JP5240814B2 - Method for detecting common DNA fragments - Google Patents

Description

  The present invention relates to a method for detecting a DNA fragment that is commonly present in a certain biological population (a method for detecting a common DNA fragment), a specific DNA fragment that is commonly present in one biological population and not present in another biological population Detection method (specific DNA fragment detection method), a method for detecting DNA fragments common to multiple populations of organisms (a method for detecting a common DNA fragment between specific populations), and a method for detecting microorganisms present in a specific environment. The present invention relates to a method for detecting a change in DNA (a method for detecting a change in microbial DNA) and a method for hybridizing a DNA fragment possessed by individuals belonging to a certain biological population (hybridization method).

  The DNA fragment detected by the detection method of the present invention is useful for identification of species discrimination and identification of genetically modified organisms. The DNA discrimination for species discrimination is a technique for discriminating a biological population by using different portions of DNA depending on the biological population. For example, taking tuna as an example, when species identification of southern bluefin tuna and yellowfin tuna is carried out by DNA identification, it is judged unless DNA existing only in southern bluefin tuna and DNA present only in yellowfin tuna are known information. I can't. DNA testing can be performed not only for tuna but also for all animals and plants and microorganisms including humans, and has already been put into practical use in the field of forensic medicine. The DNA identification of genetically modified organisms is a technique for identifying organisms in which new DNA is artificially introduced into the genes of naturally occurring organisms, and distinguishing them from natural organisms based on DNA differences.

  The method for detecting changes in microbial DNA of the present invention is useful for analyzing changes over time in a microbial ecosystem in a specific environment.

  A technique for obtaining DNA that exists only in a specific biological population used for DNA identification for species discrimination is performed by the following method.

  Conventionally, first, for example, the DNA base sequence of a plurality of SBT is read for each individual by a method based on the analysis of the DNA base sequence (for example, see Non-patent Document 1), and the homology analysis between the base sequences of a plurality of animals is performed. The non-common DNA that is not the same as the common DNA having the same base sequence in all of the plurality of animals is obtained. Thereby, it can be seen that the common DNA is DNA common to SBT, and the non-common DNA is SBT-specific DNA that varies from individual to individual. Similarly, for yellowfin tuna, a plurality of DNA base sequences are read for each individual and the same homology analysis is performed to obtain common DNA and non-common DNA. Furthermore, to find specific DNA for each species that is present in southern bluefin tuna but not in yellowfin tuna, or present in yellowfin tuna, but not in southern bluefin tuna, the common DNA of southern bluefin tuna and common DNA of yellowfin tuna can be obtained. In comparison, the same DNA is common to these tuna, and the different DNA is obtained by the specific DNA of southern bluefin tuna or yellowfin tuna. However, this method is time consuming and expensive.

  Therefore, recently, mainly based on information on the base sequence of specific DNA obtained in advance by analysis of the DNA base sequence, mainly by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP (for example, see Non-patent Document 2)). Species discrimination is performed. For example, if a restriction enzyme is applied to a DNA that cuts SBT specific DNA but not yellowfin tuna specific DNA, and a specific DNA cleaved pattern is obtained, SBT is not cut. If it is obtained, it is a method for distinguishing yellowfin tuna. This method has much simpler and fewer steps than DNA base sequence analysis, so that results can be obtained in a short time and at a low cost, and a plurality of samples can be analyzed at the same time, which is efficient. However, this method cannot be used without a restriction enzyme capable of cleaving specific DNA, so the range of use is limited, and when the DNA is amplified by PCR, an error due to the amplification enzyme occurs, and the base sequence of the restriction enzyme cleavage site. There is a problem in that accurate species discrimination may not be possible, for example, due to changes in the area and cutting becomes impossible.

  Therefore, a technique using a DNA microarray has begun to be newly used as a technique not using a restriction enzyme. In this technique, common DNA of SBT and yellowfin tuna, specific DNA of each tuna, and irrelevant DNA are discriminated using a microarray. In this method, DNA extracted from southern bluefin tuna is hybridized to a single DNA microarray for each individual to determine the DNA possessed by the individual. This experiment was repeated for a plurality of tuna, and from the results, common DNA of southern bluefin tuna and yellowfin tuna, specific DNA of each tuna, and DNA related to each other were obtained. However, this method has a problem that it is very laborious and costly because the experiment is repeated for each individual.

  In addition, when a commercially available microarray is used for this experiment, there are many cases where DNA existing in the species whose common DNA is to be determined is not fixed on the array. There is a problem that common DNA and specific DNA cannot be obtained. Furthermore, since the hybridization operation with the DNA microarray is repeated for a plurality of animals, there is a problem that it is very difficult to set the same experimental conditions such as temperature and solution concentration each time.

Sambrook J & Russell DW Chapter12 DNA sequencing. Molecular Cloning: A laboratory manual. 3rd Ed. 2001; pp12.1-12.120. Deng G R, “A sensitive non-radioactive PCR-RFLP analysis for detecting point mutations at 12th codon of oncogene c-Ha-ras in DNAs of gastric cancer.” Nucleic Acids Res. 1988 July 11; 16 (13): 6231. Patent Publication 2004-65242 “Biological Substance Microarray and Plant Variety Identification Method Using the Same”

  The present invention has been made in view of such problems of the prior art, and in order to obtain DNA that exists in common in a certain biological population, the three problems of the conventional method using a microarray, namely, repeated Solves the trouble and cost of performing experiments, the risk that results are not obtained because the DNA required on the microarray is not fixed, and the adverse effects of experimental errors for each repeated experiment, more efficiently and accurately than before, An object of the present invention is to provide means for detecting common DNA or specific DNA of a population of organisms.

To achieve the above object, the present invention provides the following (1) to (31).
(1) A method for detecting a DNA fragment that is commonly present in a certain organism population, the step of obtaining a mixture of DNA fragments from a plurality of individuals belonging to the organism population, taking a part of the mixture of DNA fragments, A step of isolating individual DNA fragments contained in the mixture, a step of hybridizing the mixture of DNA fragments and the isolated individual DNA fragments, and a step of measuring the amount of the hybridized and bound DNA fragments. A method for detecting a common DNA fragment, comprising:

(2) A mixture of DNA fragments is obtained by collecting similar tissues from each of a plurality of individuals, mixing them by the same weight, extracting the DNA from the mixed tissues, and then fragmenting the mixture. The method for detecting a common DNA fragment according to (1).
(3) A mixture of DNA fragments is obtained by collecting similar cells from each of a plurality of individuals, mixing them by the same number of cells, extracting DNA from the mixed cells, and then performing fragmentation. The method for detecting a common DNA fragment according to (1).
(4) The mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, mixing the extracted DNA by the same amount, and fragmenting the mixed DNA as described in (1) A common DNA fragment detection method.

(5) The mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, fragmenting it by the same technique, and mixing the fragmented DNA by the same amount as described in (1) A common DNA fragment detection method.
(6) The method for detecting a common DNA fragment according to any one of (1) to (5), wherein the chain length of the DNA fragment is in the range of 10 to 10,000 bases.
(7) The method for detecting a common DNA fragment according to any one of (1) to (6), wherein the mixture of DNA fragments is labeled.

(8) The method for detecting a common DNA fragment according to any one of (1) to (6), wherein each isolated DNA fragment is labeled.
(9) The double-stranded DNA fragment formed by hybridization of the mixture of DNA fragments and the isolated individual DNA fragment is specifically labeled, (1) to (6) A method for detecting the common DNA fragment as described.

(10) A method for detecting a specific DNA fragment that is common to a certain biological population and does not exist in another biological population, and that belongs to a plurality of individuals belonging to the certain biological population and to the different biological population Obtaining a mixture of DNA fragments from each of a plurality of individuals in the same manner, taking a part of a mixture of DNA fragments obtained from a plurality of individuals belonging to the certain biological population, and individual DNA contained in the mixture Isolating fragments, hybridizing a mixture of DNA fragments obtained from one organism population with individual DNA fragments isolated, a mixture of DNA fragments obtained from another organism population, and isolation A method for detecting a specific DNA fragment, comprising the step of hybridizing each individual DNA fragment and the step of measuring the amount of DNA that has hybridized and bound thereto.

(11) A mixture of DNA fragments is obtained by collecting similar tissues from each of a plurality of individuals, mixing them by the same weight, extracting the DNA from the mixed tissues, and then fragmenting the mixture. The method for detecting a specific DNA fragment according to (10).
(12) A mixture of DNA fragments is obtained by collecting similar cells from each of a plurality of individuals, mixing them by the same number of cells, extracting DNA from the mixed cells, and then fragmenting the cells. The method for detecting a specific DNA fragment according to (10).
(13) The mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, mixing the extracted DNA by the same amount, and fragmenting the mixed DNA. A method for detecting a specific DNA fragment.

(14) The mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, fragmenting by the same technique, and mixing the fragmented DNA by the same amount. A method for detecting a specific DNA fragment.
(15) The method for detecting a specific DNA fragment according to any one of (10) to (14), wherein the chain length of the DNA fragment is in the range of 10 to 10,000 bases.
(16) The method for detecting a specific DNA fragment according to any one of (10) to (15), wherein the mixture of DNA fragments is labeled.

(17) The method for detecting a specific DNA fragment according to any one of (10) to (15), wherein each isolated DNA fragment is labeled.
(18) The double-stranded DNA fragment formed by the hybridization of the mixture of DNA fragments and the isolated individual DNA fragment is specifically labeled, according to any one of (10) to (15) The method for detecting a specific DNA fragment as described.

(19) A method for detecting a common DNA fragment between specific populations that is commonly present in a plurality of specific organism populations, comprising: a plurality of individuals belonging to the one organism population; and a plurality of individuals belonging to the other organism population. A step of obtaining a mixture of DNA fragments by the same technique from each of them, taking a part of a mixture of DNA fragments obtained from a plurality of individuals belonging to the certain biological population, and isolating individual DNA fragments contained in the mixture Mixing a mixture of DNA fragments obtained from one organism population with a mixture of DNA fragments obtained from another organism population, and hybridizing the mixture with individual DNA fragments isolated, A method for detecting a common DNA fragment between specific populations, comprising a step of measuring the amount of bound DNA.

(20) A DNA testing method comprising performing DNA testing using the DNA fragment obtained by the detection method according to any one of (1) to (19).

(21) A method for detecting a change in the DNA of a microorganism present in a specific environment, wherein a microorganism group contained in a certain volume of the specific environment is collected at a certain point in time, and DNA is extracted from the microorganism group; The step of preparing a DNA fragment mixture at the first time point by fragmentation, and collecting the second time point microbe group contained in the same volume of the specific environment at a time point different from the first time point after collecting the first time point microbe group. Extracting the DNA from the microorganism group, then fragmenting the DNA fragment mixture at the first time point by the same technique, and preparing the DNA fragment mixture at the second time point, the DNA fragment mixture at the first time point and the mixture Hybridizing the individual DNA fragments contained, hybridizing the DNA fragment mixture at the second time point and the individual DNA fragments contained in the DNA fragment mixture at the first time point A step, a step of measuring the amount of hybridized and bound DNA, and individual DNA fragments contained in the DNA fragment mixture at the first time point when hybridized with the DNA fragment mixture at the first time point and the second time point A method for detecting a change in microbial DNA, comprising the step of comparing the amount of bound DNA when hybridized with the DNA fragment mixture.

(22) The method for detecting a change in microbial DNA according to (21), wherein the DNA fragment has a chain length in the range of 10 to 10,000 bases.
(23) The method for detecting a change in microbial DNA according to (21) or (22), wherein the mixture of DNA fragments at the first time point and the mixture of DNA fragments at the second time point are labeled.
(24) The method for detecting a change in microbial DNA according to (21) or (22), wherein each DNA fragment contained in the mixture of DNA fragments at the first time point is labeled.

(25) A method of hybridizing a DNA fragment possessed by an individual belonging to a certain biological population with a probe DNA, comprising preparing a mixture of DNA fragments possessed by each of a plurality of individuals belonging to the biological population, And hybridization of probe DNA.
(26) A mixture of DNA fragments is prepared by collecting similar tissues from each of a plurality of individuals, mixing them by the same weight, extracting the DNA from the mixed tissues, and then fragmenting the mixture. The hybridization method according to (25).

(27) preparing a mixture of DNA fragments by collecting similar cells from each of a plurality of individuals, mixing the same number of cells, extracting the DNA from the mixed cells, and then fragmenting the mixture. The hybridization method according to (25), characterized in that
(28) A mixture of DNA fragments is prepared by extracting DNA from each of a plurality of individuals, mixing the extracted DNA by the same amount, and fragmenting the mixed DNA (25) The hybridization method described in 1.
(29) A mixture of DNA fragments is prepared by extracting DNA from each of a plurality of individuals, fragmenting by the same technique, and mixing the fragmented DNA by the same amount (25) The hybridization method described in 1.

(30) A method for identifying an organism contained in a mixture of a plurality of organisms, the step of preparing a DNA fragment from the mixture, the prepared DNA fragment, and specific DNA for each organism that may be contained in the mixture A method for identifying an organism contained in a mixture, comprising a step of hybridizing with a fragment and a step of detecting a DNA fragment hybridized and bound.
(31) The method for identifying an organism contained in the mixture according to (30), wherein the mixture of a plurality of organisms is a laver or a mixed feed.

The basic concept of the present invention is as follows.
First, a basic concept of the present invention will be described with reference to FIG. 1 with respect to means for obtaining specific DNA of each fish species necessary for performing species discrimination between fish species A and B having unknown base sequences. Will be explained.

  The total DNA 2 of the individual a1 of the fish species A can be distinguished into a non-common DNA 3 of the individual a1 that varies depending on the individual and a common DNA 4 that is also present in common with other individuals of the fish species A, as shown in (a). Similarly, the total DNA 6 of another individual b5 of fish species A can also be distinguished into non-common DNA 7 of individual b2 and common DNA 4 of fish species A. Considering the DNA of these individuals together, as shown in (b), a distinction is made between common DNA 4 common to both individuals, non-common DNA 3 of individual a1 and non-common DNA 7 of individual b2, which are different for each individual. be able to. When such a figure is overlapped on the total DNA of a large number of individuals of fish species A, as shown in (c), the total DNA 8 of fish species A is a fish species that is common to fish species A. A distinction can be made between two parts: a common DNA 4 of A, and a set 9 of non-common DNAs in fish species A which are different among fish species A among individuals.

  Similarly for fish species B, as shown in (d), the total DNA 11 of individual c10 includes non-common DNA 12 of individual c10 that differs depending on the individual, and common DNA 13 of fish species B that is also present in common with other individuals. The total DNA 15 of another individual d14 of the fish species B can also be distinguished into the non-common DNA 16 of the individual d14 and the common DNA 13 of the fish species B. Considering the DNA of these individuals together, as shown in (e), common DNA 13 common to both two individuals of fish species B, and in addition, non-common DNA 12 of individuals 10 and non-common of individuals 14 DNA 16 is present. When such a figure is superimposed on the total DNA of a large number of individuals of fish species B, as shown in (f), the total DNA 17 of fish species B is the fish species that are common to fish species B. A distinction can be made between two parts: a common DNA 13 of B and a set 18 of non-common DNAs in a fish species B which differs among fish species B among individuals.

  Next, the total DNA 8 of fish species A and the total DNA 17 of fish species B are compared. As shown in (g), the total DNA 8 of the fish species A includes the common DNA 4 of the fish species A that is common to the individuals of the fish species A, and the set 9 of non-common DNAs in the fish species A that are different for each individual. The total DNA 17 of the fish species B can be divided into a common DNA 13 of the fish species B that is common to the individuals of the fish species B and a set 18 of non-common DNA in the fish species B that is different for each individual. Can be distinguished. Among the common DNAs possessed by each fish species, the DNA that is common to both fish species is the interspecies common DNA 19 of fish species A and B, and among the common DNA 4 of fish species A The DNA that does not exist in the fish species B is the specific DNA 20 of the fish species A, and the DNA that does not exist in the fish species A among the common DNA 13 of the fish species B is the specific DNA 21 of the fish species B.

  Thus, when the DNA of each of the fish species A and B is hybridized to the reference DNA and the signals obtained as a result of binding by hybridization are arranged, the hybridization signal table 22 in (h) is obtained. For example, when the DNA of fish species A is used as a reference DNA, a DNA microarray is prepared using this DNA, and the DNA of fish species A is hybridized, as shown in the corresponding column of hybridization signal table 22, A common DNA 19 between specific species A and fish species B and a specific DNA 20 of fish species A hybridize and bind to the DNA microarray to give an ON signal. Among fish species A, fish having different DNA for each individual As for the non-common DNA set 9 in the species A, since the hybridized DNA is different from the reference DNA immobilized on the DNA microarray, binding by hybridization does not occur and the signal is turned off. Similarly, when DNA of fish species A is used as a reference DNA, a DNA microarray is prepared using this DNA, and DNA of fish species B is hybridized to this DNA microarray, as shown in the corresponding column of hybridization signal table 22 In addition, specific species common DNA 19 between fish species A and fish species B hybridizes to and binds to the DNA microarray and becomes an ON signal, but the specific DNA 20 of fish species A immobilized on the DNA microarray and the fish Since the DNA belonging to the non-common DNA set 9 in the species A does not exist in the hybridized fish species B, no binding occurs due to hybridization, and the signal is turned off. Conversely, when DNA of fish species B is used as a reference DNA, a DNA microarray is prepared using this DNA, and DNA of fish species A is hybridized to this DNA microarray, it is shown in the corresponding column of hybridization signal table 22. As described above, the specific species common DNA 19 between the fish species A and the fish species B hybridizes to and binds to the DNA microarray and becomes an ON signal, but the specific DNA 21 of the fish species B immobilized on the DNA microarray and Since the DNA belonging to the non-common DNA set 18 in the fish species B does not exist in the hybridized fish species A, binding due to hybridization does not occur and the signal is turned off. Similarly, when DNA of fish species B is used as a reference DNA and DNA of fish species B is hybridized to this DNA microarray, as shown in the corresponding column of hybridization signal table 22, fish species A and B Specific species common DNA 19 and fish species B specific DNA 21 hybridize to and bind to the DNA microarray to give an ON signal. Among fish species B, non-common DNA in fish species B having different DNA for each individual Since the hybridized DNA is different from the reference DNA immobilized on the DNA microarray, the binding due to the hybridization does not occur and the signal is turned off.

Thus, the DNA to be identified is the same type as the reference DNA by determining the specific DNA of a specific population of organisms as the reference DNA and determining whether or not the DNA to be identified hybridizes and binds thereto. It can be judged whether or not.
In the above method, a microarray is prepared from DNA of fish species A. However, it is possible to obtain specific DNA using a commercially available microarray instead of a microarray prepared from such specific DNA. . The advantages of the method of the present invention will be described below by comparing the current method and the method of the present invention by taking a specific method for obtaining the common DNA of fish species C as an example.

  First, the current method will be described with reference to <I> in FIG. A plurality of individuals of fish species C are collected, but the experiment is performed for each individual. First, an experiment on an individual a82 of fish species C will be described. The tissue 84 collected from the individual a82 is taken into a container 83, and the DNA 85 of the individual a is extracted and cut to obtain a DNA fragment mixture 86 of the individual a. This is labeled with a fluorescent dye or the like, and hybridization is performed on a commercially available microarray to obtain an experimental result 87 of the individual a microarray. This series of experiments is repeated for the other individuals of the same fish species C, b, c, and d as many as the number of individuals, and the microarray experimental results for each individual are obtained. Thereafter, in order to summarize the results from a to d of the fish species C, an accumulation 88 of the experiment results of those microarrays is performed, and a result 89 of the entire fish species C is obtained. In order to identify the common DNA of fish species C from among them, DNA fragments derived from all individuals of the fish species C used are hybridized, and the DNA of the microarray spot showing strong fluorescence intensity is selected. Thereafter, the above series of operations is also performed for the fish species D that need to be distinguished from the fish species C, the detected common DNAs are compared, and the common ones are compared between the fish species C and the fish species D. Inter-species common DNA, fish species C specific DNA, fish species D specific DNA are being sought.

  For this reason, in the current method, the experiment is repeated as many times as the number of individuals to be analyzed, and finally, the experiment results of the respective hybridizations are accumulated to obtain the common DNA of each of the fish species C and D, so that the cost is also increased. Is also required for the number of individuals.

  On the other hand, in the method of the present invention shown in <II> of FIG. 11, in order to obtain the common DNA of the same fish species C, a plurality of fish species C are extracted together, and the DNA and the commercially available DNA are obtained. By hybridizing with a microarray, the result can be obtained in one experiment. First, the tissue of the same part of the fish species C collected from each individual a, b, c, d of the fish species C group 90 in the common container 91 for the fish species C is put together at this stage. Make a tissue mix 92 of fish species C. From here on, the mixture is treated as one specimen, and the fish species C DNA mixture 93 is extracted from the fish species C tissue mixture 92 and cut to obtain the fish species C DNA fragment mixture 94. This is labeled with a fluorescent dye or the like, hybridized over one commercially available microarray, and an overall result 89 of fish species C is obtained. In order to identify the common DNA of fish species C, the DNA of the microarray spot showing strong fluorescence intensity is selected. In the method of the present invention, this result can be said to be the common DNA of fish species C as it is.

  According to the present invention, the above method is not performed for each animal, but a plurality of animals can be distinguished efficiently by performing a group process. The group treatment is assumed to be, for example, a plurality of SBT, 100 in this case, but by crushing the 100 fillets and mixing and extracting DNA, a DNA mixture of 100 SBT can be produced, and the DNA is restricted. Cleaved with an enzyme, microarrayed with half as a reference DNA, hybridized with the other half, and the DNA part where all the numbers were bound is the common DNA shared by SBT, and the part of DNA that only partly bound Can be distinguished from individual non-common DNA. Similarly, for example, it is assumed that there are a plurality of yellowfin tuna, for example, 100 here, but the DNA mixture obtained by crushing and mixing the 100 yellowfin tuna fillets is the same as that used for the cleavage of SBT DNA. Cleave with the restriction enzyme. By doing so, common DNA present in both SBT and yellowfin tuna can be cut at the same size in both SBT and yellowfin tuna. This yellowfin tuna DNA fragment mixture is hybridized with a microarray to which individual DNA fragments contained in the southern bluefin tuna DNA fragment mixture are immobilized. As a result, the DNA to which the total number of yellowfin tuna is bound is the common DNA of these tuna common to southern bluefin tuna and yellowfin tuna, and the DNA that has not been bound can be distinguished as specific DNA of southern bluefin tuna compared to yellowfin tuna. By conducting the same experiment by replacing the southern bluefin tuna and yellowfin tuna, the specific DNA of yellowfin tuna compared with the southern bluefin tuna can be distinguished. Using the information thus obtained, it is possible to construct a system for performing DNA identification for species discrimination of southern bluefin tuna and yellowfin tuna. This method can also construct a system for performing DNA identification for species discrimination of various organisms such as animals, plants, bacteria and viruses including humans. In addition, by applying this idea of collective treatment of a plurality of animals, it is possible to identify each of the organisms contained in a mixture of a plurality of organisms such as a laver or a mixed feed. Furthermore, by determining the amount of each specific DNA fragment, the proportion of the organism can also be determined. The method for determining the amount of DNA fragments can be determined by fluorescence intensity in a commonly used DNA microarray, real-time PCR, or the like. In addition, it is possible to construct a system for performing DNA analysis of individuals of various biological populations such as animals, plants, bacteria, and viruses including humans using information on non-common DNA obtained by the present invention. In addition, according to the present invention, a sufficient amount of DNA can be extracted for analysis without amplifying the DNA by PCR or the like by changing the number of individuals and the size of the fillet as necessary. More accurate species discrimination can be performed than when DNA is amplified, and the time and cost for amplification can be reduced.

  When a change in a common DNA fragment, a specific DNA fragment, a specific inter-group common DNA fragment, or a microbial DNA is detected according to a conventional method, analysis for each individual or analysis of a base sequence is required. Analysis of each individual and analysis of the base sequence is laborious and expensive. Especially, analysis of each individual analyzes the result of accumulation of the variation of the experimental conditions for each time and the error of the experimental results derived from it. As a result, there is a risk that the reliability of the experimental result is lowered. In the detection method of the present invention, changes in common DNA fragments, specific DNA fragments, common DNA fragments between specific populations, and microbial DNA can be efficiently detected without such troublesome analysis.

Hereinafter, the present invention will be described in detail.
(A) Method for detecting common DNA fragment The method for detecting a common DNA fragment of the present invention is a method for detecting a DNA fragment that is commonly present in a certain organism population, and comprises DNA fragments from a plurality of individuals belonging to the organism population. Taking a part of the mixture of DNA fragments and isolating individual DNA fragments contained in the mixture, hybridizing the mixture of DNA fragments and the isolated individual DNA fragments And a step of measuring the amount of the DNA fragment hybridized and bound.

  The biological population in the present invention usually means a population belonging to the same taxonomic unit (for example, eyes, family, genus, species, etc.), but also includes a population unrelated to the taxonomic unit. Including. In addition, the biological population may include two or more taxonomic units.

  A mixture of DNA fragments can be obtained by mixing tissues, cells and the like collected from each individual, extracting the DNA therefrom, and then fragmenting the DNA. Alternatively, DNA may be extracted from each tissue, cell, etc. collected from each individual, mixed, and then fragmented. Furthermore, each of the DNAs derived from each individual may be fragmented by the same technique, and mixed after fragmentation. Thus, the following advantages can be obtained by preparing a DNA fragment by mixing tissues collected from each individual. Since the current detection method conducts experiments for each individual, the amount of raw materials such as tissues and cells that can be collected from each individual is limited, and DNA extracted from a very small amount of raw materials is artificially created using PCR or the like. Amplified product is used for detection. However, in the present invention, since a plurality of individuals can be mixed to prepare a DNA mixture necessary for detection, even if the amount collected from each individual is reduced, the amount of DNA that can be detected without artificial amplification is reduced. Can be secured. In addition, in order to obtain a common DNA fragment, it is necessary to amplify DNA at all sites with the same efficiency, but this is very difficult, and usually there is a difference in amplification efficiency depending on the site, so the detection accuracy is low. There is a risk of becoming. Furthermore, there is a risk that the DNA polymerase used for artificial amplification makes a mistake with a certain probability and introduces a mutated base, which also reduces the detection accuracy. According to the present invention, it is possible to perform detection without creating a factor that causes these accuracy degradations. Of course, if there is an upper limit to the amount of tissue or cells that can be collected from an individual and the amount of DNA is insufficient even after mixing and cannot be tested without amplification, DNA polymerase with the lowest error rate is possible. Detection can also be performed using the amplified DNA.

  When mixing tissues and the like collected from each individual, it is necessary to mix so that the amount of DNA derived from each individual becomes equal. As a mixing method for equalizing the amount of DNA, an appropriate method may be selected according to the type of organism. For example, if each individual is an organism that is taxonomically approximate (for example, an organism belonging to the same genus, species, etc.), the same tissue may be collected from each individual and mixed by the same weight. Alternatively, similar cells may be collected and mixed so that the number of cells is the same. If each individual is not a taxonomic approximation, it is difficult to collect the same tissue or cells. Therefore, DNA may be collected from each individual and mixed in equal amounts.

  The technique for fragmenting DNA may be any technique with strict reproducibility, and a typical technique is cleavage with a restriction enzyme. Restriction enzymes may be used alone or in combination. However, since the DNA chain length greatly affects the state of hybridization, hybridization conditions for each DNA are made as uniform as possible and the conditions are strictly set. In order to obtain a more accurate detection result, it is desirable to use a restriction enzyme so that most of the fragments generated by cleavage have as similar chain lengths as possible. When it is desired to obtain a rough common DNA fragment over a long region, a restriction enzyme that does not have a high recognition sequence in the genome is used, and the chain length of most fragments generated by cleavage is 10 to 10,000 bases. It is desirable to cut so that it is between 10 and 1000 bases, more preferably between 10 and 100 bases. On the other hand, when it is desired to eliminate a minute difference of several bases or less and to obtain a common DNA fragment in a very strict form, it is between the bases of 10 to 30 bases of most fragments generated by cutting. It is desirable to use restriction enzymes in one or a combination of a plurality of types.

In DNA extraction, when the DNA is cleaved with a restriction enzyme later, the DNA is cut too much and the result of cleaving with the restriction enzyme cannot be obtained correctly, or there is a substance that inhibits the labeling or hybridization reaction after extraction. Any method may be used as long as it does not remain.
A method for isolating individual DNA fragments contained in a mixture of DNA fragments is not particularly limited. For example, a DNA fragment contained in the mixture is cloned into a vector to prepare a library, and then individual DNA fragments are separated from each other. If each is increased independently, individual DNA fragments can be obtained.

  Hybridization of a mixture of DNA fragments and isolated individual DNA fragments may be performed by any method, but it is preferable to hybridize the DNA fragments by immobilizing them on a DNA microarray.

  The amount of the DNA fragment hybridized and bound can be measured by labeling the DNA fragment. The label may be added either to the mixture of DNA fragments or to individual DNA fragments, or may be added before or after hybridization. A label that specifically binds to a duplex formed by hybridization of a mixture of DNA fragments and individual DNA fragments may be used. In addition, it may be added directly or indirectly to the DNA fragment, or a tag such as a hapten or a low molecular weight compound is added to the DNA fragment so that an antibody or molecule having a strong binding force can be attached. A system including a bound enzyme and a coloring material serving as a substrate for the enzyme may be used. Labels include dyes, fluorescent dyes, radioisotopes, conductors or their precursors, and the amount of signal that can be detected by the label, such as the above-mentioned tag for binding an enzyme after hybridization and causing a color reaction. Any system can be used as long as it can accurately reflect the amount of binding between each DNA fragment and the DNA fragment mixture, but any method that adds a label or tag to each end of a DNA fragment quantitatively. For example, it is easy to associate the number of DNA fragments with the number of labels.

  After measuring the amount of DNA fragments hybridized and bound, a group of DNA fragments having a relatively large amount of bound DNA is selected from the individual DNA fragments. As described above, since the amount of DNA derived from each individual in the DNA fragment mixture is equal, the amount of DNA fragments hybridized and bound depends on the number of individuals having individual DNA fragments to be hybridized. And increase. Therefore, a DNA fragment belonging to a group having a relatively large amount of bound DNA is likely to be a DNA fragment (common DNA fragment) that is commonly present in the organism population, and the amount of bound DNA is particularly large. The DNA fragment that showed the value is more likely.

(B) Specific DNA fragment detection method The specific DNA fragment detection method of the present invention is a method for detecting a specific DNA fragment that is commonly present in one biological population and not present in another biological population. Obtaining a mixture of DNA fragments from each of a plurality of individuals belonging to the certain biological population and a plurality of individuals belonging to the different biological population by the same technique, obtained from the plurality of individuals belonging to the certain biological population. Taking a part of the mixture of DNA fragments and isolating individual DNA fragments contained in the mixture, hybridizing the mixture of DNA fragments obtained from the certain biological population and the isolated individual DNA fragments A step of hybridizing a mixture of DNA fragments obtained from the other population of organisms with each individual isolated DNA fragment, and measuring the amount of hybridized and bound DNA. It includes a step of measuring.

  The specific DNA fragment detection method of the present invention is an application of the above-mentioned common DNA fragment detection method. The meaning of the biological population, the preparation method of the mixture of DNA fragments, and the individual DNA fragments contained in the mixture are simply detected. The separation method, hybridization method, and method for measuring the amount of bound DNA fragments may be the same as the common DNA fragment detection method.

  After measuring the amount of DNA fragments hybridized and bound, select a group of DNA fragments that have a relatively large amount of DNA hybridized and bound with a mixture of DNA fragments obtained from the certain population From the obtained DNA fragments, DNA fragments that do not hybridize with the mixture of DNA fragments obtained from the other organism population are selected. As described above, since the amount of DNA derived from each individual in the DNA fragment mixture is the same, the amount of the DNA fragment hybridized and bound to the mixture of DNA fragments obtained from the certain biological population is determined according to the individual target to be hybridized. The number increases according to the number of individuals having the DNA fragment. Therefore, it is highly possible that a DNA fragment belonging to a group having a relatively large amount of bound DNA is a DNA fragment that is commonly present in the certain organism population, and in particular, the amount of bound DNA showed the maximum value. DNA fragments are more likely. However, since this DNA fragment may be present in the other organism population, it cannot be a specific DNA. Therefore, in order to exclude DNA fragments that are also present in the other organism population, DNA fragments that do not hybridize with a mixture of DNA fragments obtained from the other organism population are selected.

  The specific DNA fragment detected in this way can be used to judge whether the target individual belongs to one biological population or another biological population. Such appraisal can be performed not only at the individual level, but also for subjects that do not retain the prototype of the organism, such as raw materials for processed foods.

(C) Method for detecting a common DNA fragment between specific populations The method for detecting a common DNA fragment between specific populations of the present invention is a method for detecting a common DNA fragment between specific populations that is present in common in a plurality of specific biological populations. Obtaining a mixture of DNA fragments from each of a plurality of individuals belonging to the certain biological population and a plurality of individuals belonging to the different biological population by the same technique, obtained from the plurality of individuals belonging to the certain biological population. Taking a part of a mixture of DNA fragments and isolating individual DNA fragments contained in the mixture, a mixture of DNA fragments obtained from one organism population and a mixture of DNA fragments obtained from another organism population And mixing the isolated DNA fragments with each of the isolated DNA fragments, and measuring the amount of the hybridized and bound DNA. The

The method for detecting a common DNA fragment between specific populations of the present invention is an application of the above-mentioned method for detecting a common DNA fragment, meaning the biological population, a method for preparing a mixture of DNA fragments, and individual DNA fragments contained in the mixture. The method for isolating and measuring the amount of bound DNA fragments may be the same as the method for detecting common DNA fragments.
Hybridization consists of mixing a mixture of DNA fragments obtained from one organism population and a mixture of DNA fragments obtained from another organism population, and then hybridizing the mixture of these mixtures with isolated individual DNA fragments. To do.

  After measuring the amount of DNA fragments hybridized and bound, DNA fragments belonging to a group having a relatively large amount of hybridized and bound DNA are selected. As described above, since the amount of DNA derived from each individual in the DNA fragment mixture is equal, it hybridizes with a mixture of a mixture of DNA fragments obtained from one organism population and a mixture of DNA fragments obtained from another organism population. The amount of DNA fragments bound in this manner increases depending on the number of individuals who have individual DNA fragments to be hybridized. Accordingly, a DNA fragment belonging to a group having a relatively large amount of bound DNA is likely to be a DNA fragment that is present in common in the certain organism population and the other organism population, and in particular, the amount of bound DNA. The DNA fragment that shows the maximum value is more likely. The interspecific DNA fragment detected in this way is used as a control DNA for confirming whether the hybridization reaction is properly performed in the experiment for identifying the varieties among a plurality of species having the same in common. be able to.

(D) Method for detecting change in microbial DNA The method for detecting change in microbial DNA according to the present invention is a method for detecting a change in DNA of a microorganism present in a specific environment, and at a certain point in time, a constant volume of the specific environment. Collecting a microbial group contained in the microbial group, extracting DNA from the microbial group, then fragmenting and preparing a DNA fragment mixture at the first time point, after collecting the microbial group at the first time point, a time point different from the first time point The second time point microbial group contained in the same volume of the specific environment is collected, DNA is extracted from the microbial group, and then fragmented by the same method as the first time point DNA fragment mixture, and the second time point DNA is extracted. Preparing a fragment mixture, hybridizing the DNA fragment mixture of the first time point and the individual DNA fragments contained in the mixture, the DNA fragment mixture of the second time point and the first time point The steps of hybridizing with individual DNA fragments contained in the DNA fragment mixture, measuring the amount of hybridized and bound DNA, and individual DNA fragments contained in the DNA fragment mixture at the first time point The method includes a step of comparing the amount of bound DNA when hybridized with the DNA fragment mixture at one time point and when hybridized with the DNA fragment mixture at the second time point.

The method for detecting a change in microbial DNA of the present invention is an application of the above-described method for detecting a common DNA fragment. DNA extraction method, DNA fragmentation method, hybridization method, measurement of the amount of bound DNA fragments The method may be the same as the method for detecting a common DNA fragment. In addition, the specific environment in the present invention refers to a specific water area such as a river, a lake, and the sea, a soil area in a specific area, an atmosphere in a specific area, an internal environment of a living organism, and the like.
By detecting a change in microbial DNA, it is possible to know whether or not there has been a change in the composition of the microorganism in a specific environment.

(E) Hybridization method The hybridization method of the present invention is a method of hybridizing a DNA fragment possessed by individuals belonging to a certain biological population with probe DNA, wherein each of a plurality of individuals belonging to said biological population is A mixture of DNA fragments is prepared, and this is hybridized with probe DNA. The hybridization method of the present invention is an application of the above-mentioned common DNA fragment detection method. The meaning of the biological population, the preparation method of the mixture of DNA fragments, and the hybridization method are the same as the detection method of the common DNA fragment. Good.
Any probe DNA may be used. For example, a commercially available microarray may be used.

(F) Method for identifying organism contained in a mixture of a plurality of organisms The method for identifying an organism contained in a mixture of a plurality of organisms of the present invention comprises the steps of preparing a DNA fragment from the mixture, the prepared DNA fragment And a step of hybridizing with a specific DNA fragment for each organism that may be contained in the mixture, and a step of detecting a hybridized and bound DNA fragment.

  In the identification method of the present invention, the method for hybridizing and the method for detecting the bound DNA fragment may be the same as the method for detecting the common DNA fragment. In addition, DNA extraction and DNA fragmentation in preparation of DNA fragments from the mixture may be the same as the method for detecting common DNA fragments. The specific DNA fragment for each organism used for hybridization may be obtained by the above-described method for detecting a common DNA fragment of the present invention, or may be designed from a base sequence search or the like. The mixture in the present invention refers to a board paste or a mixed feed prepared by mixing a plurality of varieties.

Since it is possible to know the composition of the organism contained in the mixture from the information on the organism obtained in this way, it is possible to perform true / false identification of the component display.
Hereinafter, preferred embodiments of the detection method of the present invention will be described with reference to the drawings.

  FIG. 2 is a diagram showing a procedure of an experiment for obtaining specific DNA of SBT in order to discriminate species of biological populations whose base sequences are not known, for example, SBT and yellowfin tuna. FIG. 3 is a diagram showing an analysis procedure for identifying specific SBT specific DNA for species discrimination between SBT and yellowfin tuna based on the experimental results obtained in FIG.

  First, in FIG. 2, a tissue 24 of the same portion of the southern bluefin tuna collected from each individual of the population 23 of a plurality of southern bluefin tuna is mixed in a single southern bluefin tuna container 25, and the DNA mixture of the southern bluefin tuna from the whole. 26 is extracted. This is cleaved with a restriction enzyme that gives a fragment of an appropriate length to obtain a SBT DNA fragment mixture 27. This is divided into two, and one of the divided SBT DNA fragment mixture 27 is cloned into an appropriate vector to prepare a library. This is taken up by the host and spread on the agar medium 28 to pick up each SBT colony or plaque 29. These are propagated independently of each other to increase the number of cloned DNA fragments, and each clone is fixed at a different position on the substrate to produce a SBT DNA microarray 30. The ends of the other SBT DNA fragment mixture 27 divided in two are labeled with a fluorescent dye to produce a SBT fluorescently labeled DNA fragment mixture 31. This SBT fluorescently labeled DNA fragment mixture 31 was hybridized with the previously prepared SBT DNA microarray 30 to remove excess DNA, and the SBT fluorescently labeled DNA fragment mixture 31 was hybridized with the SBT fluorescently labeled DNA fragment mixture 31. Read the signal. Since one fluorescent molecule is added to each end of the DNA, the intensity of the fluorescence signal of each clone that can be read on the DNA microarray 32 of the southern bluefin tuna hybridized with the southern bluefin tuna fluorescently labeled DNA fragment mixture 31 is different from that of the hybridized southern bluefin tuna. It is proportional to the number of DNA molecules, and is proportional to the number of individuals having the same DNA as the DNA fixed to the SBT DNA microarray 30 among the SBT derived from the SBT fluorescently labeled DNA fragment mixture 31. As a result, if the DNA of a specific clone immobilized on the SBT DNA microarray 30 is common DNA common to all SBT tuna, all fluorescently labeled DNAs corresponding to the common DNA clone 33 of this SBT tuna are used. Since DNA from SBT individuals hybridizes completely to this clone with the same length, the fluorescent signal of this common SBT DNA clone 33 is strong. On the other hand, if the DNA of another clone fixed on the SBT DNA microarray 30 is a non-common DNA that differs for each SBT individual, this SBT non-common in the SBT fluorescence-labeled DNA fragment mixture 31. Since a DNA having a nucleotide sequence different from that of the DNA of the DNA clone 34 does not hybridize, the fluorescence signal of the non-common DNA clone 34 of SBT is weaker than that of the common DNA clone 33 of SBT on the same microarray. If such an analysis is performed for all clones immobilized on the SBT DNA microarray 30, the DNA of each clone immobilized on the SBT DNA microarray 30 is common or non-common to SBT. You can know if it is DNA.

  Next, a tissue 36 of the same portion of yellowfin tuna collected from each group 35 of yellowfin tuna 35 is mixed in a single yellowfin tuna container 37, and a yellowfin tuna DNA mixture 38 is extracted from the whole. This is cleaved with the same restriction enzyme as that obtained by cleaving SBT DNA in FIG. 2 to obtain a yellowfin tuna DNA fragment mixture 39. The ends of this yellowfin tuna DNA fragment mixture 39 are labeled with a fluorescent dye to produce yellowfin tuna fluorescently labeled DNA fragment mixture 40. This yellowfin tuna fluorescently labeled DNA fragment mixture 40 was hybridized with the previously prepared southern bluefin tuna DNA microarray 30 to remove excess DNA, and the yellowfin tuna fluorescently labeled DNA fragment mixture 40 was hybridized with yellowfin tuna fluorescently labeled DNA fragment mixture 40. Read the fluorescent signal. Southern bluefin tuna and yellowfin tuna are fish of the same Tuna genus, and DNA base sequences are thought to have many common parts. Therefore, if the same restriction enzyme is used for cleavage, both DNA cleavage sites are considered to be common. As a result of the hybridization, if the DNA of a specific clone immobilized on the SBT DNA microarray 30 is a DNA clone 42 that is common to all yellowfin tuna, this clone from all fluorescently labeled yellowfin tuna individuals Since the corresponding DNA is completely hybridized to this clone immobilized on the SBT DNA microarray 30, the fluorescence signal of this common bluefin tuna DNA clone 42 is enhanced. On the other hand, if the DNA of another clone immobilized on the SBT DNA microarray 30 is a non-common DNA clone 43 of yellowfin tuna, which is different for each yellowfin tuna individual, Since the nucleotide sequence different from that of the clone DNA is not hybridized, the fluorescence signal of the yellowfin tuna non-common DNA clone 43 is weaker than the fluorescence signal of the common yellowfin tuna common DNA clone 42. The intensity of the fluorescent signal in this case is proportional to the number of individuals having the same base sequence DNA as the DNA fixed to the southern bluefin tuna DNA microarray 30 among yellowfin tuna derived from yellowfin tuna fluorescently labeled DNA fragment mixture 40. Further, if the DNA of yet another clone immobilized on the SBT DNA microarray 30 is a specific DNA of SBT that is different from any of the yellowfin tuna DNA, any DNA in the fluorescently labeled DNA 41 of yellowfin tuna will be included in this clone. Because it does not hybridize, this SBT specific DNA clone 44 does not emit any fluorescent signal. If such an analysis is performed for all clones immobilized on the SBT DNA microarray 30, whether the DNA of each SBT clone immobilized on the SBT DNA microarray 30 is common to yellowfin tuna. It is possible to know whether it is non-common DNA or DNA that does not exist in yellowfin tuna.

  The experimental results of southern bluefin tuna and yellowfin tuna obtained according to the flow of FIG. 2 are compared and analyzed as shown in FIG. First, with respect to the SBT DNA microarray 32 hybridized with the SBT fluorescence-labeled DNA fragment mixture 31, the fluorescence signal intensity of each clone is read using a reader such as a fluorescence scanner or a CCD camera. For example, when the line of the scant line 45 of the SBT DNA microarray 32 hybridized with the SBT fluorescence-labeled DNA fragment mixture 31 is scanned with a fluorescence scanner, and the read fluorescence signal intensity is represented in a graph along the scanline 45, the SBT A signal scan graph 46 is obtained. Based on such graph data, each clone is classified by fluorescence signal intensity. That is, clones that give strong fluorescence signal intensity by hybridizing all corresponding DNA contained in fluorescently labeled DNA are classified as group a, and part of the corresponding DNA contained in fluorescently labeled DNA is hybridized. Clones that give a weaker fluorescent signal intensity are classified as group b. In accordance with this classification, the SBT column of the comparison table 48 is created. For example, from the data of the SBT signal scan graph 46, clone number 9 and clone number 11 are classified into group b, and clone number 10 and clone number 12 are classified into group a. In this case, since SBT DNA is hybridized to SBT clones, in principle, there is no clone having no fluorescence signal. Next, for the southern bluefin tuna DNA microarray 41 hybridized with yellowfin tuna fluorescently labeled DNA fragment mixture 40, the fluorescence signal intensity of each clone is read using a reader such as a fluorescent scanner or a CCD camera. For example, when the line of the scant line 45 of the SBT DNA microarray 41 hybridized with the yellowfin tuna fluorescently labeled DNA fragment mixture 40 is scanned with a fluorescence scanner, and the read fluorescence signal intensity is represented in a graph along the scanline 45, the yellowfin tuna A signal scan graph 47 is obtained. Based on such graph data, for each clone on the SBT DNA microarray 41 hybridized with yellowfin tuna fluorescently labeled DNA fragment mixture 40, all corresponding DNA contained in the fluorescently labeled DNA was hybridized. A clone that gives the strongest fluorescence signal intensity is classified as group a, and a clone that gives a weaker fluorescence signal intensity when a part of the corresponding DNA contained in the fluorescently labeled DNA is hybridized is further classified as group b. The clones in which fluorescence-labeled DNA is not hybridized at all and no fluorescence signal is obtained are classified as group c, and a yellowfin tuna column is added to the comparison table 48 of SBT hybridization results. Over 30 Creating a comparison table 48 that can list the intensity of the fluorescent signal of the SBT and yellowfin of fixed each clone. For example, from the data of yellowfin tuna signal scan graph 47, clone number 9 and clone number 10 are classified into group c, clone number 11 is classified into group b, and clone number 12 is classified into group a. The comparison table 48 compares the scores of the experimental results of southern bluefin tuna and yellowfin tuna for each clone, and clones classified as group a for southern bluefin tuna and group c for yellowfin tuna are selected. Such a clone is common DNA of SBT and non-DNA that does not exist in yellowfin tuna, that is, SBT-specific DNA when performing species discrimination between SBT and yellowfin tuna. A column for marking only such clones is provided in the comparison table 48 so that the specific DNA of SBT can be easily found. If only such clones are picked up, a DNA microarray 49 for species discrimination with a smaller number of clones can be created, and this can be used for species discrimination of a test target that requires species discrimination of SBT or yellowfin tuna. Possible DNA identification can be performed.

  According to this method, specific DNA can be obtained without performing laborious, time-consuming and expensive base sequence analysis. In addition, a plurality of individuals are first mixed to extract DNA, and a mixture of SBT and yellowfin tuna DNA fragments is subjected to hybridization of each single DNA microarray to obtain a large number of common and non-common DNAs of SBT and yellowfin tuna. Since these can be obtained separately, the labor, time and cost of the experiment can be saved also in these respects. Also, according to this method, not only simplicity, but also by performing hybridization only once, variation in experimental conditions for each experiment and errors in experimental results derived from it can be introduced. And reliable results are obtained. Furthermore, according to this method, the amount of DNA necessary for the test can be ensured without amplifying the DNA, so that there is no risk of causing an erroneous result caused by the DNA amplification. In this respect, more reliable detection can be achieved. Can be done.

  Note that specific DNA of yellowfin tuna that is present in yellowfin tuna but not in southern bluefin tuna is not fixed on the southern bluefin tuna DNA microarray 30. Therefore, specific DNA of yellowfin tuna can be obtained using this microarray. Absent. Therefore, in order to obtain specific DNA of yellowfin tuna to be used for species discrimination between southern bluefin tuna and yellowfin tuna, it is necessary to perform the above experiments and analysis by exchanging the DNA of the southern bluefin tuna and yellowfin tuna to obtain specific DNA of yellowfin tuna. is there. If this method is applied to a large number of biological populations, the interspecies common DNA of a plurality of biological populations and the specific DNA of a specific biological population for a plurality of biological populations can be obtained one after another. Furthermore, it is also possible to create a system for identifying specific organisms in a plurality of organism populations by combining the specific DNA of each of the plurality of organism populations into one test system.

  In addition, it is possible to detect a change in the ratio of a plurality of organism groups by this method. FIG. 4 is a diagram showing an outline of a procedure for analyzing the temporal change of the microbial ecosystem in a specific water area by applying this method for detecting a specific DNA fragment.

  First, when water in a specific water area such as a river, a lake, or the sea is collected at a time point 1 that is a reference for analysis, various amounts of various microorganisms are contained therein. Let this be the water 50 of the time 1 of a specific water area, and the group 50 of the microorganisms contained in it. Similar to the process shown in FIG. 3, the microorganisms contained therein are collected together and extracted with the DNA mixed, and then fragmented with an appropriate restriction enzyme, so that the DNA fragments of the entire microorganism at the time point 1 in the specific water area. A mixture 51 is made. This is divided into two, and using the individual DNA fragments contained in the DNA fragment mixture 51 of the whole microorganism at time 1 in one specific water area, a DNA microarray at time 1 in the specific water area is prepared. Next, the DNA fragment mixture 51 of the whole microorganism at the time point 1 in the other specific water area is fluorescently labeled and hybridized with the DNA microarray 52 at the time point 1 in the specific water area, so that the DNA fragment of the whole microorganism at the time point 1 in the specific water area A DNA microarray 53 of time point 1 in a specific water area hybridized with the mixture is obtained. Thereafter, from the water collected at time 2 in the specific water area and the population 54 of the microorganisms contained therein, the microorganisms contained therein are collectively collected and extracted while being mixed, and this is extracted as microorganisms at the time point 1 in the specific water area. The entire fragmented DNA fragment mixture 51 is fragmented with the same restriction enzyme as the fragmented fragment to create a DNA fragment mixture 55 of the whole microorganism at the time point 2 in the specific water area. This is fluorescently labeled and hybridized with the DNA microarray 52 at the time point 1 in the specific water area to obtain the DNA microarray 56 at the time point 1 in the specific water area hybridized with the DNA fragment mixture of the whole microorganism at the time point 2 in the specific water area. The result of the DNA microarray 53 at the time point 1 of the specific water area hybridized with the DNA fragment mixture of the whole microorganism at the time point 1 in the specific water area and the time point 1 of the specific water area hybridized with the DNA fragment mixture of the whole microorganism at the time point 2 in the specific water area The results of the DNA microarray 56 are compared. A clone of a microbial DNA whose abundance has changed over time from time point 1 to time point 2 can be detected as having changed due to the relative difference in signal intensity in the two DNA microarrays. If water is collected periodically and the state of change is tracked, changes in the amount of specific microbial DNA in the ecosystem can be captured over time.

  It is also possible to identify organisms contained in a mixture of a plurality of organisms by applying this method. First, with respect to all the organisms that may be contained in the mixture, a specific DNA fragment for each organism is determined by the above method or the like. Using a specific DNA fragment for each organism, for example, a DNA microarray for organism identification is prepared. Next, DNA is extracted from a mixture in which it is desired to identify the contained organisms, fragmented and fluorescently labeled by the above method, and hybridized with a DNA microarray for organism identification. If the organisms from which the specific DNA hybridized with the DNA of the mixture is enumerated on the DNA microarray for organism identification are listed, that is, the organisms included in the mixture.

  Further, the detection method is not limited to that described in the present embodiment. In addition, it is needless to say that various other configurations can be adopted without departing from the gist of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, it goes without saying that the present invention is not limited to this embodiment.

  The test of the present invention determines whether or not common DNA, specific DNA and non-common DNA classified as shown in (g) of FIG. 1 can be detected in a signal on / off state as shown in (h). Investigated by the method.

  First, 10 types of 40-mer oligonucleotides having the base sequences shown in the table of FIG. 5 were synthesized. Of these, oligonucleotide B has an oligonucleotide G, oligonucleotide C has an oligonucleotide H, oligonucleotide D has an oligonucleotide I, and oligonucleotide I has a complementary nucleotide sequence, oligonucleotide A, oligonucleotide E, Oligonucleotide F and oligonucleotide J do not have a complementary base sequence among these ten kinds of oligonucleotides. First, experiments were conducted to confirm the complementarity and specificity of these 10 oligonucleotides. 1 μM aqueous solution of these 10 kinds of oligonucleotides was added to the first 96-well microtiter plate 58 and the second 96-well microtiter plate 67 (Nippon Gene) according to the layout of FIGS. 22 μl was dispensed into each corresponding well of the Tics Multi Semi-skirted PCR Plate 35801). That is, the well portion 59 of the 96-well microtiter plate into which the oligonucleotide A solution was dispensed, the well portion 60 of the 96-well microtiter plate into which the oligonucleotide B solution was dispensed, and the oligonucleotide C solution were dispensed. 96-well microtiter plate well portion 61, 96-well microtiter plate well portion 62 dispensed with oligonucleotide D solution, 96-well microtiter plate well portion dispensed with oligonucleotide E solution 63, well portion 64 of 96-well microtiter plate into which oligonucleotide F solution was dispensed, well portion 65 of 96-well microtiter plate into which oligonucleotide G solution was dispensed, solution of oligonucleotide H Of a well of a 96-well microtiter plate Each of two rows of portion 66, portion 68 of a well of a 96-well microtiter plate dispensed with oligonucleotide I solution, and portion 69 of a well of 96-well microtiter plate dispensed with oligonucleotide J solution Each well was dispensed with 22 μl of a 1 μM aqueous solution of the corresponding oligonucleotide. Of the wells of the first 96-well microtiter plate 58 and the second 96-well microtiter plate 67, only one type of oligonucleotide is dispensed with 22 μl of distilled water and the oligonucleotide. To wells that were not dispensed at all, 44 μl of distilled water was dispensed. As a result, wells in which any one of oligonucleotides A to E and any one of oligonucleotides F to J are dispensed into the same well can be prepared in all combinations of at least 4 wells, In addition, as a control, wells in which only one type of each of the oligonucleotides A to J was dispensed and wells that did not contain any oligonucleotide could be prepared in four wells in each microtiter plate.

  Next, 5.5 μl of x10TE (100 mM Tris-HCl, 10 mM EDTA, pH 8.0) was dispensed into all wells of the two microtiter plates and mixed well by pipetting each well. Cover these two microtiter plates with aluminum foil seals (As One aluminum tape AH-132) and heat-press them tightly so that the solution and vapor are not exchanged between the wells. Then, attach the microtiter plate to the thermal cycler ( Applied Biosystems Japan Co., Ltd. Gene Amp PCR System 9700) was heated at 96 ° C. for 3 minutes, and then the temperature was lowered to 25 ° C. over 17 minutes to allow oligonucleotide hybridization. The plate was wiped well and 45 μl was removed from each well of these microtiter plates and transferred to two 96-well flat bottom microtiter plates (Nunc 96 well optical bottom plate 265301), keeping the same placement. Dispense 5 μl of a 20,000-fold diluted solution of the double-stranded DNA detection reagent (SYBR Green I, TaKaRa F0513) into all wells of the two flat-bottomed microtiter plates to which the solution has been transferred, and pipette well into each well. After mixing the solution, it was covered with an aluminum foil seal (As One Aluminum Tape AH-132) and firmly heat-pressed so that the solution and vapor were not exchanged between the wells. These flat bottom microtiter plates were stored in the dark for 15 minutes at 37 ° C.

  Remove these flat-bottomed microtiter plates, and measure the fluorescence intensity with a fluoro image analyzer (Fujifilm Corporation FLA-5000) (filter: LPB, excitation wavelength: 473 nm, pmt value: 400 V). did. Among these, FIG. 7 shows images of 4 wells each subjected to hybridization with the same combination of oligonucleotides. In addition, the arithmetic average of the measured values of wells hybridized with the same combination of oligonucleotides was obtained, and the value obtained by subtracting the arithmetic average of the measured values of the wells not containing the oligonucleotide from the value as the background was obtained. After obtaining the values, the values of the two 96-well microtiter plates were corrected with the measured values of the fluorescence intensity obtained by the hybridization of the oligonucleotide H present in both, and the relative fluorescence intensity by the hybridization of each combination was obtained. The values were as shown in the table of FIG.

  As is apparent from the image of FIG. 7, the combination of oligonucleotide B and oligonucleotide G, oligonucleotide C and oligonucleotide H, and combination of oligonucleotide D and oligonucleotide I, which are combinations of oligonucleotides complementary to each other, is the same. A strong fluorescence signal can be detected only at the site in the well, and the fluorescence signal of the combination of non-complementary oligonucleotides, wells containing only one oligonucleotide, and wells containing no oligonucleotide are very Only low was detected. Further, as shown in the table of FIG. 7, similar results were obtained when the relative fluorescence intensity was digitized. Therefore, these 10 kinds of oligonucleotides are unique only in the combination of oligonucleotide B and oligonucleotide G, oligonucleotide C and oligonucleotide H, and oligonucleotide D and oligonucleotide I whose complementarity is confirmed by the base sequence. It was shown that hybridization can be detected.

  Therefore, using these oligonucleotides, a model system of DNA contained in two types of biological populations as shown in FIG. That is, as shown in FIG. 8, assuming that the total DNA 71 of the biological population (1) and the total DNA 72 of the biological population (2) are assumed, the oligonucleotide A and the oligonucleotide F are converted into non-common DNA 73, oligos of the biological population (1). Nucleotide B and oligonucleotide G are the specific DNA 74 of the biological population (1), oligonucleotide C and oligonucleotide H are the interspecies common DNA 75 of the biological populations (1) and (2), oligonucleotide D and oligonucleotide I are the biological population. Specific DNA 76, oligonucleotide E and oligonucleotide J of (2) were assumed to be non-common DNA 77 of the biological population (2). Oligonucleotide A and oligonucleotide F are both non-common DNA 73 of the biological population (1), but can be assumed to be DNA sites having different sequences because the individuals from which they are derived are different. Similarly, oligonucleotide E and oligonucleotide J can also be assumed to be DNA sites of non-common DNA 77 from different individuals of the biological population (2). In FIG. 8, oligonucleotides having complementary sequences are connected by =. According to this model system, the interspecies common DNA 75 of the biological population (1) and the biological population (2), the specific DNA 74 of the biological population (1), the specific DNA 76 of the biological population (2), the biological population (1) An experiment was conducted to detect non-common DNA 73 and non-common DNA 77 of the biological population (2) using hybridization according to the method of the present invention.

  First, equal amounts of 3 μM aqueous solutions of oligonucleotides A, B, and C were mixed to prepare a 1 μM aqueous solution of a mixture of DNA fragments obtained from one individual belonging to the biological population (1). Similarly, 3 μM aqueous solutions of oligonucleotides C, D, and E were mixed in equal amounts to prepare a 1 μM aqueous solution of a mixture of DNA fragments obtained from one individual belonging to the biological population (2).

Next, oligonucleotides F and G as DNA fragments derived from the biological population (1), oligonucleotides I and J as DNA fragments derived from the biological population (2), commonly derived from the biological populations (1) and (2) As the DNA fragments to be prepared, 5 kinds of oligonucleotides of oligonucleotide H are placed in each corresponding well of 96-well microtiter plate 78 (Nippon Genetics Multi Semi-skirted PCR Plate 35801) according to the arrangement shown in FIG. Each 22 μl was dispensed. That is, the well portion 64 of the 96-well microtiter plate into which the oligonucleotide F solution was dispensed, the well portion 65 of the 96-well microtiter plate into which the oligonucleotide G solution was dispensed, and the oligonucleotide H solution were dispensed. 96 well microtiter plate well portion 66, 96 well microtiter plate well portion 68 dispensed with oligonucleotide I solution, and 96 well microtiter plate well dispensed with oligonucleotide J solution 22 μl of a 1 μM aqueous solution of the corresponding oligonucleotide was dispensed into each well of each two rows of the portion 69.

  In this 96-well microtiter plate 78 of this experiment placed horizontally, according to the arrangement shown in FIG. 9, the organism population (1 22 μl of a 1 μM aqueous solution of a mixture of DNA fragments obtained from one individual belonging to 1) was dispensed into each well. Furthermore, in each well 80 in the third and seventh tiers, 1 μM aqueous solution of a DNA fragment mixture prepared from one individual belonging to the biological population (2) previously prepared, 22 μl in each well. Dispensed. Of the wells of the 96-well microtiter plate 78 of this experiment, 22 μl of distilled water is used for wells that dispense only one kind of oligonucleotide or mixed oligonucleotide, and wells that do not dispense either. Were each dispensed with 44 μl of distilled water.

  Next, dispense 5.5 μl of x10TE (100 mM Tris-HCl, 10 mM EDTA, pH 8.0) into all wells of the 96-well microtiter plate 78 of this experiment, and pipette well into each well. Mixed. Cover the 96-well microtiter plate 78 of this experiment with an aluminum foil seal (As One aluminum tape AH-132) and heat-press the microtiter plate tightly so that no solution or vapor is exchanged between the wells. It was set in a thermal cycler (Applied Biosystems Japan Co., Ltd. Gene Amp PCR System 9700), heated at 96 ° C. for 3 minutes, and then cooled to 25 ° C. over 17 minutes to allow oligonucleotide hybridization. The plate was thoroughly wiped and 45 μl was taken from each well of the 96-well microtiter plate 78 of this experiment and transferred to a 96-well flat-bottom microtiter plate (Nunc 96-well optical bottom plate 265301) in the same arrangement. . Dispense 5 μl of a 20,000-fold diluted solution of double-stranded DNA detection reagent (SYBR Green I, TaKaRa F0513) into all wells of a 96-well flat-bottom microtiter plate to which the solution has been transferred, and pipette well into each well. After mixing the solution, it was covered with an aluminum foil seal (As One Aluminum Tape AH-132) and firmly heat-pressed so that the solution and vapor were not exchanged between the wells. The flat bottom microtiter plate was stored at 37 ° C. for 15 minutes in the dark.

  The flat-bottomed microtiter plate was removed, and the fluorescence intensity was measured with a fluoro image analyzer (Fuji Film Co., Ltd. FLA-5000) (filter: LPB, excitation wavelength: 473 nm, pmt value: 400 V), and an image was taken. . Among these, FIG. 10 shows images of 4 wells obtained by hybridization of the same combination of oligonucleotides. In addition, the arithmetic average of the measured values of wells in which the oligonucleotides of the same combination were hybridized was obtained, and the value obtained by subtracting the arithmetic average of the measured values of the wells containing no oligonucleotide from the value as the background was obtained. The obtained relative fluorescence intensity values are as shown in the table of FIG. From both images and numerical values, strong fluorescence is obtained only in the case of each mixture of oligonucleotide (A + B + C) and oligonucleotide G and oligonucleotide H, oligonucleotide (C + D + E) and oligonucleotide H and oligonucleotide I. Signals could be detected and other non-complementary oligonucleotide combinations, wells containing only one oligonucleotide, and wells containing no oligonucleotide were detected only very low. From the above results, the mixed solution of oligonucleotide (A + B + C), that is, the total DNA of the biological population (1) and the mixed solution of oligonucleotide (C + D + E), that is, the total DNA of the biological population (2), were converted into the biological populations (1) and (2 The oligonucleotide H that hybridizes to both the DNA of the biological population (1) and the DNA of the biological population (2) and emits a fluorescent signal by hybridizing with individual DNA derived from An oligonucleotide G that hybridizes to the DNA of the biological population (1) and emits a fluorescent signal but does not hybridize to the DNA of the biological population (2) and does not emit a fluorescent signal; The DNA of the biological population (1) does not hybridize and does not emit a fluorescent signal, but the DN of the biological population (2) The oligonucleotide I that hybridizes and emits a fluorescent signal is the specific DNA of the biological population (2), the DNA from the individual of the biological population (1), but any DNA of the biological populations (1) and (2) Oligonucleotide F, which does not hybridize and does not emit a fluorescent signal, hybridizes to non-common DNA of the biological population (1), DNA derived from individuals of the biological population (2), but to any DNA of the biological populations (1) and (2). Oligonucleotide F that does not soy and emits no fluorescence signal could be detected as non-common DNA of the biological population (2).

It is the figure which showed the view which calculates | requires the specific DNA of each fish species required for the species discrimination of the fish species A and the fish species B. It is the figure which showed the procedure of the experiment which calculates | requires the specific DNA of SBT in order to perform the species discrimination of SBT and yellowfin tuna. It is the figure which showed the flow which produces | generates the DNA microarray for a seed discrimination by analyzing the result obtained in the experiment in order to perform the seed discrimination of SBT and yellowfin tuna. It is the figure which showed the outline of the procedure which analyzes the time-dependent change of the microbial ecosystem in a specific water area using the DNA of the whole microorganisms which inhabit a specific water area. It is the figure which showed the base sequence and complementarity of each oligonucleotide used for the test | inspection of an Example. It is the arrangement | positioning drawing of each DNA of the experiment which confirms the complementarity and specificity of each oligonucleotide used for the test | inspection of an Example. It is a figure of the experimental result which confirmed the complementarity and specificity of each oligonucleotide used for the test | inspection of an Example. It is the figure which showed the state which distributed each oligonucleotide used for the test | inspection of an Example as a model system according to the classification | category of FIG.1 (g). It is an arrangement | positioning figure of each DNA in the experiment of the model system of an Example. It is the figure which showed the experimental result of the model system of an Example. It is the figure which showed the comparison with the method of this invention and the method of this invention which calculates | requires common DNA of the fish species C.

Explanation of symbols

1 ... Individual a of fish species A, 2 ... Total DNA of individual a, 3 ... Non-common DNA of individual a, 4 ... Common DNA of fish species A, 5 ... Fish species A Individual b, 6 ... total DNA of individual b, 7 ... non-common DNA of individual b, 8 ... total DNA of fish species A, 9 ... aggregation of non-common DNA in fish species A, 10 ... Individual c of fish species B, 11 ... Total DNA of individual c, 12 ... Non-common DNA of individual c, 13 ... Common DNA of fish species B, 14 ... Fish species B Individual d, 15 ... total DNA of individual d, 16 ... non-common DNA of individual d, 17 ... total DNA of fish species B, 18 ... set of non-common DNA in fish species B, 19: DNA common to specific species of fish species A and B, 20 ... specific DNA of fish species A, 21 ... specific DNA of fish species B, 22 ... hybridase 23 ... a group of multiple SBT, 24 ... a tissue of the same portion of SBT collected in equal amounts, 25 ... a container for SBT, 26 ... a DNA mixture of SBT, 27 ... SBT DNA fragment mixture, 28 ... agar medium, 29 ... SBT colony or plaque, 30 ... SBT DNA microarray, 31 ... SBT fluorescently labeled DNA fragment mixture, 32. .. SBT DNA microarray hybridized with SBT fluorescently labeled DNA fragment mixture, 33 ... SBT common DNA clones, 34 ... SBT non-common DNA clones, 35 ... Multiple yellowfin tuna populations, 36 ... Same amount of yellowfin tuna collected in equal amounts Partial tissue, 37 ... Container for yellowfin tuna, 38 ... DNA mixture of yellowfin tuna, 39 ... DNA fragment mixture of yellowfin tuna, 40 ... Fluorescently labeled DNA fragment mixture of yellowfin tuna, 41 ... Yellowfin tuna Southern bluefin tuna DNA microarray hybridized with fluorescently labeled DNA fragment mixture, 42 ... yellowfin tuna common DNA clone, 43 ... yellowfin tuna non-common DNA clone, 44 ... southern bluefin tuna specific DNA clone, 45 ... Scan line, 46 ... Signal scan graph of SBT, 47 ... Signal scan graph of yellowfin tuna, 48 ... Comparison table, 49 ... DNA microarray for species discrimination, 50 ... Time point 1 of specific water area A group of water and microorganisms contained in it, 51 ... special Mixture of DNA fragments of whole microorganism at time point 1 in constant water area, 52... DNA microarray at time point 1 of specific water area, 53 ... Time point of specific water area hybridized with DNA fragment mixture of whole microorganism at time point 1 of specific water area 1 DNA microarray, 54... Water at a time point 2 in a specific water area and a group of microorganisms contained in the water, 55... DNA fragment mixture of all microorganisms at a time point 2 in a specific water area, 56. DNA microarray at time point 1 in a specific water region hybridized with a mixture of DNA fragments of all microorganisms, 57 ... Oligobase sequence and characteristics table, 58 ... First 96-well microtiter plate, 59 ... A well portion of a 96-well microtiter plate into which the oligonucleotide A solution was dispensed, 60... The oligonucleotide B solution was dispensed 96 Well part of a well of a micro-titer plate, 61... Well part of a 96-well micro-titer plate into which a solution of oligonucleotide C is dispensed, 62... 96-well micro-titer plate with a solution of oligonucleotide D dispensed , 63 ... well part of 96-well microtiter plate dispensed with oligonucleotide E solution, 64 ... well part of 96-well microtiter plate dispensed with oligonucleotide F solution 65 ... well part of 96-well microtiter plate dispensed with oligonucleotide G solution, 66 ... well part of 96-well microtiter plate dispensed with oligonucleotide H solution, 67 ..・ Second 96-well microtiter plate, 68 ... Dispensing oligonucleotide I solution Well part of 96-well microtiter plate, 69 ... well part of 96-well microtiter plate into which oligonucleotide J solution was dispensed, 70 ... for confirmation of complementarity and specificity of oligonucleotide Table of experimental results of 71, 71 ... total DNA of biological population (1), 72 ... total DNA of biological population (2), 73 ... non-common DNA of biological population (1), 74 ... Specific DNA of biological population (1), 75... Common DNA between biological population (1) and biological population (2), 76... Specific DNA of biological population (2), biological population (2) Non-common DNA, 78 ... 96-well microtiter plate for model experiments, 79 ... well part of 96-well microtiter plate into which a mixed solution of oligonucleotides A, B, and C is dispensed, ..Oligonule Well part of 96-well microtiter plate into which a mixed solution of tides C, D, and E was dispensed, 81 ... Table of experimental results of model system, 82 ... Individual a of fish species C, 83 ... Container, 84 ... Tissue collected from individual a, 85 ... DNA of individual a, 86 ... DNA fragment mixture of individual a, 87 ... Experimental result of microarray, 88 ... Experimental result of microarray 89 ... Fish species C overall result, 90 ... Fish species C population, 91 ... Common container for fish species C, 92 ... Fish species C tissue mixture, 93 ... DNA mixture of fish species C, 94 ... DNA fragment mixture of fish species C

Claims (24)

  A method for detecting a DNA fragment commonly present in a population of organisms, the step of obtaining a mixture of DNA fragments from a plurality of individuals belonging to the population of organisms, taking a part of the mixture of DNA fragments, and in the mixture Isolating individual DNA fragments contained, hybridizing the mixture of DNA fragments with the isolated individual DNA fragments, and measuring the amount of the hybridized and bound DNA fragments. A method for detecting a common DNA fragment.   2. A mixture of DNA fragments is obtained by collecting similar tissues from each of a plurality of individuals, mixing them by the same weight, extracting DNA from the mixed tissues, and then fragmenting the same. The method for detecting a common DNA fragment according to claim 1.   The mixture of DNA fragments is obtained by collecting cells from each of a plurality of individuals, mixing them in equal amounts, extracting DNA from the mixed cells, and then fragmenting the cells. A common DNA fragment detection method.   The common DNA according to claim 1, wherein a mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, mixing the extracted DNA in the same amount, and fragmenting the mixed DNA. Fragment detection method.   2. The common DNA according to claim 1, wherein the mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, fragmenting the DNA by the same technique, and mixing the fragmented DNA by the same amount. Fragment detection method.   The method for detecting a common DNA fragment according to any one of claims 1 to 5, wherein the chain length of the DNA fragment is in the range of 10 to 10,000 bases.   The method for detecting a common DNA fragment according to any one of claims 1 to 6, wherein the mixture of DNA fragments is labeled.   The method for detecting a common DNA fragment according to any one of claims 1 to 6, wherein each isolated DNA fragment is labeled.   The double-stranded DNA fragment formed by hybridization of a mixture of DNA fragments and an isolated individual DNA fragment is specifically labeled, according to any one of claims 1 to 6, A method for detecting a DNA fragment.   A method for detecting a specific DNA fragment that is common to a certain biological population and does not exist in another biological population, comprising a plurality of individuals belonging to the certain biological population and a plurality of individuals belonging to the different biological population A step of obtaining a mixture of DNA fragments by the same method from each of the above, taking a part of the mixture of DNA fragments obtained from a plurality of individuals belonging to the certain biological population, and individually singling individual DNA fragments contained in the mixture. Separating the mixture of the DNA fragments obtained from the one biological population with the isolated individual DNA fragments, the mixture of the DNA fragments obtained from the other biological population, and the isolated individual fragments. A method for detecting a specific DNA fragment, comprising a step of hybridizing with a DNA fragment and a step of measuring the amount of DNA hybridized and bound.   The mixture of DNA fragments is obtained by collecting similar tissues from each of a plurality of individuals, mixing them by the same weight, extracting DNA from the mixed tissues, and then fragmenting the same. A method for detecting a specific DNA fragment according to claim 1.   The mixture of DNA fragments is obtained by collecting cells from each of a plurality of individuals, mixing them in equal amounts, extracting DNA from the mixed cells, and then fragmenting the cells. A method for detecting a specific DNA fragment.   The specific mixture according to claim 10, wherein the mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, mixing the extracted DNA in the same amount, and fragmenting the mixed DNA. A method for detecting a DNA fragment.   11. The specific mixture according to claim 10, wherein the mixture of DNA fragments is obtained by extracting DNA from each of a plurality of individuals, fragmenting the DNA by the same method, and mixing the fragmented DNA by the same amount. A method for detecting a DNA fragment.   The method for detecting a specific DNA fragment according to any one of claims 10 to 14, wherein the chain length of the DNA fragment is in the range of 10 to 10,000 bases.   The method for detecting a specific DNA fragment according to any one of claims 10 to 15, wherein the mixture of DNA fragments is labeled.   The method for detecting a specific DNA fragment according to any one of claims 10 to 15, wherein each isolated DNA fragment is labeled.   The specificity according to any one of claims 10 to 15, characterized in that a double-stranded DNA fragment formed by hybridization of a mixture of DNA fragments and an isolated individual DNA fragment is specifically labeled. Of detecting a typical DNA fragment.   A method for detecting a common DNA fragment between specific populations common to a plurality of specific organism populations, the same from each of a plurality of individuals belonging to the one organism population and a plurality of individuals belonging to the other organism population A step of obtaining a mixture of DNA fragments by the method of, taking a part of a mixture of DNA fragments obtained from a plurality of individuals belonging to the certain biological population, and isolating individual DNA fragments contained in the mixture, Mixing a mixture of DNA fragments obtained from one organism population and a mixture of DNA fragments obtained from another organism population, and then hybridizing the mixture to individual DNA fragments isolated; A method for detecting a common DNA fragment between specific populations, which comprises a step of measuring the amount of the DNA.   A DNA identification method, wherein DNA identification is performed using a DNA fragment obtained by the detection method according to any one of claims 1 to 19. A method for detecting a change in DNA of a microorganism present in a specific environment, collecting a group of microorganisms contained in a certain volume of the specific environment at a certain point in time, extracting DNA from the group of microorganisms, then fragmenting, Preparing a first time point DNA fragment mixture, taking a portion of the first time point DNA fragment mixture and isolating individual DNA fragments contained in the mixture, after collecting the first time point microbial population, Collect a group of microorganisms at the second time point included in the same volume of the specific environment at a time point different from the first time point, extract DNA from the group of microorganisms, and then perform fragmentation using the same method as the DNA fragment mixture at the first time point. Preparing a DNA fragment mixture at the second time point, hybridizing the DNA fragment mixture at the first time point with individual DNA fragments contained in the mixture, and DNA fragmentation at the second time point. A step of hybridizing the mixture with individual DNA fragments contained in the DNA fragment mixture at the first time point, a step of measuring the amount of hybridized and bound DNA, and an individual contained in the DNA fragment mixture at the first time point And a step of comparing the amount of bound DNA when hybridized with the DNA fragment mixture at the first time point and when hybridized with the DNA fragment mixture at the second time point. A method for detecting changes in DNA.   The method for detecting a change in microbial DNA according to claim 21, wherein the chain length of the DNA fragment is in the range of 10 to 10,000 bases.   The method for detecting a change in microbial DNA according to claim 21 or 22, wherein the mixture of the DNA fragments at the first time point and the mixture of the DNA fragments at the second time point are labeled.   The method for detecting a change in microbial DNA according to claim 21 or 22, wherein each DNA fragment contained in the mixture of DNA fragments at the first time point is labeled.