method for determining the full-length base sequence of mitochondrial DNA.

17. The method for determining the full-length base sequence of mitochondrial DNA according to Claim 15, comprising: preparing, as the DNA sample, a sample containing mitochondrial DNA derived from a plurality of types of organisms A method for determining the full-length base sequence of mitochondrial DNA.

18. The method for determining the full-length base sequence of mitochondrial DNA according to Claim 14, comprising: preparing, as the DNA sample, a sample containing genomic DNA different from mitochondrial DNA in addition to mitochondrial DNA A method for determining the full-length base sequence of mitochondrial DNA.

19. A method for determining the full-length nucleotide sequence of mitochondrial DNA according to claim 15, wherein as the DNA sample, in addition to mitochondrial DNA, a sample containing genomic DNA different from mitochondrial DNA is prepared A method for determining the full-length nucleotide sequence of mitochondrial DNA.

20. A primer set design device for determining the full-length nucleotide sequence of mitochondrial DNA, comprising: an acquisition unit that acquires the full-length sequences of known mitochondrial DNAs of a plurality of species belonging to a specific biological classification; an extraction unit that aligns the full-length sequences of the mitochondrial DNAs acquired by the acquisition unit and extracts a conserved region, which is a region that satisfies a predetermined criterion as a criterion indicating a high degree of nucleotide conservation, from the full-length sequences of the mitochondrial DNAs; a setting unit that sets primer candidate sequences using the nucleotide sequences of the respective conserved regions extracted by the extraction unit; a specifying unit that specifies, as a primer set candidate, a combination of primer candidate sequences that, when used to amplify mitochondrial DNA as a primer set, results in three or more and six or fewer fragments that cover the full length of the mitochondrial DNA and a desired length as the length of the overlapping region with an adjacent fragment; A primer set design device comprising the above.

21. A computer program for designing a primer set for determining the full-length nucleotide sequence of mitochondrial DNA, comprising: an acquisition function that acquires the full-length sequences of known mitochondrial DNAs of a plurality of species belonging to a specific biological classification; an extraction function that aligns the full-length sequences of the mitochondrial DNAs acquired by the execution of the acquisition function and extracts a conserved region, which is a region that satisfies a predetermined criterion as a criterion indicating a high degree of nucleotide conservation, from the full-length sequences of the mitochondrial DNAs; a function that sets primer candidate sequences using the nucleotide sequences of the respective conserved regions extracted by the execution of the extraction function; a function that specifies, as a primer set candidate, a combination of primer candidate sequences that, when used to amplify mitochondrial DNA as a primer set, results in three or more and six or fewer fragments that cover the full length of the mitochondrial DNA and a desired length as the length of the overlapping region with an adjacent fragment; A computer program that causes a computer to execute.

22. A storage medium, which stores the computer program according to claim 21, the storage medium.

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