JP6646120B1 - DNA identification method capable of individual identification with degraded DNA - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify a DNA marker which is indistinguishable by a current DNA identification method but has a different base sequence even if the chain length is the same, identifies a DNA marker using a small amount of DNA, and identifies a DNA marker using degraded DNA. It is an object of the present invention to determine whether a DNA marker derived from the same individual is contained is used to determine the match or mismatch of a DNA marker with a base sequence, and to provide the same as a judgment material for DNA identification. [MEANS FOR SOLVING PROBLEMS] The present invention is characterized in that the base sequences of DNA markers determined by a DNA sequencer are compared between samples, and a match or mismatch of the DNA markers is determined based on the base sequences. [Selection diagram] FIG.

Description

  TECHNICAL FIELD The present invention relates to a DNA identification method that allows individual identification using degraded DNA.

  In the DNA test used by police, a region containing a short tandem repeat (STR) having individual differences in the number of repetitions is selected and used as a DNA marker for individual identification. By using (locus), the detection rate of individual differences is increased. The locus region and number used for this purpose vary depending on the region. Currently, in Japan, 16 loci amplified by the AmpFlSTR Identifiler PCR Amplification Kit (Thermo Fisher Scientific) are used (Non-Patent Document 1). It is said that the GlobalFiler PCR Amplification Kit (Thermo Fisher Scientific) using 24 loci will be changed soon (Non-Patent Document 2).

  As a specific test operation, DNA amplification is performed by PCR using a unique primer set for each locus of a DNA marker of DNA extracted from a sample, and electrophoresis of the amplified product is performed. As a result, the identification is performed by comparing the pattern of the DNA marker in which the number of types of the amplified DNA fragments and the relative migration distance are matched for each locus.

  Normally, the human genome is diploid, with one locus having two alleles (alleles), each of which is independently derived from the father and mother. In the same locus, when the number of repetitions of the STR of the allele derived from each of the parents is different, the STR analysis detects DNA fragments of two types of chain length. In the same locus, when the number of repetitions of the STR of the allele derived from each parent is the same, a DNA fragment having only one chain length is detected by the STR analysis.

  Considering the above facts, for example, if the pattern of the suspect's DNA marker and the pattern of the offender's DNA marker are the same in all loci, it is determined that the probability that the suspect is the same as the offender is high.

  In order to perform a highly accurate DNA test, it is necessary to secure a sufficient number and type of DNA markers to ensure the detection sensitivity and accuracy of the test. There is a case where it is difficult to secure a sufficient amount of a sample for performing a test, or a case where only a sample in a state of deterioration due to environmental conditions is obtained.

AmpFlSTR Identifiler PCR Amplification Kit User Guide, Thermo Fisher Scientific GlobalFiler PCR Amplification Kit User Guide, Thermo Fisher Scientific

  Current methods for discriminating the pattern of the DNA marker based on the results of electrophoresis include a gel electrophoresis method which is identified by the ladder pattern of the band in FIG. 1 and a capillary electrophoresis method which is identified by the waveform pattern in FIG. Both methods use the difference in the chain length of DNA fragments to separate bands and waveforms in the same sample and identify the generated pattern. If they are the same, they are identified as the same band or waveform, so that there is a problem that the difference cannot be detected.

  Also, in poor environments, such as those often encountered in criminal investigations, DNA is cut at random locations and gradually decomposed into shorter DNA fragments. Even if the DNA in such a state is PCR-amplified by the current method, the pattern of the DNA marker cannot be detected.

The reason will be described below.
First, one allele of locus 1 of a DNA marker having a structure as shown in FIG.

  When the DNA marker loci 1 was amplified by PCR using undegraded DNA as a template as shown in FIG. 3 (2), the entire loci region containing the base sequences of the two primers 1 and 2 set at both ends of the loci region was obtained. The DNA strands in both directions (fragment 1a1 and fragment 1b1) are logarithmically grown, and if there is a sufficient amount of the original DNA serving as a template, an amount of double-stranded DNA fragment capable of detecting a band or waveform by electrophoresis is generated. . Similarly, for the other allele and all other loci, if the amount of the original DNA serving as the template is sufficient, an amount of double-stranded DNA fragment capable of detecting a band or waveform by electrophoresis is generated, and all loci Can be determined.

  However, when PCR is performed using DNA degraded in a poor environment or the like as a template as shown in FIG. 3 (3), the resulting allele DNA fragment group of the locus is an example of the locus 1 in FIG. As shown in the figure, a mixture of various DNA fragments of the following 1) to 3) is obtained. The thickness of each fragment in the figure represents the amount of each fragment after the PCR reaction as an image.

  1) The DNA of allele 1 of locus 1 which has two primer sequences at both ends and whose DNA is not degraded is usually a very small number of DNAs extracted from a deteriorated sample, and even if they are logarithmically grown, However, the amount of DNA fragments that can be clearly distinguished by bands or waveforms cannot be obtained.

  2) The positions of cleavage due to DNA degradation are usually random and differ for each DNA molecule as shown by the degraded DNA (1) and DNA (2) in FIG. Among these DNA fragments, single-stranded DNA fragments (fragment 1a1 and fragment 1c2) containing any one of the base sequences of the two primers undergo a DNA extension reaction using their complementary strand as a template. Therefore, the number of those single-stranded DNA fragments increases in proportion to the number of cycles of PCR. As a result, as a PCR product of a single-stranded DNA fragment containing any one of the base sequences of the two primers, a single-stranded DNA fragment having a different chain length increased by the number of PCR cycles depends on the cutting position of each template DNA. A mixture of strand DNA fragments (fragment 1a1 and fragment 1c2) is obtained. However, the amount of each single-stranded fragment is extremely small, and the single-stranded chain has various three-dimensional structures, so that the electrophoresis distance is not constant even with the same chain length. As a result, none of the single-stranded DNA fragments can be clearly identified by bands or waveforms.

  3) Since the DNA fragment cut so as to have both ends inside the primer sequence of the locus of the DNA marker has no primer binding sequence, the number of fragments does not increase at all and cannot be detected as a band or a waveform.

  Similarly, a mixture of various DNA fragments is produced for another allele and all other loci.

  When multiplex PCR of all loci is performed using DNA extracted from one sample as a template, DNA fragment groups having various chain lengths, which are a mixture of the PCR products of all loci, are generated. When this was electrophoresed, no clear band was recognized in gel electrophoresis, and a smear image with a density corresponding to the amount of DNA was obtained, or in capillary electrophoresis, an unanalyzable pattern in which many weak signals overlapped was observed. This results in a problem in that DNA analysis cannot be performed.

  In the present invention, in order to solve these problems, a DNA base sequence of DNA is read, a DNA marker having the same DNA chain length and a different base sequence is identified, and a DNA marker having only a trace amount of DNA is identified, and degraded DNA is identified. In order to perform DNA identification by identifying a DNA marker by using a DNA marker, it is characterized by determining whether the same DNA exists in the target DNA and the sample DNA.

  In addition, since DNA sequencers have made remarkable technical progress in recent years and can acquire a large amount of nucleotide sequence data from a very small amount of DNA, using such equipment to acquire the nucleotide sequence data of DNA markers has And DNA markers can be identified.

  In the present invention, the degraded DNA and a DNA sequencer capable of reading a large amount of base sequence in one experiment are used.

  By comparing the base sequences of the obtained DNA markers among the samples and performing DNA identification, it is possible to identify DNA marker fragments having the same chain length but different base sequences.

  Further, even when the degraded DNA is used as a sample, the partial DNA of the DNA fragment cleaved in the middle of the locus is compared and analyzed by comparing the partial DNA chain length and / or the base sequence between the samples. It is characterized in that DNA markers can be identified.

  By preparing a list of DNA markers for each sample and each locus obtained as described above and a list summarizing the results of their mutual comparison, the same DNA that cannot be detected and identified by the current DNA identification method is used. DNA marker analysis that is useful for identifying individuals with DNA marker base sequences that differ in their chain lengths, for individuals with extremely small amounts of DNA markers, and for individuals with only degraded DNA with DNA markers It is characterized by being able to provide information.

  In addition, since a human is a diploid organism as described above, if a DNA marker containing three or more nucleotide sequences is detected in a criminal specimen, for example, the specimen may be a mixture derived from a plurality of individuals. Come out.

  Although it cannot be denied that there are human individuals having three alleles in some loci such as trisomy individuals, since multiple loci of DNA markers for human DNA identification are not usually triploid, When three or more subgroups are detected in a plurality of loci, the possibility that DNAs from a plurality of individuals are mixed in the sample specimen can be more strongly indicated.

  On the other hand, in the case of an individual in which the homologues of two bases of alleles are exactly the same for a specific DNA marker, only one base sequence is detected for the DNA marker. Therefore, for example, in a sample in which DNAs derived from two individuals are mixed, when two individuals are homozygous for the DNA marker and the two individuals have different base sequences, In the case where the individual is a homozygote different from the DNA marker, and the third individual is a heterozygote having a nucleotide sequence corresponding to the nucleotide sequence of each of the two individuals, etc., the subordinate belonging to the locus of the DNA marker is used. There may be two groups. Therefore, even if there are two subgroups, the specimen may contain DNAs derived from a plurality of individuals.

  Thus, when there are two subgroups of the DNA marker, it is difficult to distinguish whether the specimen contains only DNA from one individual or a mixture of DNAs from multiple individuals. However, by using a plurality of DNA markers for identification, a DNA marker containing three or more nucleotide sequences may be detected by other DNA markers, and depending on the result, DNAs derived from a plurality of individuals may be mixed. Sex can be presented.

  Therefore, by using the list of DNA markers for each sample and each locus and the list summarizing the mutual comparison results, identification of individuals not only between samples but also in the same sample, that is, identification of individuals in the same sample from a plurality of individuals Provides information to determine whether or not two samples (eg, a suspect (group) sample and a criminal (group) sample) contain DNA from the same individual. It also features that it can be done.

The present invention includes the following [1] to [4].
[1] A method for determining whether a target DNA and a sample DNA are DNAs derived from the same individual, which comprises the following steps:
(1) determining the base sequence of the DNA fragment of the target DNA,
(2) extracting a DNA fragment containing a DNA marker or a partial base sequence thereof from the DNA fragment whose base sequence has been determined in step (1);
(3) a step of dividing the DNA fragments extracted in the step (2) into groups for each identical locus (locus groups) based on the nucleotide sequence;
(4) For each locus group divided in step (3), DNA fragments having the same base sequence in the locus group are divided into subgroups, and the longest DNA fragment among the subgroups is a subgroup representative fragment. The process of selecting as
(5) determining the base sequence of the DNA fragment of the sample DNA;
(6) a step of extracting a DNA fragment containing a DNA marker or a partial base sequence thereof from the DNA fragment whose base sequence has been determined in step (5),
(7) a step of dividing the DNA fragments extracted in the step (6) into groups (locus groups) for each same locus based on the nucleotide sequence;
(8) For each locus group divided in step (7), DNA fragments having a common base sequence in the locus group are divided into subgroups, and the longest DNA fragment among the subgroups is a subgroup representative fragment. The process of selecting as
(9) comparing the subgroup representative fragment of the target DNA selected in step (4) with the subgroup representative fragment of the sample DNA selected in step (8) for each locus;
(10) A step of determining, for each locus, whether the same DNA exists in the target DNA and the sample DNA from the result of the comparison in the step (9).

[2] In the step (9), the base sequences of the subgroup representative fragment of the target DNA selected in the step (4) and the subgroup representative fragment of the sample DNA selected in the step (8) are calculated on a per subgroup basis. The DNA identification method according to [1], wherein the method is performed for each locus.

[3] The subgroup representative fragment according to [2], wherein in step (9), the subgroup representative fragments are compared based on the base sequence of the entire length of the shorter DNA fragment of the two subgroup representative fragments. DNA identification method.

[4] In steps (4) and (8), DNA fragments in the locus are divided into subgroups for each identical base sequence, so that when the number of subgroups is 2 or more, DNAs of a plurality of individuals are mixed. The DNA identification method according to [1], which can determine that there is a possibility.

  According to the method of the present invention configured as described above, 1) identification of DNA markers having the same base length but different base sequences, 2) identification of DNA markers using a small amount of DNA, and 3) degraded DNA 4) Provision of data for detection of the mixture of multiple individuals in the same sample, and 5) Provision of data for identification of whether two samples contain DNA from the same individual Becomes possible.

The figure which shows the example of the gel electrophoresis result of the DNA which has not been decomposed | disassembled. The figure which shows the example of the result of the capillary electrophoresis of the DNA which is not decomposed | disassembled. The figure which shows the fragment of a DNA marker. The figure which shows the PCR product of the locus of the decomposed DNA marker. The figure which shows the flow of the base sequence comparison based on the large amount base sequence data of a DNA marker. The figure which shows the example of the base sequence group which belongs to the locus subgroup 1 among the base sequences containing the locus 1 of the DNA extracted from the test substance, its common part, and the longest base sequence which has a common part. The figure which shows the flow of creation of the database of all marker fragment base sequence groups of the decomposed DNA. The figure which shows the selection to a DNA marker base sequence group from all marker fragment base sequence group databases. The figure which shows the selection flow to the DNA marker base sequence group database for every sample. The figure which shows the comparative analysis of the DNA marker base sequence group of STR. Comparative example schematic diagram of target and sample with PCR The figure which shows the flow (without PCR) of the database containing the marker-containing fragment base sequence group of the degraded DNA. The figure which shows selection (without PCR) from a marker containing fragment base sequence group database of a sample DNA to a DNA marker base sequence group. The figure which shows the selection flow (no PCR) to the DNA marker base sequence group database for each sample. The figure which shows the comparative analysis (no PCR) of a DNA marker base sequence group.

Hereinafter, the present invention will be described in detail with reference to FIG.
The DNA identification method of the present invention can be performed based on a DNA marker comparison table. This DNA marker comparison table shows the results of comparison of the patterns of both DNA markers in order to determine whether the target DNA and the sample DNA contain DNA derived from the same individual. It can be prepared by a method including steps (A) to (L).

  In the step (A), a base sequence group including a locus of a DNA marker of the target DNA is determined using a DNA sequencer, and a database in which all of them are registered is created. As a preparation step before this step, a larger amount of base sequence using a DNA sequencer, such as a step of amplifying a sample DNA by a PCR reaction or a step of adding an adapter to single-stranded DNA by Adaptase technology (Swift Bioscience), etc. In some cases, a step for obtaining data with high accuracy is added.

  The DNA marker is not particularly limited. For example, the DNA marker may include STR, or may not include a repetitive sequence such as STR. Further, the DNA marker may include a sequence in which a difference in base sequence is observed between individuals. Examples of loci of the DNA marker containing STR include 16 loci amplified by AmpFlSTR Identifiler PCR Amplification Kit (manufactured by Applied Biosystems).

  The locus of the DNA marker to be assessed may be only one, but is preferably two or more. When the number of loci of the DNA marker is two or more, it is preferable that setting for performing multiplex PCR is possible.

  The method for determining the base sequence is not particularly limited, but it is preferable to use a next-generation DNA sequencer or a next-generation DNA sequencer capable of obtaining a large amount of base sequence data in one experiment.

  In the step (B), all the nucleotide sequences including the DNA marker and / or a partial nucleotide sequence thereof are extracted from the nucleotide sequence group registered in the step (A).

  In the step (C), the base sequence extracted in the step (B) is divided into groups for each locus (locus groups).

  In the step (D), in each of the locus groups created in the step (C), a base group (locus subgroup) is divided for each base sequence having a common base sequence.

  In step (E), the longest nucleotide sequence (locus subgroup representative nucleotide sequence) in each of the locus subgroups created in step (D) is selected. The concept of the longest nucleotide sequence in the locus subgroup is as shown in FIG.

  In the step (F), the base sequence including the locus of the DNA marker of the sample DNA is determined using a DNA sequencer, and a database in which all of them are registered is created.

In the step (G), all the nucleotide sequences including the DNA marker and / or a partial nucleotide sequence thereof are extracted from the nucleotide sequence group registered in the step (F).

  In the step (H), the DNA fragments extracted in the step (G) are divided into groups for each locus (locus groups).

  In the step (I), in each of the locus groups created in the step (H), each base sequence having a matching base sequence is divided into subgroups (locus subgroups).

  In step (J), the longest nucleotide sequence (locus subgroup representative nucleotide sequence) in each of the locus subgroups created in step (I) is selected. The concept of the longest nucleotide sequence in the locus subgroup is as shown in FIG.

  In the step (K), the locus representative base sequence selected in the step (E) and the locus subgroup representative base sequence selected in the step (J) are combined with the locus subgroup in the corresponding locus group of the target DNA and the sample DNA. Brute force comparison.

  In the step (L), the comparison results of all the locus subgroup representative base sequences of the target and the sample performed in the step (K) are summarized in a list (Table 2).

  As a result of the comparison, when there is a mismatch between the representative base sequence of the locus subgroup of the target DNA and the representative base sequence of the locus subgroup of the sample DNA in the corresponding locus subgroups of the target DNA and the sample DNA, those locus The subgroup can be determined to be mismatched. The concept of the common part is the same as the concept shown in FIG.

  The determination of the nucleotide sequence match or mismatch may be performed by any method. However, when the locus of the DNA marker contains STR, as shown in Table 1 below, the length and the nucleotide sequence of the DNA fragment are determined. It is preferable to carry out based on this. On the other hand, when the locus of the DNA marker does not contain STR, it is preferable to perform the determination based on the base sequence of the entire chain length of the shorter DNA fragment of the two DNA fragments.

  Steps (A) to (E) relate to a target DNA, and steps (F) to (J) relate to a sample DNA. Steps (A) to (E) and steps (F) to (J) may be performed simultaneously and in parallel, or one may be performed first and the other may be performed later.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

  In many cases, DNA testing in criminal investigations involves a plurality of DNA markers each of DNA extracted from a suspected criminal sample (target DNA) and DNA extracted from a suspect sample (sample DNA). Is performed by comparing the pattern of the chain length and the number of DNA fragments between the target DNA and the sample DNA.

  According to the present invention, as shown in FIG. 5, the whole image of DNA marker comparison data by base sequence comparison is prepared by: 1) the base sequence group of the target DNA fragment read by a DNA sequencer and the base sequence group of the sample DNA fragment; 2) a marker sequence extracting step of extracting the DNA marker and / or a part of the nucleotide sequence from 1), and 3) a group for each locus of the nucleotide sequence selected in 2) In the locus group creating step of creating the loci, and in the respective locus groups created in 3), the step of creating a locus subgroup having a base sequence having a common part, and 5) each of the locus subgroups created in 4) For selecting the longest DNA fragment in a locus subgroup , 6) a comparison step of comparing the loci of the loci subgroups extracted in 5) with respect to the target DNA and the sample DNA for each loci of the DNA marker, and 7) all the loci of the loci of the target DNA and the sample DNA for each loci It consists of a process called a table preparation step of compiling a comparison result of the locus subgroup representative base sequences into a list.

The configuration of the present invention in the case of using DNA amplification of a DNA marker by PCR will be described with reference to FIG. Since this PCR reaction is an extension reaction from one pair of two types of primer sequences located at both ends of the DNA marker, all DNA fragments that increase exponentially or in proportion to the number of PCR cycles are all 5 of them. At the 'end, there is one kind of base sequence of the two kinds of primer sequences or at least a part thereof.
[Example 1] Creation of a DNA marker base sequence group database from base sequence groups of DNA fragments of a target sample and a sample sample read by a DNA sequencer (when PCR is included in the pre-process)

  First, as a preparation step, multiplex PCR is performed on all loci of DNA markers using DNA (target DNA and sample DNA) extracted from two specimens (target and sample) to be subjected to DNA analysis as templates. A DNA marker PCR product group including the locus PCR product is obtained for each of the target DNA and the sample DNA. In addition, in these fragment groups, of the DNA of the target or the sample used as a template for PCR, DNA of a site which was not an object of PCR amplification because it was not any of the locus sites was mixed (FIG. 7). (Corresponds to the upper diagram). For all the DNA fragments of these targets and samples, the nucleotide sequence was determined using a DNA sequencer capable of obtaining a large amount of nucleotide sequence data in one experiment, and all the information was obtained as shown in the middle part of FIG. It is registered in the database as a base sequence group of a DNA marker PCR product for each sample DNA.

  Next, all of these base sequences are compared with the base sequences of all the loci of the DNA marker for DNA identification, and a base sequence corresponding to all or a part of any of the loci is extracted, and the target DNA or sample is extracted. It is registered in the database as a group of all marker fragment base sequences of DNA (corresponding to the lower diagram in FIG. 7).

  Subsequently, as shown in the upper to middle diagrams of FIG. 8, from the registered base sequences, only the base sequences containing either or both of the two types of primer sequences were extracted for each locus of the DNA marker, and Are classified into groups for each locus and each primer, and a database of base sequences for each locus / primer of a target or sample is created.

  Further, for each specimen, within the group of the same locus and the same primer, base sequences belonging to the group are compared with each other, and base sequences having the same base sequence are collected to form a subgroup. The processing in FIG. 8 will be described with reference to FIG.

  (010) In order to start the process from locus 1 of the DNA marker, N = 1 is set as the initial value of the locus number.

  (020) P = 1 is set as the initial value of the primer number in order to process from the base sequence containing the base sequence of the primer number 1.

  (030) From the group of all marker fragment base sequences shown in FIG. 8, all the base sequences in which the base sequence of primer No. 1 exists at locus No. 1 are extracted.

  (040) A name consisting of the locus number N and the primer number P is assigned to the extracted base sequence group. (E.g., 012 if N = 1, P = 2, etc.) The locus number N and the primer number P are according to the locus and primer numbers set in the DNA marker for DNA identification, and if not set, Give them in an easy-to-understand order, such as following the order of positions on the genome. As a result, as shown in the middle diagram of FIG. 8, a database of the base sequence group for each locus / primer for each sample is created.

  (050) For each group NP of the base sequences created in (040), all base sequences belonging to the group are compared with each other, and base sequences having the same common part are collected to form a subgroup. If the specimen contains a single individual, the number of subgroups in the same primer group of the same locus is usually 2 or less, reflecting that human individuals are usually diploid. If the sample contains a plurality of individuals, the number of subgroups in the same primer group of the same locus may be 3 or more.

  (060) It is necessary to distinguish whether the specimen for which the subgroup is created is a target or a sample in a later operation. If the sample being worked on is the target, the process proceeds to (070), and a name that indicates that the sample is a subgroup of the target is given. If the specimen in operation is a sample, the process proceeds to (080), and a name that indicates that the sample is a subgroup of the sample is given.

  (070) When the operation target is the base sequence of the target sample, the subgroup numbers U are assigned to all the subgroups included in the group of the same primers of the same locus created in (050) in order from 1 respectively. Further, a subgroup name TNPU to which T is added first (for example, T0121 if N = 1, P = 2, U = 1) to clearly indicate that the sample is the target is given to each subgroup.

  (080) When the work target is the base sequence of the sample specimen, the subgroup numbers V are assigned to all the subgroups included in the same primer group of the same locus created in (050) in order from 1 respectively. Further, a subgroup name SNPV to which S is first added to clearly indicate that the specimen is a sample (for example, S0121 if N = 1, P = 2, V = 1) is given to each subgroup.

  (090) The subgroups of the nucleotide sequences named in (070) and (080) are moved to the DNA marker nucleotide sequence group database of the corresponding specimen (target or sample) while maintaining the group structure.

  (100) Next, it is necessary to determine whether or not it is necessary to perform the operations (030) to (090) for different primer sequences P of the same locus. Since there are two types of primer sequences in one locus, it is determined whether the current primer number is 1 or 2. In the case of 1, go to (120), change the primer number to P = 2, and repeat the process from (030). In the case of 2, go to (110).

  (110) Usually, there are a plurality of loci of DNA markers for DNA identification, and the above operation is performed for all of them. Therefore, it is necessary to determine the presence or absence of a loci number larger than the current loci number N. If there is a locus number larger than the current locus number N, the flow goes to (130), the setting of the locus number N is changed to N + 1, and the flow returns to (020). If there is no locus number larger than the current locus number N, the operation ends.

  By the processing so far, as shown in the lower diagram of FIG. 8, the base sequence including the primer base sequence of the DNA marker is divided into groups for each locus and each primer sequence, and further, for each subgroup of the same base sequence having the same common part. In this state, it is registered in the database of the DNA marker base sequence group for each sample.

  In addition, the base sequence extracted under the condition of the smaller primer number by the above process is deleted from the database of the original base sequence group by the process of (040). The base sequence of the full-length DNA marker possessed is classified into a group having primer number 1.

  This operation is performed for each of the two samples to be subjected to the DNA test, and two databases of the DNA marker base sequence group of the target sample and the DNA marker base sequence group of the sample sample are created.

  In the present embodiment, the base sequence of the fragment containing the base sequence of any of the primers for each locus was extracted. However, as a method of extracting the base sequence when creating a database of DNA marker base sequence groups of the target specimen or the sample specimen, Is a method for extracting the base sequence of a fragment containing not the entire base sequence of the primer but a part of the base sequence, or a method for extracting the base sequence of a fragment containing the whole or a part of the base sequence for each locus Can be selected, and the present invention is not limited to these methods.

[Example 2] Flow of preparing a comparison table of representative base sequences of DNA markers having STR (when PCR is included in the pre-process)
This processing is performed using the two databases of the DNA marker base sequence group of the target specimen and the DNA marker base sequence group of the sample specimen created above. The processing performed here will be described with reference to FIG.

  (100) In order to start processing from the locus 1 of the DNA marker, N = 1 is set as an initial value of the locus number.

  (200) P = 1 is set as the initial value of the primer number in order to process from the nucleotide sequence in which the nucleotide sequence of the primer 1 exists.

  (300) In order to start processing from the target subgroup 1, U = 1 is set as an initial value of the subgroup number.

  (400) A group of DNA marker nucleotide sequences of subgroup number U is selected from a database of target DNA marker nucleotide sequence groups.

  (500) The selected subgroups of DNA marker base sequences are DNA marker base sequences of various chain lengths having the same base sequence at the 5 ′ end beginning with the base sequence P of the primer and having the same base sequence at the common part. Group. DNA chain lengths are compared with each other in this group, and the longest DNA marker base sequence is defined as a locus subgroup representative base sequence TRNPU. (For example, TR0112)

  (600) In order to start processing from the subgroup 1 of the sample, V = 1 is set as an initial value of the subgroup number.

  (700) A group of DNA marker nucleotide sequences of subgroup number V is selected from the database of DNA marker nucleotide sequence groups of the sample.

  (800) The subgroups of the selected DNA marker nucleotide sequences include DNA marker nucleotide sequences having the same nucleotide sequence whose 5 ′ end starts with the nucleotide sequence P of the primer and having the same nucleotide sequence at a common portion. Group. The DNA chain lengths in this group are compared with each other, and the longest DNA marker base sequence is defined as a locus subgroup representative base sequence SRNPV. (For example, SR0212)

(900) The nucleotide sequences of TRNPU and SRNPV are compared as shown in the table below. The base sequence names N and P to be compared always match between the target and the sample.

  Since it is necessary to perform the steps (700) to (900) for all subgroups in the group of the same locus and the same primer sequence of the (1000) sample, other subgroups in the group of the same locus and the same primer sequence are required. Determine if there are any subgroups. If there is another subgroup, the procedure goes to (1500) to change the subgroup number V of the sample to V + 1, and repeat the operations of (700) to (900). If there are no other subgroups, go to (1200).

  (1100) It is necessary to perform the steps (400) to (1000) for all subgroups in the same target locus and the same primer sequence group. It is determined whether there is a subgroup. If there is another subgroup, go to (1600), change the subgroup number U of the target to U + 1, and repeat the operations of (400) to (1000). If there is no other subgroup, go to (1200).

  (1200) Next, it is necessary to determine whether or not it is necessary to perform the operations (300) to (1100) for different primer sequences P of the same locus. Since there are two types of primer sequences in one locus, it is determined whether the current primer number is 1 or 2. In the case of 1, go to (1700), change the primer number to P = 2, and repeat the process from (300). In the case of 2, go to (1300).

  (1300) It is checked whether the processing of all the loci has been completed. If not completed, go to (1800) to process the next locus, change the setting of the locus number N to N + 1, and repeat the process from (200). If completed, go to (1400).

  (1400) A list of the comparison results of the nucleotide sequences of TRNPU and SRNPV is created for each of all the locus subgroups, for example, as follows (Table 2).

Table 2 shows a part of an example in which each of the target and the sample is a specimen containing a single individual. FIG. 11 shows a schematic diagram serving as a basis for this table.

  In the example of FIG. 11, for the locus 1 DNA marker, primers 1 and 2 each have two subgroups, and for the locus 2 DNA marker, primers 1 and 2 each have one subgroup. It is a specimen to have. On the other hand, the sample is a specimen having two types of subgroups for the primers 1 and 2 for the locus 1 DNA marker and two types of subgroups for the primers 1 and 2 for locus 2 respectively. According to the nomenclature of FIG. 10, the representative base sequence 1 of the locus subgroup 1 in the group of the target locus 1 and the primer number 1 is TR0111, the representative base sequence 2 of the locus subgroup 2 is TR0112, the sample locus 1 and the primer number 1 The representative nucleotide sequence 1 of the locus subgroup 1 in the group of No. is SR0111 and the representative nucleotide sequence 2 of the locus subgroup 2 is SR0112. A representative base sequence for each locus subgroup is obtained. Similarly, the targets of locus 2 include TR0211 and TR0221, and the samples include SR0211, SR0212, SR0221, and SR0222, respectively.

  Based on this, the target and the sample are compared in a group where the locus number and the primer number match. For example, for the group of locus number 1 and primer number 1, the four comparisons of TR0111 and SR0111, TR0111 and SR0112, TR0112 and SR0111, and TR0112 and SR0112 were performed. The conclusion is entered in each column where the comparison result of the locus subgroup representative base sequence applies. This operation is performed for all the loci subgroup representative nucleotide sequences, and Table 2 is completed.

Table 2 is an example in which both the target and the sample contain only one specimen, and therefore the number of subgroups of each locus for each primer is usually either 1 or 2. However, when three or more subgroups are included in the group of the same locus and the same primer of one sample, the sample may be a mixture of DNAs from a plurality of individuals as described above. If a table similar to Table 2 is created for such specimens, for one or both specimens of the target and the sample, which loci include the subgroup number of 3 or more, You can also see at a glance how often the number of groups is 3 or more. Examples are shown in Table 3.

  In Table 3, at the locus number 1 of the target sample, there are two subgroups for both primer numbers 1 and 2. Primers usually define a DNA fragment of one allele in pairs of 1 and 2. Considering that there are usually two alleles in human diploid organisms, the number of individuals contained in the target sample is There is no contradiction, considering that is 1. On the other hand, since there are three subgroups in the locus number 1 of the sample specimen, both the primer numbers 1 and 2, there is a possibility that the sample specimen is a mixed specimen of a plurality of individuals as described above. Furthermore, when three or more subgroups are detected in a plurality of loci in the list, it is more strongly suggested that the sample specimen may contain DNAs from a plurality of individuals.

  As described above, by providing the comparison result table of the locus subgroup representative base sequences, it is understood that there is a possibility that DNAs from a plurality of individuals are mixed in the sample.

  Further, comparison results of loci subgroup representative nucleotide sequences as shown in Table 3 for a sample in which DNAs derived from a plurality of individuals are mixed and a sample containing only DNA of a single individual, or a sample in which DNAs derived from a plurality of individuals are mixed. When the table is created, it is possible to indicate the possibility that the DNA of an individual common to both of the samples to be compared is included.

  Therefore, according to the comparison result table of the locus subgroup representative base sequences, 1) identification of DNA markers having the same base length but different base sequences, 2) identification of DNA markers using trace DNA, and 3) identification of degraded DNA Identification of the DNA marker used, 4) Provision of data for detection of the mixture of multiple individuals in the same sample, and 5) Determination of DNA judgment on whether two samples contain DNA derived from the same individual Becomes

  Next, the configuration of the present invention when the DNA amplification of the DNA marker by PCR is not used will be described with reference to FIGS.

[Example 3] Creating a database of DNA marker base sequence groups from base sequence groups of DNA fragments of a target sample and a sample sample read by a DNA sequencer (when PCR is not included in the pre-process)
The DNA extracted from the target specimen and / or the sample specimen is a mixture of DNA fragments of various chain lengths that have been cleaved and decomposed at an arbitrary site (corresponding to the upper diagram in FIG. 12). With respect to all of these DNA fragments, the nucleotide sequence was determined using a DNA sequencer capable of obtaining a large amount of nucleotide sequence data in a single experiment, and all of the information was obtained as shown in the middle part of FIG. Is registered in the database as a base sequence group.

  Next, all of these base sequences are compared with the base sequences of all the loci of the DNA marker for DNA identification, and a base sequence containing a base sequence portion corresponding to all or a part of any of the loci is extracted. Then, it is registered in the database as a marker-containing fragment base sequence group of the target DNA or sample DNA (corresponding to the lower diagram in FIG. 12).

  Subsequently, as shown in the upper to middle upper diagrams of FIG. 13, from each of the base sequences registered in the marker-containing fragment base sequence group of the sample DNA, only the portion corresponding to the base sequence of the contained DNA marker was extracted. Then, it is registered in the database as a base sequence group of all marker fragments of the sample DNA. (Figure in the upper middle of FIG. 13)

  All registered marker fragment base sequence groups are classified into groups for each locus, and the group is moved to a database of base sequence groups for each locus, which is created for each sample, while maintaining the groups. (Figures from upper middle to lower middle in FIG. 13)

  Subsequently, all the base sequences belonging to the same locus group of the moved locus-specific base sequence group are compared with each other, and a subgroup having a common portion having the same sequence is created, and this subgroup configuration is maintained. As it is, the data is moved to the database of DNA marker base sequences of the target or sample created for each specimen. (Drawing from bottom to bottom in FIG. 13) The processing of FIG. 13 will be described with reference to FIG.

  (010) For all of the base sequences registered in the database of the marker-containing fragment base sequence group of the target DNA or sample DNA at the top of FIG. 13, the base sequences are compared with all the DNA markers for DNA identification, and any DNA The entire base sequence or only a part of the base sequence corresponding to a part of the base sequence of the marker is extracted.

  (020) All of the nucleotide sequences extracted in (010) are moved to the database of all marker fragment nucleotide sequences of the corresponding target DNA or sample DNA.

  (030) For each base sequence of the transferred base sequence group, the sequence is grouped for each locus of the corresponding DNA marker for DNA identification, and if a locus number is set for the DNA marker for DNA identification, the sequence is followed accordingly. In addition, if not set, the locus number N is given as a name for each locus in an easy-to-understand order such as following the order of positions on the genome. When all the base sequences are grouped for each locus, and a locus number is given to each locus group, all the base sequences are moved to the database of the locus-specific base sequence group for each sample while maintaining the group structure.

  (040) In order to start the processing from locus 1 of the DNA marker, N = 1 is set as the initial value of the locus number.

  (050) For each locus group created in (030), all base sequences belonging to the group are compared with each other, and base sequences having the same common part are collected to form a subgroup. As described above, if a single individual is included in the sample, the number of subgroups in the same primer group of the same locus is usually 2 or less, reflecting that human individuals are usually diploid. Become. If the sample contains a plurality of individuals, the number of subgroups in the same primer group of the same locus may be 3 or more.

  (060) It is necessary to distinguish whether the specimen for which the subgroup is created is a target or a sample in a later operation. If the sample being worked on is the target, the process proceeds to (070), and a name that indicates that the sample is a subgroup of the target is given. If the specimen in operation is a sample, the process proceeds to (080), and a name that indicates that the sample is a subgroup of the sample is given.

  (070) When the work target is the base sequence of the target sample, the subgroup numbers U are assigned to all the subgroups included in the same locus group created in (050) in order from 1 respectively. Further, a subgroup name TNU (for example, T = 11 if N = 1 and U = 1) to which T is added first to specify that the sample is a target is given to each subgroup.

  (080) When the operation target is the base sequence of the sample specimen, the subgroup numbers V are assigned to all the subgroups included in the same locus group created in (050) in order from 1 respectively. Further, a subgroup name SNV to which S is first added to clearly indicate that the sample is a sample (for example, N = 1, if V = 1, S011) is given to each subgroup.

  (090) The subgroups of the nucleotide sequences named in (070) and (080) are moved to the DNA marker nucleotide sequence group database of the corresponding specimen (target or sample) while maintaining the group structure.

  (100) Usually, there are a plurality of loci of DNA markers for DNA identification, and it is necessary to judge the presence or absence of a loci number larger than the current loci number N in order to perform the above operation on all of them. If there is a locus number larger than the current locus number N, go to (110), change the setting of the locus number N to N + 1, and return to (050). If there is no locus number larger than the current locus number N, the operation ends.

  By the processing so far, the base sequence including the full length or a part of the base sequence of the DNA marker was grouped for each locus and for each subgroup of the same base sequence as shown in the lower part of FIG. In this state, it is registered in the database of the DNA marker base sequence group for each sample.

  This operation is performed for each of the two samples to be subjected to the DNA test, and two databases of the DNA marker base sequence group of the target sample and the DNA marker base sequence group of the sample sample are created.

  In this example, first, after extracting all the nucleotide sequences including any one of the nucleotide sequences of all the loci for DNA marker evaluation, the nucleotide sequence of the fragment for each locus or a part thereof was extracted. Any one of the sequences or only a part of the sequences may be extracted, and the method is not limited to these methods.

[Example 4] Flow of DNA identification by comparison of representative nucleotide sequences of DNA markers (when PCR is not performed)
This processing is performed using the two databases of the DNA marker base sequence group of the target specimen and the DNA marker base sequence group of the sample specimen created above. The processing performed here will be described with reference to FIG.

  (100) In order to start processing from the locus 1 of the DNA marker, N = 1 is set as an initial value of the locus number.

  (200) In order to start processing from the subgroup 1 of the target sample, U = 1 is set as an initial value of the subgroup number of the target.

  (300) A group of DNA marker base sequences of subgroup number U is selected from a group of DNA marker base sequences of locus number N in the DNA marker base sequence group of the target sample.

  (400) A DNA marker nucleotide sequence having the longest chain length is selected from the DNA marker nucleotide sequence group of the selected target sample, and this is defined as a locus subgroup representative nucleotide sequence TRNU. (For example, the locus subgroup representative base sequence of locus number 1 and subgroup number 1 is TR011, etc.)

  (500) In order to start the process from the subgroup 1 of the sample specimen, V = 1 is set as an initial value of the subgroup number of the sample.

  (600) A group of DNA marker base sequences of subgroup number V is selected from the group of DNA marker base sequences of locus number N in the DNA marker base sequence group of the sample specimen.

  (700) A DNA marker nucleotide sequence having the longest chain length is selected from the group of DNA marker nucleotide sequences of the selected sample specimen, and this is defined as a locus subgroup representative nucleotide sequence SRNV. (For example, the locus subgroup representative base sequence of locus number 1 and subgroup number 1 is SR011, etc.)

(800) The locus representative nucleotide sequences TRNU and SRNV selected above are compared based on Table 4.

  (900) It is necessary to perform the steps (600) to (800) for all subgroups in the same locus and the same primer sequence group of the sample specimen, so that other subgroups in the same locus group of the sample specimen are required. Determine if there are any subgroups. If there is another subgroup, the procedure goes to (1300) to change the subgroup number V of the sample sample to V + 1 and repeat the operations of (600) to (800). If there are no other subgroups, go to (1000).

  (1000) It is necessary to perform the steps (300) to (900) for all subgroups in the same locus and the same primer sequence group of the target sample. It is determined whether there is a subgroup. If there are other subgroups, go to (1400), change the subgroup number U of the target to U + 1, and repeat the operations of (300) to (900). If there are no other subgroups, go to (1100).

  (1100) It is checked whether the processing of all the loci has been completed. If not completed, go to (1500) to process the next loci, change the loci number N to N + 1, and repeat the process from (200). If completed, go to (1200).

(1200) A list of the comparison results of the base sequences of TRNU and SRNV is prepared for each locus subgroup, for example, as follows (Table 5), and the process is completed when completed.

  In Table 5, there are two subgroups for locus number 1 of the target sample. On the other hand, there are three subgroups in locus number 1 of the sample specimen. For this reason, as described above, it is conceivable that DNAs derived from a plurality of individuals are mixed in the sample specimen. Further, when three or more subgroups are detected in a plurality of loci, this possibility can be more strongly presented.

  As described above, by providing the comparison result table of the locus subgroup representative base sequences, it can be seen that there is a possibility that DNAs from a plurality of individuals are mixed in the sample.

  Furthermore, a comparison result of the locus subgroup representative base sequences as shown in Table 5 for a sample in which DNAs derived from a plurality of individuals are mixed and a sample containing only DNA of a single individual, or a sample in which DNAs derived from a plurality of individuals are mixed. When the table is prepared, it is understood that there is a possibility that the DNA of an individual common to both of the samples to be compared is included.

  Therefore, according to the comparison result table of the locus subgroup representative nucleotide sequences, 1) identification of DNA markers having the same nucleotide length but different nucleotide sequences, 2) identification of DNA markers using a small amount of DNA, and 3) identification of degraded DNA Identification of the DNA marker used, 4) Provision of data for detection of the mixture of multiple individuals in the same sample, and 5) Determination material for DNA identification as to whether two samples contain DNA from the same individual Becomes

  Since the present invention provides a data table contributing to DNA testing, it can be used not only in fields related to crime-related humans but also in industrial fields related to DNA testing such as judging animal and plant varieties.

Claims (4)

A DNA marker containing a sequence showing a difference in base sequence between individuals, and using a DNA marker containing STR , data for judging whether the target DNA and the sample DNA are DNAs derived from the same individual. A method for providing a method for providing a material for DNA identification, which comprises the following steps:
(1) using the target DNA as a template, performing PCR using primers that amplify the locus of the DNA marker, and then determining the base sequence of the DNA fragment;
(2) extracting a DNA fragment containing a DNA marker or a partial base sequence thereof from the DNA fragment whose base sequence has been determined in step (1);
(3) dividing the DNA fragments extracted in step (2) into groups (locus groups) for each same locus based on the nucleotide sequence;
(4) For each locus group divided in step (3), DNA fragments having the same base sequence in the locus group are divided into subgroups, and the longest DNA fragment among the subgroups is a subgroup representative fragment. The process of selecting as
(5) using the sample DNA as a template, performing PCR using primers for amplifying the locus of the DNA marker, and then determining the base sequence of the DNA fragment;
(6) a step of extracting a DNA fragment containing a DNA marker or a partial base sequence thereof from the DNA fragment whose base sequence has been determined in step (5),
(7) a step of dividing the DNA fragments extracted in the step (6) into groups (locus groups) for each same locus based on the nucleotide sequence;
(8) For each of the locus groups divided in step (7), DNA fragments having a common base sequence in the locus group are divided into subgroups, and the longest DNA fragment among the subgroups is a subgroup representative fragment. The process of selecting as
(9) comparing the subgroup representative fragment of the target DNA selected in step (4) with the subgroup representative fragment of the sample DNA selected in step (8) for each locus;
(10) a step of determining, for each locus, whether the same DNA exists in the target DNA and the sample DNA based on the following criteria (a) to (g):
(A) When both the subgroup representative fragment of the target DNA and the subgroup representative fragment of the sample DNA are full-length, and if both fragments have the same full-length and sequence, both fragments are judged to be identical.
(B) When both the subgroup representative fragment of the target DNA and the subgroup representative fragment of the sample DNA are full-length, and when the total lengths of both fragments are different, it is determined that both fragments do not match.
(C) If the subgroup representative fragment of the target DNA is full length and the subgroup representative fragment of the sample DNA is a partial sequence, and if the full length of the sample DNA is unknown, it is determined that the determination cannot be made (however, If the sequences of both fragments do not match, it is determined that the two fragments do not match)
(D) When the subgroup representative fragment of the target DNA is full length and the subgroup representative fragment of the sample DNA is a partial sequence, the subgroup representative fragment of the sample DNA is longer than the subgroup representative fragment of the target DNA. If both fragments do not match,
(E) If the subgroup representative fragment of the target DNA is a partial sequence and the subgroup representative fragment of the sample DNA is full length, and the total length of the target DNA is unknown, it is determined that the determination cannot be made (however, If the sequences of both fragments do not match, it is determined that the two fragments do not match)
(F) When the subgroup representative fragment of the target DNA is a partial sequence and the subgroup representative fragment of the sample DNA is full length, the subgroup representative fragment of the target DNA is longer than the subgroup representative fragment of the sample DNA If both fragments do not match,
(G) If both the subgroup representative fragment of the target DNA and the subgroup representative fragment of the sample DNA are partial sequences, it is determined that the determination is impossible (however, if the sequences of both fragments do not match, the two fragments match. Judge not to).   In step (9), the base sequences of the subgroup representative fragment of the target DNA selected in step (4) and the subgroup representative fragment of the sample DNA selected in step (8) are determined for each locus in the entire subgroup. The method for providing a DNA identification material according to claim 1, wherein the comparison is performed.   The DNA fragment for DNA identification according to claim 2, wherein in the step (9), the subgroup representative fragments are compared based on the base sequence of the entire shorter DNA fragment of the two subgroup representative fragments. How to provide materials.   In step (4) and step (8), DNA fragments in the locus are divided into subgroups for each identical base sequence, so that when the number of subgroups is 2 or more, the DNA of a plurality of individuals may be mixed. 2. The method according to claim 1, wherein the information can be obtained.