JP2002350401A - Genomic DNA analysis program - Google Patents

Abstract

(57) [Summary] [Problem] To create a two-dimensional electrophoresis pattern of a control to be compared easily and under various conditions, and to perform quick and accurate analysis. A computer generates a control two-dimensional electrophoresis pattern based on genomic base sequence information, a two-dimensional electrophoresis pattern using the control two-dimensional electrophoresis pattern, and a genomic DNA to be analyzed. A procedure for comparing the two-dimensional electrophoresis pattern of the analysis target obtained by performing the above, and a procedure for detecting a difference in the spot position between the two-dimensional electrophoresis pattern of the control and the two-dimensional electrophoresis pattern of the analysis target Is executed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a program for analyzing a two-dimensional electrophoresis pattern using genomic DNA using a computer, and to a genomic DNA analysis method using a two-dimensional electrophoresis pattern.

[0002]

2. Description of the Related Art In recent years, the development of genome scanning technology has been rapidly progressing in order to analyze genomes of higher organisms faster. Genome scanning is a technique in which a locus (locus) or its copy number is analyzed at a high speed at a time, and the physical state of genomic DNA is searched over the entire genome. According to genome scanning, the position of the gene on the genetic map of the genome, or the D encoding one gene product
Regarding the section on the chromosome occupied by the NA region, the signal can be detected at high speed to determine the presence or absence. In this genome scanning, what kind of landmark should be made on the genome, what kind of landmark on the genome should be detected as what kind of signal, and how to detect and analyze more landmarks It is extremely important to decide. These landmarks are called landmarks, and analyzing the chromosomal location information of landmarks is a great basic technique for creating a linkage map or physical map of a genome. Therefore, when genome scanning is applied to two or more types of genomic DNA and compared,
Changes in genomic DNA such as deletions, amplifications, and translocations can be detected.

[0003] By the way, RLGS (Restriction Landmark G)
enomic scanning) method is genomic DNA cut with restriction enzymes.
Is labeled as a landmark, and the labeled genome
In this method, a DNA fragment is developed using two-dimensional electrophoresis and detected using the obtained pattern. This RLGS method can detect many landmarks at once,
It can be said that this technology can detect changes in genomic DNA at high speed.

[0004] The RLGS method requires a two-dimensional electrophoresis pattern as a control in order to compare with a two-dimensional electrophoresis pattern obtained from genomic DNA to be analyzed. A two-dimensional electrophoresis pattern serving as a control can be obtained by performing two-dimensional electrophoresis in the same manner using genomic DNA extracted from wild-type cells. RLGS
In the method, a step for obtaining a control two-dimensional electrophoresis pattern is troublesome, and comparison using the obtained control two-dimensional electrophoresis pattern is troublesome. In addition, spot cloning has the problem of requiring significant labor and complicated techniques.

[0005]

Accordingly, the present invention provides a two-dimensional electrophoresis using genomic DNA, which can easily produce a two-dimensional electrophoresis pattern of a control to be compared under various conditions. It is an object of the present invention to provide a computer-readable recording medium on which a program for analyzing an electrophoresis pattern is recorded. Another object of the present invention is to provide a genomic DNA analysis method that can perform a quick and reliable analysis by easily creating a control two-dimensional electrophoresis pattern under various conditions. .

[0006]

The present invention that has achieved the above-mentioned object includes the following. (1) A procedure for creating a control two-dimensional electrophoresis pattern on a computer based on genomic base sequence information, and performing two-dimensional electrophoresis using the control two-dimensional electrophoresis pattern and the genomic DNA to be analyzed. A procedure for comparing the two-dimensional electrophoresis pattern of the analysis target obtained by performing the procedure, and a procedure for detecting a difference in spot position between the two-dimensional electrophoresis pattern of the control and the two-dimensional electrophoresis pattern of the analysis target The program to be executed.

(2) In the procedure for creating the two-dimensional electrophoresis pattern of the control, the recognition sequence of the first restriction enzyme and the recognition sequence of the second restriction enzyme in the genome base sequence information are detected, and The base sequence sandwiched between the recognition sequence of the restriction enzyme and the recognition sequence of the second restriction enzyme, and the base sequence sandwiched between the recognition sequence of the first restriction enzyme and the recognition sequence of the first restriction enzyme The program according to (1), wherein a two-dimensional electrophoresis pattern of the control is created based on the program.

(3) The program according to (2), wherein the first restriction enzyme and the second restriction enzyme are selected from methylation-insensitive restriction enzymes. (4) The first restriction enzyme and the second restriction enzyme are selected from methylation-sensitive restriction enzymes (2).
The program described. (5) The program according to (1), further including a step of acquiring the genome base sequence information via a communication network.

(6) In the procedure for creating the control two-dimensional electrophoresis pattern, a plurality of spots are created on the basis of genome base sequence information, and the plurality of spots are linked to locus information on the genome. The program according to (1), which is characterized in that: (7) The program according to (1), wherein in the procedure for creating a control two-dimensional electrophoresis pattern based on the genome base sequence information, abnormal information included in the genome base sequence information is detected. (8) The program according to (7), wherein the detected abnormality information is stored in association with spots included in the created two-dimensional electrophoresis pattern.

(9) A step of preparing a two-dimensional electrophoresis pattern by performing two-dimensional electrophoresis using genomic DNA to be analyzed, and a control prepared based on the two-dimensional electrophoresis pattern and genomic base sequence information. Comparing the two-dimensional electrophoresis pattern of the control with the two-dimensional electrophoresis pattern of the control, and detecting the difference in spot position between the two-dimensional electrophoresis pattern of the control object and the two-dimensional electrophoresis pattern of the analysis target Method. (10) The genomic DN according to (9), which comprises a step of extracting the genomic DNA to be analyzed from plant cells.
A analysis method.

(11) The genomic DNA analysis method according to (9), wherein the genomic DNA to be analyzed is derived from a higher organism. (12) the following steps, (a) a step of treating genomic DNA with a first restriction enzyme, (b) a step of adding a label to the restriction enzyme cleavage site, and (c) electrophoresis of the obtained DNA fragment. A step of performing one-dimensional fractionation, (d) a step of treating the DNA fragment fractionated in step (c) with a second restriction enzyme, and then performing a two-dimensional fractionation, (e) a step of performing step (d). (9) The step of detecting a spot of the labeled labeled DNA fragment, wherein the step of creating a two-dimensional electrophoresis pattern of the genomic DNA to be analyzed is performed.
DNA analysis method.

(13) After the step (b), the step (c) is carried out after treating with a restriction enzyme different from the first restriction enzyme and the second restriction enzyme (12).
The genomic DNA analysis method according to the above. (14) detecting the recognition sequence of the first restriction enzyme and the recognition sequence of the second restriction enzyme in the genome base sequence information, and creating a two-dimensional electrophoresis pattern of the control based on these cleavage sites. Features (1
2) The method for analyzing genomic DNA according to the above.

(15) The method according to (12), wherein the step (b) is performed by connecting one of the adapters to the restriction enzyme cleavage portion and adding the label to the other of the adapters. Genomic DNA analysis method. (16) The two-dimensional electrophoresis pattern of the control has a plurality of spots created based on genomic base sequence information, and the plurality of spots are linked to loci information on the genome. (9) The method of analyzing genomic DNA according to (9).

(17) The method according to (9), wherein prior to creating a control two-dimensional electrophoresis pattern based on the genomic base sequence information, abnormal information contained in the genomic base sequence information is detected. Genomic DNA analysis method. (18) The program according to (17), wherein the detected abnormality information is associated with spots included in the created two-dimensional electrophoresis pattern.

(19) Involved in a mutant gene and / or trait characterized by detecting a mutation site in a genomic DNA to be analyzed by the genomic DNA analysis method according to any one of (9) to (18). Gene identification method. (20) A DNA fragment containing the displacement site detected by the identification method described in (19) is isolated from the two-dimensional electrophoresis pattern, and a mutated gene and / or a gene involved in a mutant trait is isolated. Release method.

(21) A two-dimensional electrophoresis pattern prepared based on genomic base sequence information and having a plurality of spots estimated based on the genomic base sequence information. (22) The two-dimensional electrophoresis pattern according to (21), wherein the plurality of spots are linked to locus information on a genome.

(23) The two-dimensional electrophoresis pattern according to (21), wherein the genome base sequence information is base sequence information obtained from a nuclear genome of a plant. (24) The two-dimensional electrophoresis pattern according to (21), wherein the plurality of spots are associated with abnormal information retrieved from the genome base sequence information. (25) The genomic base sequence information is base sequence information obtained from a genome of a higher organism (2).
The two-dimensional electrophoresis pattern according to 1).

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The program according to the present invention is, for example, recorded on an information recording medium such as a flexible disk, an optical disk, a magneto-optical disk, a phase-change optical disk, and a hard disk, or distributed from a server via a communication network such as the Internet. It may be something. The present program recorded on the information recording medium or the present program distributed from the server can cause a computer to execute the following processing.

This program includes the following procedure. Procedure for creating a control two-dimensional electrophoresis pattern based on genome base sequence information (hereinafter, Procedure 1). A procedure for comparing the two-dimensional electrophoresis pattern of the control with a two-dimensional electrophoresis pattern obtained by performing two-dimensional electrophoresis using genomic DNA to be analyzed (hereinafter, procedure 2).

That is, this program uses a computer to compare a two-dimensional electrophoresis pattern obtained from genomic DNA extracted from an object to be analyzed with a two-dimensional electrophoresis pattern serving as a control. In this program, in procedure 1, first, a two-dimensional electrophoresis pattern serving as a control is obtained. In this program, in the procedure 2, the obtained two-dimensional electrophoresis pattern to be analyzed is separately used, and the two-dimensional electrophoresis pattern is compared with a control two-dimensional electrophoresis pattern.

First, a method for obtaining a two-dimensional electrophoresis pattern of genomic DNA to be analyzed will be described. The two-dimensional electrophoresis pattern is obtained by using a restriction enzyme recognition site as a landmark, and detecting the landmark as a signal (Restriction Landmark Genomic Sca).
nning method (RLGS method); Hatada, I. et al., Proc. Natl.
Acad. Sci., USA, 88, 9523-9527, 1991) is applied as a basic concept. For analysis of plant genomic DNA, see Mat
suyama et al. Plant.Mol. Biol. Rep. 18: 331-338,
Apply 2000 as a basic philosophy. Genome to be analyzed
The two-dimensional electrophoresis pattern of DNA can be obtained according to the following method.

(1) Treatment of Genomic DNA with First Restriction Enzyme The genomic DNA to be subjected to the analysis method of the present invention is not particularly limited. Bacteria, yeast, etc.). Genomic DNAs derived from higher organisms can also be used. Examples of plant-derived genomic DNAs include those derived from rice, tobacco, Arabidopsis, wheat, tomato, and the like, and those derived from rice or Arabidopsis are preferred. Examples of animal-derived genomic DNA include those derived from humans, mice, nematodes, and the like.
Examples of the genomic DNA derived from bacteria include those derived from Bacillus subtilis, Escherichia coli, and the like.

In the program and the DNA analysis method according to the present invention, it is particularly preferable to analyze the above-mentioned plant-derived genomic DNA. Genomic DNA from plants
Is a known method (Dllaport ret al. Plant Mo
l. Biol. Rep. 1: 19-21, 1983), and can be obtained by subjecting plants to protein removal treatment, polysaccharide removal treatment, and the like.

In the "treatment of genomic DNA with first restriction enzyme", first, the extracted genomic DNA is treated with a first restriction enzyme (an enzyme that cuts the position of "A" in FIG. 1 (1). (Referred to as enzyme A). As the restriction enzyme A, those having an average fragment length of more than 100 kb generated upon cleavage, that is, a restriction enzyme that recognizes restriction enzyme sites that exist only at intervals exceeding an average of 100 kb, and
A so-called rare cutter restriction enzyme that recognizes a base is exemplified.

The restriction enzyme A is cleaved such that the 5 ′ side of the restriction enzyme site generated upon cleavage is a protruding sticky end (referred to as a 5 ′ protruding type), and the 3 ′ side is a protruding sticky end. Any of those which are cut likewise (referred to as 3 'protruding type) can be used. In addition, the length of the protrusion is
Enzymes that have one or more bases are preferred. In order to obtain a sufficient signal intensity, an enzyme that cuts so that the length of the protruding portion is two or more bases is preferable. As such a restriction enzyme A, 5 'protrusive For example Not I, Bss HII, Acc III, and the like, 3' for projecting type eg Bst XI, Bgl I, Mwo
I.

Here, especially when the analysis target is genomic DNA derived from a plant cell, it is preferable to use a methylation-insensitive restriction enzyme as the first restriction enzyme. By using a methylation-insensitive restriction enzyme, even genomic DNA having methylated cytosine, such as genomic DNA derived from plant cells, can be reliably cleaved. Examples of methylation-insensitive restriction enzymes include, for example,
Acc III and Pac I can be mentioned.

(2) Labeling of Restriction Enzyme Cleavage Site Next, a label is added to the restriction enzyme A cleavage site. The label is added by filling in a base (labeling sequence) previously labeled with a sequenase (T7 DNA polymerase) at the cleavage site of restriction enzyme A. Therefore, in this case, in order to be a reaction target of the sequenase, it is necessary to use, as the above-mentioned restriction enzyme A, one that cleaves such that the 5 ′ side of the cleavage site is a sticky end protruding (referred to as 5′-protruding type) preferable.

As the labeling substance, [α- ³² P] dCTP, [α- ³² P]
Radioisotopes such as [P] dGTP, tetramethyl-rhodamine-6-
Examples thereof include fluorescent dyes such as dUTP and fluorescein-12-dUTP, and can be arbitrarily selected. When a label is added, an adapter may be added to the cleavage site of restriction enzyme A, and a label may be added to the adapter. The adapter is designed on one side as a sequence that can be ligated to the restriction enzyme A cleavage site (referred to as a linking sequence), and on the other side as a sequence for labeling the adapter (referred to as a labeling sequence). I do. By making the labeling sequence of the adapter a cohesive end protruding on the 5 ′ side, it can be used as a reaction target by the sequenase, and a label can be added to the labeling sequence.

When a label is added via an adapter as described above, the restriction enzyme A is one that cleaves such that the 3 ′ side of the cleavage site is a protruding sticky end, ie, 3 ′.
Preferably, a protruding mold is used. In this case, the restriction enzyme
Fill-in by the sequenase (T7 DNA polymerase) can be prevented at the cleavage site by A. In other words, the connecting sequence of the adapter is preferably a cohesive end protruding on the 5 ′ side. Here, the linking sequence and the labeling sequence are single-stranded. Further, between the linking sequence and the labeling sequence is composed of a double strand consisting of 5 to 45 bases,
Considering the easiness of cloning, those consisting of 20 to 35 bases are preferable. The linking sequence can be designed corresponding to the above-mentioned recognition sequence for restriction enzyme A. The labeling sequence and the sequence of the double-stranded region can be arbitrarily designed.

Since the length of the labeling sequence can be set arbitrarily, the longer the length of the labeling sequence, the higher the detection sensitivity. However, if the labeling sequence is too long, DNA
May be formed (for example, a stem-loop structure or a hairpin structure). Therefore, the labeling sequence is preferably 2 to 10 bases so as not to form these secondary structures. As described above, when a label is directly added to the cleavage site of restriction enzyme A, since the spot detection ability for tobacco, Arabidopsis, etc. is low, all the genomes
Difficult to apply to DNA. Therefore, by adding a label via an adapter, all genomic DNA
And the detection ability can be improved.

When an adapter is used, the restriction enzyme A includes N (N represents A, G, C or T) in the recognition sequence.
Bst XI having the following may be used. If it is a 3 'protruding type such as Bst XI, it is necessary to use a sequenase (T7 DNA
Polymerase), and can be prevented from labeling fragments other than the target DNA fragment. In addition, such an enzyme having a 3 ′ protruding type and a protruding portion of 2 bases or more is
Bst XI, that much other Bgl I and MWO I (e.g. Alw
NI, Ban II, Bsi EI, Bsi HKAI, Bsl I, Bsm I, Bsp 1286I, B
sr DI, Dra III, Drd I, Pfl MI, Sfi I, Tsp RI, Bpm I, Bse R
I, Bsg I, Eco 57I), and any of these enzymes can be applied in the method of the present invention. For example, using Bst XI as restriction enzyme A, Bst XI recognizes the following sequence,
The region between the 8th N and the 9th N (position indicated by *) is cut from the 5 'end so that the 3' side protrudes 4 bases (underlined portion) (Fig. 2 (1)). Sense strand: 5'-CCAN NNNN * NTGG-3 '(SEQ ID NO: 1) Antisense strand: 3'-GGTN * NNNN NACC-5'

Since N may be any of A, G, C and T,
The four sequences of adapter overhangs are all combinations (4
⁴ = 256 ways). In this case, Genome D
Depending on the type of NA and the purpose of analysis (specifically, detection of a mutated region in an Arabidopsis mutant, etc.), the four bases of the protruding portion can be defined to be an appropriate number of combinations. For example, in FIG. 2 (2), “GGGC” is shown as a connecting sequence of the adapter. In this case, the adapter is a fragment of Bst XI that is one type of fragment that has the “CCCG” sequence as a protruding sequence ( That is, the protrusion is "CCC
G ") (Fig. 2 (3)). Similarly, the connecting sequence of the adapter is "GGGS"
(S represents G or C), the adapter is Bst XI
Are ligated to two types of fragments having the "CCCG" or "CCCC" sequence as a protruding sequence among the fragments cut by the above. Therefore, when Bst XI is used as restriction enzyme A, 1 to 256 types of landmark sites can be arbitrarily selected depending on the method of selecting a connecting sequence of an adapter. In addition, the preparation of the adapter is performed using a conventional chemical synthesis device (for example, PE Applied B
iosystems DNA / RNA synthesizer model 394). Then, the adapter is treated with DNA ligase and ligated to genomic DNA.

(3) Other Restriction Enzyme Treatment When the fragment digested with restriction enzyme A has a length of 100 kb or more, fractionation by electrophoresis or the like cannot be performed as it is. Therefore, another restriction enzyme treatment is performed to reduce the fragment digested with restriction enzyme A to a shorter fragment (FIG. 1 (4)). Other restriction enzymes are those that have an average fragment length of several to several tens of kb generated upon cleavage, that is, a restriction enzyme that recognizes restriction enzyme sites that are present at intervals of an average of several to several tens of kb.
One that recognizes 6 bases can be used (an enzyme that cleaves position B in FIG. 1 and is referred to as restriction enzyme B). Examples of the restriction enzyme B include Eco RV, Dra I and the like.
The average number of fragments cut by restriction enzyme A to several tens of k
In the case of b, fractionation by electrophoresis can be performed as it is, so that there is no need to perform treatment with restriction enzyme B.

(4) One-dimensional fractionation After treatment with restriction enzyme B, one-dimensional fractionation is performed for each fragment (FIG. 1 (5)). One-dimensional fractionation is 3-4.5 in diameter
This is performed by using an elongated capillary tube having a length of about 60 cm and a length of about 60 mm. The fragment treated in the above section (4) is injected into the origin of the tube and fractionated. The one-dimensional fraction is preferably agarose electrophoresis of about 0.7 to 1.0%, and in the presence of 5% sucrose at room temperature (more preferably 22%).
-26 ° C) for 20-48 hours. In the one-dimensional fractionation step, the fragment digested with the restriction enzyme B is electrophoresed so that the length of the DNA fragment gradually decreases from the origin (the starting point of the one-dimensional fractionation) toward the end (FIG. 1). (5) enlarged view). For example, in the enlarged view of FIG. 1 (5), fragment 1 with a long length migrates closer to the origin and fragment 2 with a shorter length migrates away from the origin. Therefore, the position from the origin reflects the length of the fragment.

(5) Treatment with Second Restriction Enzyme After the fractionation, the tube is immersed in a second restriction enzyme solution, and the one-dimensional fractionated product is treated with the restriction enzyme. The second restriction enzyme is an enzyme having a higher cleavage frequency than restriction enzymes A and B, and recognizes a restriction enzyme site having an average fragment length of several hundred bp at the time of cleavage, for example, an interval of about 300 bp. (This is an enzyme that cuts the position of “C” in FIG. 1 and is referred to as restriction enzyme C.) The restriction enzyme C can be used 4-6 base recognition ones, for example the Mbo I, can be employed Hin fI like.

(6) Two-dimensional fractionation When the fragment is treated with the restriction enzyme C, the restriction enzyme recognition sites A and B
The fragment sandwiched between and (AB fragment) is the fragment sandwiched between the restriction enzyme recognition sites A and C (the AC fragment), and the fragment sandwiched between the restriction enzyme recognition sites C and B (the BC fragment).
The resulting DNA fragments each have an average chain length of several hundred bp or less. Two-dimensional fractionation is performed on these fragments. Examples of the two-dimensional fractionation method include a method by 5% polyacrylamide gel electrophoresis (2 v / cm for 20 to 24 hours). Alternatively, a more accurate pattern can be obtained by performing 5% polyacrylamide gel electrophoresis containing 6 M urea.

(7) Spot Detection Spot detection can be performed by employing a method according to the type of the labeling substance. For example, when ³² P is used as a labeling substance, detection by autoradiography can be mentioned, and when a fluorescent dye is used, detection by a fluorescence image analyzer (for example, FMBIO II Multi-View, TaKaRa) can be mentioned. Since the BC fragment after the two-dimensional fractionation is not labeled, no spot is generated.

The position of each spot obtained in this way is defined as the distance in the X and Y directions (X ₁ , Y ₁ ), (X ₂ , Y ₂ ),.
_n , Y _n ), the X coordinate is the distance from the recognition site of restriction enzyme A to the recognition site of restriction enzyme B (AB fragment), and the Y coordinate is the recognition site of restriction enzyme C from the recognition site of restriction enzyme A. It reflects the distance (AC fragment) to the site (Fig. 1 (6)). Therefore, by analyzing the spot pattern, it is possible to identify a mutation site in the genome or to detect a mutation due to DNA modification. For example, since the spot density reflects the copy number of the detected fragment, it can be seen that the specific area overlaps. In the spot pattern shown in FIG. 1 (6), the position (X ₂ , Y _{When 2} ) is shifted or disappears, it is understood that a part of the base sequence of the DNA fragment has been mutated (deletion, substitution, addition, etc.).

Next, a program for analyzing the two-dimensional electrophoresis pattern of the genomic DNA to be analyzed obtained as described above will be described. FIG. 3 is a block diagram showing a configuration of a system including a computer device that executes the program. FIG. 4 is a flowchart showing a specific process of a program using this system.

The computer that executes the program is
CPU 501 for controlling the entire operation, RAM 503 for temporarily storing necessary program data and the like, input unit 504 for inputting various information by an operator, and data transmission / reception to / from external devices via the Internet A transmitting / receiving unit 505 including a communication modem and the like, and an output unit 506 for outputting data to be displayed and the like.
HDD 507 in which data, programs, etc. are recorded,
A CD-ROM drive 508 for reading data written on the CD-ROM 509 is provided. The system also includes a database 510 connected to a computer via a communication network such as the Internet. In addition,
This program can be recorded on any recording medium, such as a tape-shaped or disk-shaped magnetic recording medium, an optical recording medium, and any other information recording medium generally used for an external storage device of a computer.

The CPU 501 has a RAM 503, an HDD 507, or a CD-ROM 509.
In accordance with the program recorded in the system, the entire system is controlled to execute processing described later. The input unit 504 is, for example, a scanner, a mouse, a keyboard, or the like, and is operated when inputting conditions and the like necessary for executing processing. The output unit 506 is, for example, a display, a printer, or the like, and can display data processed under the control of the CPU 501. The transmission / reception unit 505 stores the data recorded in the database 510 under the control of the CPU 501 in the RAM.
503, HDD507 and so on.

In this process, as shown in FIG. 4, step S1, step S2 and step S3 can be processed in parallel. In this processing, step S1 may be performed prior to steps S2 and S3, or steps S2 and S3 may be performed prior to step S1. In step S1, the two-dimensional electrophoresis pattern obtained from the genomic DNA to be analyzed as described above is input. Specifically, the position (X _n , Y _n ) of each spot obtained in “(7) Spot detection” can be input as digital data. Also, a two-dimensional electrophoresis pattern can be input as image data by a scanner or the like.

In step S2, genomic base sequence information recorded in the database 510 is obtained via a communication network such as the Internet. Here, the genome base sequence information is a base sequence registered as a wild type of the genomic DNA to be analyzed. Examples of the database 510 include Genbank, EMBL, DDBJ, TAIR, and KAOS. The present program is not limited to those recorded in the database 510 as genomic nucleotide sequence information, and uses, for example, genomic nucleotide sequence information recorded in the HDD 507, CD-ROM 509, and other various information recording media. be able to. In step S2, it is preferable to detect abnormal information included in the genome base sequence information. Specifically, in step S2, abnormal information included in the genome base sequence information is detected, and the abnormal information is sorted and temporarily stored in, for example, the RAM 503.

Here, the abnormal information is information indicating that the base at a predetermined position is undetermined. For example, “m”, “r”, “w”, “s”,
"Y", "k", "v", "h", "d", "b" and "n"
Etc. are included as character information. “M” means that the position is A or C. "R" indicates that the position is G
Or A. “W” means that the position is A or T or U. "S" means that the position is G or C. "Y" is T or
Means U or C. “K” means that the position is G or T or U. "V" means that the position is A or G or C. "H" means that the position is A or C or T or U.
"D" means that the position is A or G or T or U. "B" means that the position is G or C or T or
U means. "N" means that the position is A or G or C or T or U, or that the position is unknown.

In step S3, a control two-dimensional electrophoresis pattern is created using the genomic base sequence information obtained from the database 510. When creating a control two-dimensional electrophoresis pattern, first, a recognition sequence of restriction enzyme A and a recognition sequence of restriction enzyme B are searched from genome base sequence information, and AA fragments and AB fragments are predicted. Next, when the two-dimensional electrophoresis pattern to be analyzed uses an adapter, the base sequence number of the adapter is added to the predicted AA fragment and AB fragment. Thereby, a plurality of putative AA fragments and putative AB fragments corresponding to the plurality of AA fragments and AB fragments obtained in “(3) Other restriction enzyme treatment” can be predicted from the genome base sequence information.

Next, based on the number of base sequences of a plurality of putative AA fragments and putative AB fragments and the estimated isoelectric point, etc.,
Predict the migration pattern estimated when “(4) One-dimensional fractionation” is performed. This electrophoresis pattern is based on Southern, EM
(Measurement of DNA Length by Gel Electrophoresis,
Analytical Biochemistry: 100, 319-323, 1979). The electrophoresis pattern is the concentration and type of gel, the voltage for electrophoresis,
It can be estimated by inputting various conditions such as migration time and temperature as parameters. In other words,
In the present program, it is preferable to provide in advance a correction value for estimating the migration pattern using these conditions as parameters. Next, the recognition sequence of the restriction enzyme C in the putative AA fragment and the putative AB fragment is searched to predict the AC fragment. Thereby, a plurality of putative AC fragments corresponding to the plurality of AC fragments obtained in “(5) Second restriction enzyme treatment” can be predicted from the genome base sequence information.

Next, based on the number of base sequences of a plurality of estimated AC fragments and the estimated isoelectric point, etc., “(6) Two-dimensional fractionation”
The two-dimensional electrophoresis pattern estimated in the case of performing is performed. The two-dimensional electrophoresis pattern can be estimated by inputting various conditions such as the type of gel, voltage for performing electrophoresis, electrophoresis time and temperature as parameters, similarly to the case of predicting the “electrophoresis pattern” described above. . In other words, in the present program, it is preferable to provide in advance a correction value for estimating a two-dimensional electrophoresis pattern using these conditions as parameters.

In the present program, the position of the spot to be predicted can be obtained as the estimated coordinates (X _n , Y _n ) based on the obtained two-dimensional electrophoresis pattern of the control. At this time, X-coordinate (X _n) indicates the size fractionation position of AB fragments obtained by adding the number of base sequences in the adapter, Y-coordinate (Y _n), the size of the AC fragment fraction obtained by adding the number of base sequences in the adapter Indicates the image position. Also, in this step S3,
Based on the obtained estimated coordinates (X _n , Y _n ), a two-dimensional electrophoresis pattern of the control can be obtained as image data.

In the above description, the electrophoresis pattern was created based on the putative AB fragment. However, when creating the two-dimensional electrophoresis pattern to be analyzed, it is assumed that “(3) Other restriction enzyme treatment” is not performed. Are restriction enzymes A and C
The migration pattern may be created based on the putative AA fragment and the putative AC fragment estimated to be obtained by the processing.
When an adapter is not added when creating a two-dimensional electrophoresis pattern to be analyzed, it is not necessary to consider the number of base sequences of the adapter in the above-described processing.

Further, it is preferable that the two-dimensional electrophoresis pattern of the control is prepared by excluding a predetermined region in the genome base sequence information. Examples of the region to be excluded include a telomere region at the end of genomic DNA and a highly repetitive base sequence such as near the centromere of genomic DNA. Specifically, before the third restriction enzyme treatment, a fragment having a recognition sequence of restriction enzyme A only at one end,
It is not identified as a spot because it is derived from the telomere region at the end of the genomic DNA or from the gap region at the time of base sequence determination.

In the present method, when abnormal information included in the genomic base sequence information is detected in step S2, the genomic base sequence information other than the abnormal information is searched for the recognition sequence of the restriction enzyme A or the like. Search for. That is, the base at the position searched as abnormal information is excluded from the target when searching for a recognition sequence such as restriction enzyme A.In addition, in the present method, the abnormal information contained in the genomic base sequence information is detected in step S2. If so, the predicted spot is associated with the abnormality information. Specifically, whether or not the abnormal information is included in the DNA fragment included in the predicted spot and the type of the abnormal information included in the predicted spot are associated with each spot.

Next, in step S4, the control two-dimensional electrophoresis pattern obtained based on the genome base sequence information is compared with the two-dimensional electrophoresis pattern to be analyzed. Specifically, comparing spot coordinates (X _n, Y _n) input in step S1 and, spot obtained in step S3 estimated coordinates (X _n, Y _n) and. In step S4, the image data of the two-dimensional electrophoresis pattern obtained in step S1 can be compared with the image data of the control two-dimensional electrophoresis pattern obtained in step S3.

Next, in step S5, the result of the comparison performed in step S4 is displayed on, for example, a display. As a comparison result, a spot that does not match the two-dimensional electrophoresis pattern to be analyzed in the control two-dimensional electrophoresis pattern can be identified, and the coordinates and base sequence information of the spot can be displayed. Since the two-dimensional electrophoresis pattern of the control is created based on the genomic base sequence information obtained from the database 510, the base sequence information of each spot can be associated.

In particular, when a cell having a chromosomal structure such as a higher organism is to be analyzed, highly repetitive cells such as a telomere region at the end of genomic DNA or a centromere which shows unclear data when determining a nucleotide sequence are used. If a base sequence is to be compared, it causes noise. Therefore, by using the two-dimensional electrophoresis pattern of the control excluding the telomere region at the end of the genomic DNA and the highly repetitive base sequence such as the vicinity of the centromere of the genomic DNA, it is possible to compare spots that cause noise. Can be prevented. On the other hand, in the first stage, both ends of each clone are marked so that noise appearing in the pattern can be clearly identified. As a result, a noise component can be reduced and a more accurate comparison can be performed.

In step S5, step S2
In the case where abnormal information contained in the genome base sequence information is detected, a spot that does not match the two-dimensional electrophoresis pattern to be analyzed in the control two-dimensional electrophoresis pattern is identified, and the spot contains the abnormal information. Determine whether or not. As a result, if the identified spot contains abnormal information, the abnormal information is corrected and the control two-dimensional electrophoresis pattern is reconstructed. For example,
When the identified spot contains "m" as anomalous information, the two-dimensional electrophoresis pattern of the control when the location of the anomaly information is A and the two-dimensional electrophoresis of the control when the location of the anomaly information is C Reconstruct the pattern.

Then, in step S5, the two-dimensional electrophoresis pattern of the reconstructed control and the two-dimensional electrophoresis pattern to be analyzed are compared again. For example, the identified spot contains “m” as abnormal information, and the two-dimensional electrophoresis pattern of the control when the position of the abnormal information is A and the control when the position of the abnormal information is C When the two-dimensional electrophoresis pattern is reconstructed, the two-dimensional electrophoresis pattern of these two types of controls is compared with the two-dimensional electrophoresis pattern to be analyzed. Thereby, the two-dimensional electrophoresis pattern to be analyzed can be analyzed with higher accuracy by reflecting the abnormal information included in the genome base sequence information.
If the abnormality information cannot be corrected, information that the identified spot contains abnormality information is associated with the spot.

As described above, according to the program according to the present invention, a two-dimensional electrophoresis pattern of the control is created using the genomic base sequence information recorded in the database 510 and the like, and the two-dimensional electrophoresis pattern of the control is created. The two-dimensional electrophoresis pattern to be analyzed is analyzed using the pattern. Thus, according to this program, the genome
When analyzing a two-dimensional electrophoresis pattern obtained from DNA, it is not necessary to create a two-dimensional electrophoresis pattern as a control using genomic DNA extracted from a wild-type cell line. The program also eliminates the need for tremendous effort and cumbersome technology. Therefore, this program can analyze genomic DNA to be analyzed very simply and quickly.

In particular, in the present program, it is preferable to use genomic DNA in which a mutation such as deletion of a base has been induced by a physical mutagen. That is, in this program,
It is preferable to analyze a genomic DNA whose length is changed as compared with a wild-type genomic DNA. Specifically, genomic DNA into which the mutation has been introduced is analyzed using a technique of inducing mutation by irradiating a heavy ion beam (see Japanese Patent Application Laid-Open No. 9-28220).

In particular, when genomic DNA in plant cells is analyzed by applying this program, plant genomic DNA
Is often methylated, it is preferable to use a methylation-insensitive restriction enzyme as the first restriction enzyme. That is, when plant genomic DNA is to be analyzed, the use of a methylation-insensitive restriction enzyme enables reliable cleavage without being affected by methylation. Examples of the methylation-insensitive restriction enzyme used in the first restriction enzyme treatment include, for example,
Acc III and Pac I can be mentioned. Examples of the methylation-insensitive restriction enzyme used in the second restriction enzyme treatment include Mbo I that recognizes four bases. By using these restriction enzymes, even when analyzing plant genomic DNA, many spots can be obtained without being affected by methylation, and more precise analysis can be performed. Can be. When analyzing genomic DNA, a methylation-sensitive restriction enzyme may be used. When a methylation-sensitive restriction enzyme is used as the first restriction enzyme, it is possible to detect and analyze an epigenetic mutation region due to DNA methylation.

[0060]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples. Example 1: Arabidopsis chloroplast DNA < Preparation of control two-dimensional electrophoresis pattern> In this example, chloroplast DNA of Arabidopsis was analyzed.

Arabidopsis thaliana ,
(Columbia strain)) The base sequence of the chloroplast DNA has been determined to have a circular genomic DNA of 154,478 bp (Sato, S. et al. 1999, DNA RESEARCH 6,238-29).
0), and its base sequence data can be obtained from DDBJ as accession number AP000423. Based on the nucleotide sequence data, a control two-dimensional electrophoresis pattern was created through the above-described program step.

In this example, Acc III was used as the first restriction enzyme,
Mbo I as the third restriction enzyme without using the second restriction enzyme
A two-dimensional electrophoresis pattern was prepared when was used. This <br/> these Acc III and the Mbo I are both non-sensitive to 5-methyl cytosine cytosine is methylated (5mC). The two-dimensional electrophoresis pattern of the obtained control is shown in FIG. In FIG. 5, the display of the number of bases shows the mobility when the molecular weight marker is actually migrated, and the horizontal axis shows the one-dimensional fractionation and the vertical axis shows the development of the two-dimensional fractionation.

The base sequence of spot CP3-a in the two-dimensional electrophoresis pattern of the control prepared in this example (FIG. 5) is shown in SEQ ID NO: 2, the base sequence of spot CP6-b is shown in SEQ ID NO: 3, and spot CP16 The nucleotide sequence of spot CP19-a is shown in SEQ ID NO: 5, the nucleotide sequence of spot CP19-a is shown in SEQ ID NO: 6, and the nucleotide sequence of spot CP19-b is shown in SEQ ID NO: 5. Shown in 7 and spot
The nucleotide sequence of CP22-a is shown in SEQ ID NO: 8, and spot CP25-b
The nucleotide sequence of spot CP28-a is shown in SEQ ID NO: 10, the nucleotide sequence of spot CP28-b is shown in SEQ ID NO: 11, and the nucleotide sequence of spot CP310-a is shown in SEQ ID NO: 12. The nucleotide sequence of spot CP310-b is shown in SEQ ID NO: 13, the nucleotide sequence of spot CP15-a is shown in SEQ ID NO: 14, and the nucleotide sequence of spot CP15-b is shown in SEQ ID NO: 1.
It is shown in FIG.

When a spot is selected in the control two-dimensional electrophoresis pattern shown in FIG. 5, the base sequence of the DNA fragment contained in the selected spot is displayed. In FIG. 5, the spot CP25-b is selected, and the nucleotide sequence of the DNA fragment contained in the spot CP25-b is displayed.

<Preparation of Two-Dimensional Electrophoresis Pattern for Detection> For chloroplast DNA of Arabidopsis thaliana (Columbia strain), the two-dimensional electrophoresis pattern was actually obtained using Acc III and Mbo I as follows. Created. First, the extracted chloroplast DNA is digested with Acc III, and
Labeling was performed with [α- ³² P] dCTP or [α- ³² P] dGTP for 30 minutes. Then, 0.8% agarose gel electrophoresis was performed at 2 V / cm for 4 hours.
The reaction was performed at room temperature (24 ° C.) for 8 hours. In addition, Mbo I as the third restriction enzyme in the gel
Performed at 37 ° C. for 2 hours in nuits. Thereafter, two-dimensional fractionation was performed by 5% acrylamide electrophoresis. 5% acrylamide electrophoresis conditions are 2V / cm, 22 hours, room temperature (24 ° C)
And After drying the gel, the two-dimensional electrophoresis pattern
After exposure at −80 ° C. for a day, development was performed. FIG. 6 shows a two-dimensional electrophoresis pattern obtained by analyzing chloroplast DNA. In FIG. 6, white triangles indicate positions of spots to be observed.

<Spot Identification from Comparison of Both Patterns>
The two-dimensional electrophoresis pattern of the control shown in FIG. 5 and the two-dimensional electrophoresis pattern of the analysis object shown in FIG. 6 were compared. As is apparent from FIGS. 5 and 6,
It can be seen that the positions of the spots in each two-dimensional electrophoresis pattern almost coincide.

Of the spots shown in FIG. 6, CP3-
a (SEQ ID NO: 2) and a DNA fragment extracted from spots corresponding to the putative spots represented by CP15-b (SEQ ID NO: 15)
The clone was sequenced. As a result, CP3-a and C in the genome sequence information obtained from the database
It was completely consistent with the information on the nucleotide sequence of P15-b. This clearly shows that the two-dimensional electrophoresis pattern of the actual analysis target can be analyzed using the two-dimensional electrophoresis pattern of the control created using the genome base sequence information.

Further, from the two-dimensional electrophoresis pattern of the analysis object shown in FIG. 6, it can be seen that spots show strong signal intensity when chloroplast DNA is used. The spots show strong signal intensity because of the large number of chloroplast DNA copies. From this, it was found that the two-dimensional electrophoresis pattern to be analyzed can be displayed with a strong signal intensity in proportion to the amount of the genomic DNA to be analyzed.

Example 2: Arabidopsis thaliana nuclear genomic DNA < Preparation of control two-dimensional electrophoresis pattern> In this example, Arabidopsis thaliana nuclear genomic DNA was analyzed. Arabidopsis thaliana ( Arabidopsis thaliana , (Co
lumbia strain)) has a gap region (undetermined region) of about 130 Mb, but almost the entire nucleotide sequence has already been determined (The Arabidopsis Genome).
Initiative, Nature.408 796-815 (2000), The A
rabidopsis Information resource (TAIR, ww
w.arabidopsis.org/home.html)). Based on the nucleotide sequence data, a control two-dimensional electrophoresis pattern was created through the above-described program step.

In this example, a two-dimensional electrophoresis pattern was prepared using Not I as the first restriction enzyme, Eco RV as the other restriction enzyme, and Mbo I as the second restriction enzyme. These Eco RV and Mbo I are both insensitive to 5-methylcytosine (5 mC) formed by cytosine methylation, and N
ot I is sensitive. FIG. 7 shows a two-dimensional electrophoresis pattern of the obtained control. In FIG. 7, the display of the number of bases indicates the mobility when the molecular weight marker was actually migrated,
The horizontal axis shows the development of the one-dimensional fraction, and the vertical axis shows the development of the two-dimensional fraction. In FIG. 7, a character string corresponding to each spot indicates a locus name including a Not I site in genomic DNA. The numbers 1 to 5 at the left end indicate the chromosome number, `` -a '' and `` -b '' at the right end indicate the upstream and downstream sides of the Not I site, respectively, and the other character strings are used when determining the base sequence. Means the name of the clone created. For example, the spot linked by “4FCA1-b” is derived from chromosome 4 and is the DN downstream of the NotI site in the FCA1 clone.
This means a DNA fragment including the A fragment and having a determined nucleotide sequence. In FIG. 7, black circles (●) indicate spots derived from chromosome 1, black diamonds (◆) indicate spots derived from chromosome 2, and solid triangles (▲) indicate chromosome 3 The crosses (x) are spots derived from chromosome 4, and the solid squares (■) are spots derived from chromosome 5.

<Preparation of Two-Dimensional Electrophoresis Pattern for Analysis> Genomic DNA of Arabidopsis thaliana (Columbia strain) which has been mutated by X-ray irradiation was analyzed, Not I was used as the first restriction enzyme, and the second restriction enzyme was used. Eco RV as enzyme,
A two-dimensional electrophoresis pattern was actually created in the same manner as in Example 1 except that Mbo I was used as the third restriction enzyme.
The obtained two-dimensional electrophoresis pattern is shown in "Mutant" in FIG.

In FIG. 8, "Control" is a part of the control two-dimensional electrophoresis pattern extracted and enlarged. By comparing these “Mutant” and “Control”, it was confirmed that 4FCA1-b had disappeared by X-ray irradiation. In addition, when the presence or absence of Not I site spots on both sides of 4FCA1-b was examined, upstream 4T15F16-b,
Since the spots of downstream 4T16H5-a and 4T8O5-a can be confirmed, the longest between 4FCA1-b and 4T16H5-a is about 2.4
It was suggested that a deletion of about Mb had occurred. To verify this deletion, 4F13C5 between 4FCA and 4T16H5
Southern analysis was performed using a part of the region as a probe. As a result, as shown in "Southern analysis" in FIG. 8, deletion of this region could be confirmed. Note that “Sou
In the thern analysis, C lane is a sample not subjected to X-ray irradiation, and M lane is a sample subjected to X-ray irradiation.

As described above, it was revealed that the mutant region in the mutant can be detected by using the two-dimensional electrophoresis pattern of the control shown in FIG. That is, by comparing the two-dimensional electrophoresis pattern of the analysis target and the two-dimensional electrophoresis pattern of the control, it became clear that the mutant region in the mutant can be efficiently detected in a very short time.

By the way, in the obtained two-dimensional electrophoresis pattern (“Mutant” in FIG. 8), some spots in the control two-dimensional electrophoresis pattern shown in FIG. 7 could not be confirmed. This is because Not I is sensitive to methylation and no spot appears due to the effect of methylation on chromosomal DNA.
When the chromosomal DNA is hypomethylated by the demethylation treatment, these spots can be confirmed. Therefore, it was revealed that, by using the two-dimensional electrophoresis pattern of the control shown in FIG. 7, not only the mutant region in the mutant but also the DNA modification such as DNA methylation can be detected.

<Search for Abnormal Information> In this example, first, in the above <Creation of control two-dimensional electrophoresis pattern>, abnormal information in the base sequence information on the Arabidopsis thaliana nuclear genome was searched. Specifically, character strings other than A, G, C, and T included in the base sequence information were searched, and the extracted character strings other than A, G, C, and T were regarded as abnormal information. As a result, as an example, as shown in FIG. 9, Accession No. U95973 (DDBJ: h
ttp: //ftp2.ddbj.nig.ac.jp: 8000 / getstart-j.html), the "r", which is the abnormal information, was found at the 27421st position.

In this case, in the two-dimensional electrophoresis pattern of the control shown in FIG. 7, it means that the spot derived from the T19D16 clone contains abnormal information. Therefore, before comparing the two-dimensional electrophoresis pattern of the control shown in FIG. 7 with the two-dimensional electrophoresis pattern of the analysis target, the spot derived from the T19D16 clone is associated with the abnormal information. Therefore, in the above comparison, when a mismatch is found for the spot derived from the T19D16 clone, it is first understood that the abnormal information is associated with the spot derived from the T19D16 clone.

Next, it is determined whether or not the mismatch of the spot derived from the T19D16 clone is a mismatch caused by abnormal information. To determine whether there is a mismatch due to the abnormal information, correct the abnormal information “r” to “G” or “A” to create a two-dimensional electrophoresis pattern of the two types of control, The two-dimensional electrophoresis pattern is compared with the two-dimensional electrophoresis pattern to be analyzed. When the spot derived from the T19D16 clone in the two-dimensional electrophoresis pattern to be analyzed matches the spot derived from the T19D16 clone in the two-dimensional electrophoresis pattern of any of the controls, it was determined that there was a mismatch due to abnormal information. Prove. In this way, by searching for abnormal information in the nucleotide sequence information related to the Arabidopsis thaliana nuclear genome and relating the abnormal information to spots in the control two-dimensional electrophoresis pattern, the two-dimensional electrophoresis pattern to be analyzed is analyzed in more detail. I was able to.

[0078]

As described in detail above, according to the present invention, a control two-dimensional electrophoresis pattern for comparing genomic DNAs to be analyzed can be easily prepared under various conditions. Can be. Therefore, according to the present invention, it is possible to provide a recording medium on which a program capable of easily analyzing a two-dimensional electrophoresis pattern created using genomic DNA is recorded. Further, according to the present invention, it is possible to provide a genomic DNA analysis method by which a control two-dimensional electrophoresis pattern is created based on genomic nucleotide sequence information, thereby enabling quick and reliable analysis.

[0079]

[Sequence List] SEQUENCE LISTING <110> RIKEN <120> A recording media having analysis program for genomic DNA <130> RJH13-047 <150> JP2000-327632 <151> 2000-10-26 <150> JP2001-84370 <151 > 2001-03-23 <160> 15 <170> PatentIn Ver. 2.0 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <220> <221> modified base <222> 4, 5, 6, 7, 8 and 9 <223> n represents a, g, colt <400> 1 ccannnnnnt gg 12 <210> 2 <211> 207 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 2 tccggataat atccataaat gacttgtttt tggtaagagg tggacattac tatatgtaaa 60 ttcgggtgca tgggacacat cagtactcca gtgcatttcg ccctctgagt cagaataaat 120 atattttcta accctctctt taaaatgaaa agtgtatgtt ccctcgcgaa tctcagcaat 180 cacttgttct gattccacat attgatc 207 <210> 3 <211> 163 <212> DNA < 213> Arabidopsis thaliana (Columbia) <400> 3 gatcttttat actgtgtccg attccaaagt tcgttctata catatgaccc gcaatgagga 60 aaagaattgc gatagctaaa tgatgatgtg ccatatcggt tagccataaa ctttgcgttt 120 gtggatggaa tcccc caaga agggttagaa tggcagttcc gga 163 <210> 4 <211> 131 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 4 tccggaatat gagtgtgtga cttgttagaa ttgaccctat ggatagtaca gagaatgggg 60 tctgtcatct ttatcaagat ggttg ctag tgg ctagtggtcattggttagtggtcattggtcattggtcattggctagtgg <211> 134 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 213> Arabidopsis thaliana (Columbia) <400> 6 tccggaggat gccttatata tatattaata tatattatat caaaaagatg gacaatcaaa 60 tctatttctc gattcaatag aagtccaacc aaagaggtga atagggtccc aaataacgag 120 agatatgtaa aaagtaggtc agatttcgcc tattcctaat cctaaatgga atgtaacgac 180 gtagggatc 189 <210> 7 <211> 232 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 7 gatcagccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 60 ttttccgcaa tgggcgaaag cctgacggag caatgccgcg tggaggtaga aggcct acgg 120 gtcctgaact tcttttccca gagaagaagc aatgacggta tctggggaat aagcatcggc 180 taactctgtg ccagcagccg cggtaataca gaggatgcaa gcgttatccg ga 232 <210> 8 <211> 597 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 8 tccggagatt cccgaatagg ttaacctttt gaactgctgc tgaatccatg ggcaggcaag 60 agacaacctg gcgaactgaa acatcttagt agccagagga aaagaaagca aaagcgattc 120 ccgtagtagc ggcgagcgaa atgggagcag cctaaaccgt gaaaacgggg ttgtgggaga 180 gcaaaaaaag cgtcgtgctg ctaggcgaag cggtggagtg ccgcacccta gatggcgaga 240 gtccagtagc cgaaagcatc actagcttat gctctgaccc gagtagcatg gggcacgtgg 300 aatcccgtgt gaatcagcaa ggaccacctt gcaaggctaa atactcctgg gtgaccgata 360 gcgaagtagt accgtgaggg aagggtgaaa agaaccccca tcggggagtg aaatagaaca 420 tgaaaccgta agctcccaag cagtgggagg agccctgggc tctgaccgcg tgcctgttga 480 agaatgagcc ggcgactcat aggcagtggc ttggttaagg gaacccaccg gagccgtagc 540 gaaagcgagt cttcataggg caattgtcac tgcttatgga cccgaacctg ggtgatc 597 <210> 9 <211> 592 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 9 gatcacccag gtt cgggtcc ataagcagtg acaattgccc tatgaagact cgctttcgct 60 acggctccgg tgggttccct taaccaagcc actgcctatg agtcgccggc tcattcttca 120 acaggcacgc ggtcagagcc cagggctcct cccactgctt gggagcttac ggtttcatgt 180 tctatttcac tccccgatgg gggttctttt cacccttccc tcacggtact acttcgctat 240 cggtcaccca ggagtattta gccttgcaag gtggtccttg ctgattcaca cgggattcca 300 cgtgccccat gctactcggg tcagagcata agctagtgat gctttcggct actggactct 360 cgccatctag ggtgcggcac tccaccgctt cgcctagcag cacgacgctt tttttgctct 420 cccacaaccc cgttttcacg gtttaggctg ctcccatttc gctcgccgct actacgggaa 480 tcgcttttgc tttcttttcc tctggctact aagatgtttc agttcgccag gttgtctctt 540 gcctgcccat ggattcagca gcagttcaaa aggttaacct attcgggaat ct 592 <210> 10 <211> 232 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 10 tccggataac gcttgcatcc tctgtattac cgcggctgct ggcacagagt tagccgatgc 60 ttattcccca gataccgtca ttgcttcttc tctgggaaaa gaagttcagg acccgtaggc 120 cttctacctc cacgcggcat tgctccgtca ggctttcgcc cattgcggaa aattccccac 180 tgctgcctcc cgtaggagtc tgggccgtgt ctcag tccca gtgtggctga tc 232 <210> 11 <211> 190 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 11 gatccctacg tcgttacatt ccatttagga ttaggaatag gcgaaatctg acctactttt 60 tacatatctc tcgttatttg ggaccctatt cacctctttg gttggacttc tattgaatcg 120 agaaatagat ttgattgtcc atctttttga tataatatat attaatatat atataaggca 180 tccttccgga 190 <210> 12 <211> 134 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 12 tccggaagca gatgattatt cattcgcttc tcacgttccg tgaatagccg gggcattgag 60 gaatagccag aaaggtattt agggaatcgg tctgattcttcct tctgattcttcttcttcttcttcttcttcttcttcttcattcttcttcttcattcttcttcttcttcttcttcttcttctttattc <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 13 gatcttgtgg gaagagtgtt gccaaattta tactgtccgc tatctcatag gtggctacgg 60 gccgcaccaa aacaaaaaac tttttcttgg ttggtgtaat ccgttggaca taaatccaat 120 ttttaaattt tttggattcc ttcgagtttt tttttcctct tcctggcggt atcaagatgc 180 cactgtgtcg ggatatttta tctgtcttgt ccgga 215 <210> 14 <211> 618 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 14 tccggaggag cacgaatgca agaaggaagt ttaa gtttga tgcaaatggc taaaatttct 60 tcggttttat gtgattatca atcaagtaaa aagttattct atatatcaat tcttacatct 120 cctactaccg gtggagtgac agctagtttt ggtatgttgg gggatatcat tattgccgaa 180 ccctatgcct atattgcatt tgcgggtaaa agagtaattg aacaaacatt gaaaaaagcc 240 gtgcctgaag gttcacaagc ggctgaatct ttattacgta agggcttatt ggatgcaatt 300 gtaccacgta atcttttaaa aggtgttctg agcgagttat ttcagctcca tgcttttttt 360 cctttgaaca caaattaaat aaaatagaac ggttagttta tcagaattaa acgaaaaccc 420 agaaaaatgc atttttcttt caaatcattt ttttttatcg atattcttgt ttactactca 480 gtaaacctct atcaacaagc taaaaagtga atttttttgg gggggaagtt caaattagac 540 tagacaaaca aaaaaaagtt cattttcctc ccttgcttgc atatgtatag ataattcaaa 600 tatagataga tgcagatc 618 <210> 15 <211> 323 <212> DNA <213> Arabidopsis thaliana (Columbia) <400> 15 gatctggtcc aagaagcact actgtaggga agttattgaa accattgaat tccgaatatg 60 gtaaagtagc tcccggatgg ggaacgaccc ctttgatggg tgttgcaatg gctctatttg 120 cggtattcct atctattatt ttggagattt ataattcttc tgttctactg gatggaattt 180 cagtgaatta gactgagaag aatcttg aag ttctagcttt tagctcgata caaaaaagta 240 aagtatgcag gtctaacaat tttagcctat tctcctttgg tagttcgacc gcgaaatttt 300 tttctgcatt gtatatttcc gga 323

[0080]

[Sequence List Free Text] SEQ ID NO: 1: n represents a, g, c or t (location: 4th to 9th bases)

[Brief description of the drawings]

FIG. 1 is a diagram showing a step of creating a two-dimensional electrophoresis pattern using a genomic DNA to be analyzed.

FIG. 2 is a view showing a step of performing labeling using an adapter.

FIG. 3 is a configuration diagram schematically showing a system for executing a recording medium on which a program according to the present invention is recorded.

FIG. 4 is a flowchart illustrating processing for executing a program.

FIG. 5 is a diagram showing a two-dimensional electrophoresis pattern of a control prepared based on chloroplast DNA sequence information of Arabidopsis thaliana.

FIG. 6 is a photograph showing a two-dimensional electrophoresis pattern of an analysis target prepared using chloroplast DNA of Arabidopsis thaliana.

FIG. 7 is a diagram showing a two-dimensional electrophoresis pattern of a control prepared based on the nucleotide sequence information of chromosomal DNA of Arabidopsis thaliana.

FIG. 8 is a schematic diagram showing an example in which actual analysis was performed using a two-dimensional electrophoresis pattern of a control.

FIG. 9 is a schematic diagram showing an example of abnormal information obtained as a result of searching for abnormal information in the nucleotide sequence information on the Arabidopsis thaliana nuclear genome.

[Explanation of symbols]

501 CPU, 503 RAM, 504 input section, 505 transmission / reception section, 506 output section, 507 HDD, 508 CD-ROM drive, 50
9 CD-ROM, 510 database

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 27/26 325 (72) Inventor Tomoko Abe 2-1, Hirosawa, Wako-shi, Saitama Pref. ) Inventor Shunichi Ebisaki 2-1, Hirosawa, Wako-shi, Saitama F-term in RIKEN (reference)

Claims (25)

[Claims] 1. A computer, comprising: a procedure for creating a control two-dimensional electrophoresis pattern based on genomic base sequence information; a two-dimensional electrophoresis pattern using the control two-dimensional electrophoresis pattern; and a genomic DNA to be analyzed. A procedure for comparing the two-dimensional electrophoresis pattern of the analysis target obtained by performing the electrophoresis, and a procedure for detecting a difference in a spot position between the two-dimensional electrophoresis pattern of the control and the two-dimensional electrophoresis pattern of the analysis target And a program to make it run. 2. In the step of creating a two-dimensional electrophoresis pattern of the control, a recognition sequence of a first restriction enzyme and a recognition sequence of a second restriction enzyme in the genomic base sequence information are detected, and the first and second restriction enzymes are detected. The base sequence sandwiched between the recognition sequence of the restriction enzyme and the recognition sequence of the second restriction enzyme, and the base sequence sandwiched between the recognition sequence of the first restriction enzyme and the recognition sequence of the first restriction enzyme 2. The program according to claim 1, wherein a two-dimensional electrophoresis pattern of the control is created based on the control. 3. The program according to claim 2, wherein the first restriction enzyme and the second restriction enzyme are selected from methylation-insensitive restriction enzymes. 4. The program according to claim 2, wherein the first restriction enzyme and the second restriction enzyme are selected from methylation-sensitive restriction enzymes. 5. The method according to claim 1, further comprising a step of obtaining said genomic base sequence information via a communication network.
The program described. 6. The step of creating a two-dimensional electrophoresis pattern of the control includes creating a plurality of spots based on genomic base sequence information and linking the plurality of spots with locus information on a genome. The program according to claim 1, wherein: 7. The step of creating a control two-dimensional electrophoresis pattern based on genomic nucleotide sequence information,
2. The program according to claim 1, wherein abnormal information included in the genome base sequence information is detected. 8. The program according to claim 7, wherein the detected abnormal information is stored in association with spots included in the created two-dimensional electrophoresis pattern. 9. A step of creating a two-dimensional electrophoresis pattern by performing two-dimensional electrophoresis using genomic DNA to be analyzed, and a control created based on the two-dimensional electrophoresis pattern and genomic nucleotide sequence information. Comparing a two-dimensional electrophoresis pattern with a two-dimensional electrophoresis pattern, and detecting a difference in a spot position between the two-dimensional electrophoresis pattern of the control and the two-dimensional electrophoresis pattern of the analysis target. . 10. The genomic DNA analysis method according to claim 9, further comprising the step of extracting the genomic DNA to be analyzed from plant cells. 11. The genomic DNA analysis method according to claim 9, wherein the genomic DNA to be analyzed is derived from a higher organism. 12. The following steps: (a) a step of treating genomic DNA with a first restriction enzyme; (b) a step of adding a label to the restriction enzyme cleavage site; and (c) electrophoresis of the obtained DNA fragment. One-dimensional fractionation by electrophoresis, (d) step (c)
Treating the DNA fragment fractionated by the above with a second restriction enzyme, and then performing a two-dimensional fractionation, (e) detecting a spot of the labeled DNA fragment fractionated by the step (d), The genomic DNA analysis method according to claim 9, wherein the step of creating a two-dimensional electrophoresis pattern of the genomic DNA to be analyzed is performed. 13. The genome according to claim 12, wherein after the step (b), the step (c) is performed after treating with a restriction enzyme different from the first restriction enzyme and the second restriction enzyme. DNA analysis method. 14. A two-dimensional electrophoresis pattern of the control is created based on the cleavage sites by detecting the recognition sequence of the first restriction enzyme and the recognition sequence of the second restriction enzyme in the genome base sequence information. The genomic DNA analysis method according to claim 12, wherein 15. The method according to claim 12, wherein the step (b) is carried out by linking one of the adapters to the restriction enzyme cleavage part and adding the label to the other of the adapters. Genomic DNA analysis method. 16. The control two-dimensional electrophoresis pattern has a plurality of spots created based on genomic nucleotide sequence information, and links the plurality of spots with locus information on a genome. The genomic DNA analysis method according to claim 9, wherein: 17. The method according to claim 9, wherein prior to generating a control two-dimensional electrophoresis pattern based on the genomic base sequence information, abnormal information included in the genomic base sequence information is detected. Genomic DNA analysis method. 18. The program according to claim 17, wherein the detected abnormal information is associated with spots included in the created two-dimensional electrophoresis pattern. 19. Identification of a mutated gene and / or a gene involved in a mutated trait, characterized by detecting a mutated portion in a genomic DNA to be analyzed by the genomic DNA analysis method according to any one of claims 9 to 18. Method. 20. A mutated gene and / or a gene involved in a mutated trait, wherein a DNA fragment containing a displacement site detected by the identification method according to claim 19 is isolated from the two-dimensional electrophoresis pattern. Isolation method. 21. A two-dimensional electrophoresis pattern which is created based on genomic base sequence information and has a plurality of spots estimated based on the genomic base sequence information. 22. The two-dimensional electrophoresis pattern according to claim 21, wherein the plurality of spots are linked to locus information on a genome. 23. The two-dimensional electrophoresis pattern according to claim 21, wherein the genomic base sequence information is base sequence information obtained from a nuclear genome of a plant. 24. The two-dimensional electrophoresis pattern according to claim 21, wherein the plurality of spots are associated with abnormal information retrieved from the genome base sequence information. 25. The two-dimensional electrophoresis pattern according to claim 21, wherein the genomic base sequence information is base sequence information obtained from a genome of a higher organism.