JP2003038160A - Device and method for designing probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect the possibility of an unknown gene (or DNA fragment) mixing in a test sample in a DNA chip even in case the gene is contained in the sample. SOLUTION: A plurality of auxiliary probes are designed and then disposed in the DNA chip in advance; thereby, even in case an unexpected gene (or DNA fragment) is contained in the test sample, the possibility of the gene's mixing therein is judged; wherein the policy of designing the auxiliary probes is the following: it is assumed that, in the whole DNA sequence to be identified, there is high possibility of the presence of similarity to an unknown DNA sequence, and such a probe as to cover the known DNA sequence and serve as a similar proximity is selected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA chip technique for discriminating a plurality of types of DNA contained in a sample.

[0002]

2. Description of the Related Art With the recent development of gene analysis technology,
The function and structure of genes have gradually become clear. Above all, the technology of a DNA chip or a DNA microarray (hereinafter, collectively referred to as a DNA chip) has attracted attention as an effective means for gene analysis.

A DNA chip is a high density array of many different DNAs (probes) on the surface of a substrate made of glass, silicon, plastic or the like. As the probe, usually, cDNA, short-chain nucleotides of about 20 to 30 bases, etc. are used. The principle of the DNA chip is that in the four bases A (adenine), T (thymine), G (guanine), and C (cytosine) that make up DNA, A and T, and G and C are hydrogen-bonded to each other. It is based on hybridization. The target is captured by hybridizing DNA or RNA (target) labeled with a fluorescent substance or the like on this DNA chip. The captured target is detected as a fluorescence signal from each spot, and by analyzing the data with a computer, it becomes possible to observe the status of thousands to tens of thousands of DNAs in the target at once.

As one of the methods of using a DNA chip, by capturing a target gene (or DNA fragment), it is possible to observe whether or not the target DNA to be investigated is contained in the sample, and to determine the sequence of the captured DNA. Usage (SBH method) to read and examine DNA polymorphisms such as SNP
There is. For example, DN in clinical tests and food tests
The bacterial identification using the A chip will be described. Bacterial DNA
Contains a 16S ribosomal RNA gene (16S rDNA) in common. This base sequence is different for each bacterium,
As of 1997, the nucleotide sequences of about 90% of the bacteria that have been identified at present have been clarified, and the effective use of this nucleotide sequence information enables accurate identification of the taxonomic position of all bacteria. It may be possible to decide (Hiraishi,
A .: Bulletin of Japanese Society of Microbial Ecolog
y 10, (1), 31-42, 1995).

In the identification of bacteria using a DNA chip, sequence information of 16S rDNA of each bacterium is obtained from a DNA sequence database or the like, a bacterium-specific portion is selected, and this is placed on the DNA chip as a probe. On the other hand, bacteria are extracted from blood or sputum collected from a patient, 16S rDNA is excised and specifically amplified by PCR to target it.

FIG. 1 is a diagram schematically showing this. Specific subsequences are selected from the DNA sequences of 20 types of bacteria such as bacteria P, Q, and R, and placed on a DNA chip. The positions of these probes are horizontal No. (1-5) and vertical No.
Expressed as (1 to 4). As a result of reacting the sample 110 containing the bacterial DNA extracted from the patient's blood, sputum, etc. with the probe on the DNA chip 100, two corresponding to (horizontal No1, vertical No2) and (horizontal No3, vertical No5) Suppose a signal is observed from one spot. At this time, from the table that manages the position information of the probe, the bacteria [Actinobacillus actinomycete
mcomitans] (horizontal No1, vertical No2) and bacteria [Klebsiella oxy
toca] (horizontal No3, vertical No5) is mixed (possibly) in the sample.

[0007]

However, unexpected bacterial mutants appear in actual clinical and food testing laboratory sites. For this reason, the target DNA fragment extracted at the experimental site cannot be detected because it does not have the probe of the corresponding complementary sequence on the DNA chip prepared in advance. In addition, when the type of bacteria or food that has been assumed in advance is determined, unknown hybridization results may be added, which may cause confusion.

[0008] At the inspection site, the information that a new bacterium has been generated can be quickly transmitted via the Internet and the necessary data can be obtained, but a new DNA chip that can detect a new bacterium is required. It seems that it will take some time and cost to obtain it. For this reason, it is desirable to be able to determine the possibility of contamination with new bacteria by using the DNA chip at hand. If a DNA chip is prepared so that the contamination of new bacteria can be determined, for example, a rapid service for bacterial tests as shown in FIG. 2 can be realized. In Figure 2, information on new bacteria collected at overseas bacterial information center X is transmitted to the domestic bacterial information center Y via the Internet. What kind of experimental results can be observed by the DNA chip? "And is applied to the judgment of the inspection result at the laboratory B.

On the other hand, as a technique for measuring a mismatch between a DNA fragment of a known target and a probe of a DNA chip, for example, cross-hybridization is performed by Michael D. Kane et al .: Assessment of the sens.
itivity and specificity of oligonucleotide (50mer)
microarrays: Nucleic Acids Res., 28 (22), 4552-4557,
There are 2000 reports. Also, as an attempt to prevent cross-hybridization, a method of selecting a probe specific to a sequence (Ken-ichi Kurata et al .:
Probe Design for DNA Chips: Genome Informatics 19
99, 225-6, 1999). In addition, a method of predicting how much the original fluorescence signal is from the cross hybridization to some extent (Mitsuteru Nakao et al .: Quantitative Estimation)
of Cross-Hybridization in DNAMicroarrays Based on
a Linear Model: Genome Informatics 2000, 231-232,2
000) is also proposed.

[0010] However, these techniques are for measuring the experimental accuracy with respect to a target DNA in a range assumed in advance, and are a method for handling DNA fragments obtained from unexpected unknown bacteria and new kinds of foods. Not not. Assuming detection of an unknown DNA sequence, for example, if a probe having a length of about 25 bases is used, it is necessary to prepare 4 to the 25th power probe according to a general method. However, this is not a practical method, considering that the upper limit of the number of spots that can be arranged on the DNA chip is about tens of thousands. Furthermore, it is necessary to narrow down the probe candidates and select a collection of probes for efficient unknown sequence detection.

The present invention has been made in view of the above problems of the prior art.
An object of the present invention is to provide a method for maximizing the possibility of contamination of a DNA chip even when an unknown target gene (or DNA fragment) is contained in a test sample.

[0012]

[Means for Solving the Problems] In order to achieve the above object, in the present invention, an unknown gene (or DNA fragment) is provided on a DNA chip in addition to a probe for a bacterial species or the like which is a target to be identified in advance. By designing multiple auxiliary probes for capturing DNA and placing them on the DNA chip in advance,
Even if the target contains an unexpected gene (or DNA fragment), the possibility of contamination can be determined.

The auxiliary probe is selected according to the following procedure. First, by limiting the region in the genome of the observation target, the range of objects (bacteria and food types) to be identified and their range.
Determine the DNA sequence. Next, the region in this genome is divided into two types. From the data of the DNA to be identified, the part having the common sequence is selected and used as the common region.
On the other hand, a part having a kind that is slightly different is defined as a mutation region. For the common area, multiple coated probes are selected to cover it. On the other hand, for each mutated region, a plurality of neighboring probes having high homology to this probe are prepared based on the probe for identifying bacteria mutated therein. Auxiliary probes are determined based on these coated probes and neighboring probes and placed on the DNA chip to determine the possibility of contamination of unknown genes (or DNA fragments) from the hybridization reaction results.

That is, the present invention provides a probe design apparatus for designing an auxiliary probe set, which is similar to a plurality of known biopolymers to be identified and hybridizes with biopolymers not included in the identification target, The base sequence of a known biopolymer is provided with a common sequence region determination processing unit that divides into a common region having the same sequence regardless of the type of biopolymer and a mutation region containing a mutation. It is characterized in that auxiliary probes are designed separately for the determined common region and mutation region.

This probe designing device also selects a plurality of auxiliary probes that cover the common region and overlap each other, and assume that the mutation region does not contain a mutation. A coated probe selection processing unit is provided which determines a sequence and covers a plurality of sequences and selects a plurality of auxiliary probes overlapping each other. Also,
For each mutated partial sequence in the mutated region, a neighboring probe selection processing unit is provided for selecting a plurality of auxiliary probes similar to the partial sequence. Furthermore, a base exchange adjustment processing unit that changes a part of the base sequence in the selected auxiliary probe is provided so that hybridization does not occur with respect to the biopolymer to be identified.

The probe designing method according to the present invention is a probe designing method for designing an auxiliary probe set which is similar to a plurality of types of known biopolymers to be identified and which hybridizes with biopolymers not included in the identifications. In, in the known biopolymer, the base sequence of the biopolymer is divided into a common region having the same sequence regardless of the type of biopolymer and a mutation region containing a mutation, and the common region and the mutation region separately. Designing an auxiliary probe.

The steps of separately designing auxiliary probes for the common region and the mutant region include selecting auxiliary probes that cover the common region and overlap each other, and for the mutant region. , A step of obtaining an array assuming that the mutation is not included,
Selecting sub-probes that cover sequences conforming to the consensus sequence and that overlap each other. Further, for each mutated partial sequence in the mutation region, a step of selecting a plurality of auxiliary probes similar to the sequence may be included. Further, it is preferable to include a step of changing a part of the base sequence in the selected auxiliary probe so that hybridization does not occur with respect to the biopolymer to be identified.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, one mode for carrying out the present invention will be specifically described with reference to the drawings. Figure 3
FIG. 3 is a block diagram showing a configuration example of a probe design apparatus of the present invention for designing a probe. This probe design device includes a sequence database 301 in which information on target DNA used for designing a probe DNA sequence is stored, an input screen for inputting necessary information and a display device 302 for displaying a calculation result, etc. An input means such as a keyboard 303 and a mouse 304 for inputting and selecting values, a central processing unit 305 for performing various probe selection processing, and a program memory 306 for storing programs necessary for the processing in the central processing unit 305. It is equipped with. The program memory 306 stores the common array region determination processing program 30.
7, identification probe selection processing program 308, coated probe selection processing program 309, neighboring probe selection processing program 310, base exchange adjustment processing program 31
1, a sequence generation processing program with mutation 312, and an auxiliary probe adjustment processing program 313 are provided. These programs are CD-ROM, DVD-ROM, M
It can be provided by being stored in a recording medium such as O or a floppy disk, or can be provided via a network. The DNA sequence database 301 may be held by a storage device connected to the central processing unit 305, or may be managed by a server computer installed in a remote place, and the gene data is transferred from the database via a network or the like. May be acquired.

In the present invention, unlike a normal DNA chip, the probe to be placed on the DNA chip captures an unknown gene (or a DNA fragment) in addition to a probe for a bacterial species which is a target to be identified in advance. Auxiliary probe for This auxiliary probe is selected according to the following procedure.

FIG. 4 shows a schematic flow of selecting an auxiliary probe. First, the region in the genome of the observation target is designated (step 400), and the range of the object (the type of bacteria or food) to be identified and the sequences of those DNAs are input from the sequence database 301 (step 401). For example, 16S ribosomal RNA is a region in the bacterial genome.
When the gene (16S rDNA) is selected, this region has been revealed by reading the sequence of various bacteria, which is convenient when preparing a DNA chip for bacterial identification.

It is assumed that the unknown DNA sequence to be detected is a part of any one of the entire DNA sequence input in step 401 which is mutated. Therefore, it is considered that there is a high possibility that the entire input DNA sequence may be similar to the mutant sequence, and an auxiliary probe effective for detecting an unknown DNA sequence will be selected as follows.

Next, the common sequence region determination processing program 3
According to 07, the region in the genome is divided into two types (step 402). From the data of the DNA to be identified, a portion having a common sequence in all DNAs is selected and used as a common region. On the other hand, a part having a kind that is slightly different is defined as a mutation region.

FIG. 5 is a diagram schematically showing a common region and a mutation region of 16S rDNA in the case of identifying a plurality of bacteria targeting 16S rDNA. Here, 16S rDNA of 8 kinds of bacteria
On the other hand, the union of the parts with mutations was taken, and its complement was set as the common region. In addition, regions with a large number of mutations were collected, and the regions linked therein were designated as mutation regions A, B, and C, respectively.
As can be seen from the figure, all the bacteria 1 to 8 have the same sequence for the common region. On the other hand, not all bacteria 1-8 have the same sequence for the mutated region.

Next, for the common area, the coating probe selection processing program 309 selects a plurality of coating probes that cover the common area (step 403). Figure 6
Shows two ways of taking a coated probe for the common region. Each probe represents one that hybridizes to a position in the corresponding common region. The upper part shows how to take a coated probe that overlaps by one-half, and the lower part shows how to overlap by two-thirds. These are DN as the degree of overlap increases.
It is preferable because the identification accuracy of the A sequence is improved. However D
Since the number of probes that must be prepared on the NA chip also increases, it is necessary to determine the degree of overlap in consideration of the number of spots that can be arranged and the cost.

Further, the base exchange adjustment processing program 311
Thus, some of the bases of these coated probes at the positions shown in FIG. 6 are replaced and adjusted so as not to hybridize with the common region. This may be done by replacing some base sequences at the center of the probe. By this adjustment, the coated probe does not hybridize to all the bacterial species to be identified at the position in the common region. If the coated probe is adjusted so that it will not hybridize to other genomic regions, if even one signal is observed from the coated probe, it can be judged that the variant may be mixed. .

On the contrary, if some of the bases of each coated probe are selected as they are without being replaced, hybridization occurs without fail even if any species to be identified is contained, and a signal is observed from the corresponding spot. It Therefore, in the experiment conducted on the isolated species, it can be judged that there is a possibility that some kind of mutant species is contaminated if at least one signal is not observed from the specific coated probe. On the other hand, for the mutated region, more probes than the common region are selected depending on the situation of the mutation.
(Steps 404 and 405).

First, for the mutation region, the common sequence region determination processing program 307 determines a sequence (a sequence corresponding to the common sequence) when it is assumed that no mutation is contained. This is a sequence that is common to most of the bacterial and food species to be identified. Since each mutation region is a region where at least one mutation is found in the bacterial species or food species to be identified, it is not possible to find a sequence common to all species, but it is generally specified. When attention is paid to the location, it is considered that most of them are not mutated, so it is possible to determine a sequence that conforms to such a common sequence. For example, in FIG. 5, the sequences of Bacterium 3, Bacterium 5, and Bacterium 8 are mutated in the mutation region B, but the same sequence is the same in other bacteria.
Therefore, in the case of the example of FIG. 5, the sequence conforming to the common sequence of the mutation region B is the sequence of the mutation region B possessed by bacteria 1, 2, 4, 6, 7.

The same processing as that for the above-mentioned common region is performed on the sequence conforming to this common sequence, and the coating probe is selected by the coating probe selection processing program 309 (step 404). Furthermore, for each mutated region, based on a probe for identifying a bacterium that is mutated in the mutated region, a neighborhood probe having a high homology to this probe is selected by the neighborhood probe selection processing program 310. A plurality is prepared (step 405). Figure 7
Is a detailed flow of this step 405.

First, for each bacterial species or food species that is actually mutated in each mutated region, the discrimination probe selection processing program 308 discriminates them by using the mutation in this region. Find the probe (step 700). This can be calculated by applying the conventional probe design algorithm only to this mutation region.

Next, for each identification probe,
The sequence with mutation generation program 312 embeds this into a sequence that conforms to the common sequence in the mutation region to create a sequence with mutation (step 701). With respect to this sequence with mutation, the coated probe selection processing program 309 creates a coated probe that covers the particularly embedded identification probe portion (step 702). With each of the coated probes as an axis, a part of the bases is replaced by the base exchange adjustment processing program 311, and a plurality of neighboring probes are obtained by the neighboring probe selection processing program 310 (step 703).

The following policy can be adopted when obtaining this neighborhood probe by the neighborhood probe selection processing program 310. (1) The original mutated portion is preserved, and the common sequence portion is selected so as to have variations. (2) The common sequence part is preserved, and the original mutation part is selected so as to have variation. (3) The original mutant portion and the common sequence portion are not distinguished from each other and selected so as to have variations as a whole.

FIG. 8 is a diagram showing a method of creating a neighbor probe candidate according to the flow shown in FIG. Taking the mutation region B of bacterium 3 shown in FIG. 5 as an example, the identification probe b
3 schematically shows a procedure for creating a candidate for a probe near to. Similarly, candidates for neighboring probes are created for the other identification probes a and c.

These neighboring probes created in accordance with the flow of FIG. 7 further prevent the hybridization from occurring in all the identification objects including the identification objects of the original identification probe so that the base exchange adjustment processing program 31
Adjust by 1. Then, if a signal is observed from at least one of the neighboring probes, it can be determined that there is a possibility that the mutant species is contaminated, and that any bacterial or food species and DNA
Information on whether the sequences are similar is also obtained.

Furthermore, it is effective to select similar neighboring probes for the partial sequences in the common region. Also, from biological knowledge, in a particular sequence
If it is known that there is a high possibility that DNA will be mutated, and if it can be determined that the sequence is contained in the region of the observed genome, it is advisable to select neighboring probes with the sequence as the axis. desirable.

Since the coated probe and the neighboring probe selected by the above procedure include the procedure of exchanging a part of the bases, a plurality of candidates can be considered. Therefore,
When selecting a probe, several candidates are always selected and retained, and finally, the auxiliary probe adjustment processing program 31
According to 3, the coated probe and the neighboring probe are combined, and each probe is selected so as not to hybridize with the entire region of the genome to be identified. The probes obtained as a result are combined and used as an auxiliary probe (step 406).

The auxiliary probe obtained by the above procedure is DNA
When placed on a chip and tested, no signal should be observed at the spot for the auxiliary probe when the target species for which identification is assumed is limited and predicted within the range of the genomic region of interest. Therefore, assuming that the following results are obtained as experimental data, DNA contamination of an unknown species is presumed.

(1) When a signal is observed at a spot where a coating probe for the common region is arranged (2) When a signal is observed at a spot where a coating probe for a common sequence of a certain mutation region is observed (3) A mutation When a signal is observed in a spot where a probe near the specific mutation part of the region is placed, in each of these cases, in the common region, in the common sequence in the mutation region, in the mutation part in the mutation region, the species to be identified It can be judged that there is a possibility of contamination by new bacteria having a close sequence.

[0038]

As described above, according to the present invention,
By placing the auxiliary probe selected as described above on the DNA chip and preparing the test result data for the bacterial species or food species that are the identification targets in advance, When the data of is obtained, it is possible to judge the possibility that a gene (or DNA fragment) of an unknown bacterial species or food species is mixed in the test sample. Furthermore, since the possibility can be estimated from the information of the mutation site, the possibility can be confirmed by comparing with external information obtained from the Internet or the like.

[Brief description of drawings]

FIG. 1 For the purpose of identifying the bacteria contained in the sample
Schematic diagram when using a DNA chip.

FIG. 2 is an explanatory diagram of information transmission regarding a new type of bacterium via the Internet.

FIG. 3 is a block diagram showing a configuration example of a probe designing device according to the present invention.

FIG. 4 is a schematic processing flowchart of the present invention.

FIG. 5 is a diagram showing a genomic region of interest, a common region and a mutation region in the genomic region.

FIG. 6 is a diagram showing how to take a coated probe for a common region.

FIG. 7 is a detailed flowchart of selection of a proximity probe.

FIG. 8 is a diagram showing a procedure for creating a candidate for a nearby probe by focusing on a mutated portion.

[Explanation of symbols]

100 ... DNA chip, 110 ... Sample, 301 ... Sequence database, 302 ... Display device, 305 ... Central processing unit,
306 ... Program memory

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 F (72) Inventor Yasuyuki Nozaki 6-chome, Onoue-cho, Naka-ku, Yokohama-shi, Kanagawa 81 Hitachi Software Engineering Co., Ltd. In-house (72) Inventor Toshiko Matsumoto 6-81, Onoe-machi, Naka-ku, Yokohama-shi, Kanagawa Hitachi Software Engineering Co., Ltd. In-house (72) Inoue Shino Onoe-machi, Naka-ku, Yokohama-shi, Kanagawa 6-81 Hitachi Software Engineering Co., Ltd. In-house (72) Inventor Takuro Tamura 6-81 Onoue-cho, Naka-ku, Yokohama-shi Kanagawa Hitachi Software Engineering Co., Ltd. Internal F-term (reference) 4B024 AA11 AA20 CA01 CA09 HA14 HA19 4B029 AA07 AA21 AA23 BB20 CC03 FA15 4B063 QA01 QA 12 QA17 QA18 QQ06 QQ42 QR32 QR38 QR56 QR69 QR80 QR82 QS24 QS25 QS28 QS34 QS39 QX02

Claims (8)

[Claims] 1. A probe design apparatus for designing an auxiliary probe set, which is similar to a plurality of types of known biopolymers to be identified and hybridizes with biopolymers not included in the identification target, The base sequence of the macromolecule is provided with a common sequence region determination processing unit that divides into a common region having the same sequence regardless of the type of biopolymer and a mutation region containing a mutation, and is determined by the common sequence region determination processing unit. A probe designing device, characterized in that auxiliary probes are separately designed for the common region and the mutation region. 2. The probe design apparatus according to claim 1, wherein a plurality of auxiliary probes that cover the common region and overlap each other are selected, and the mutation region contains a mutation. A probe designing apparatus comprising: a covered probe selection processing unit that obtains an array when it is assumed that there is no such array, and covers the array and selects a plurality of auxiliary probes that overlap each other. 3. The probe designing apparatus according to claim 1, further comprising: for each mutated partial sequence in the mutated region, a neighboring probe selection processing unit that selects a plurality of auxiliary probes similar to the partial sequence. A probe design device characterized in that 4. The probe designing apparatus according to claim 1, 2 or 3, wherein a part of the base sequence in the auxiliary probe selected is selected so that hybridization does not occur with the biopolymer to be identified. A probe design apparatus comprising a base exchange adjustment processing unit to be changed. 5. A probe design method for designing an auxiliary probe set, which is similar to a plurality of types of known biopolymers to be identified and hybridizes with biopolymers not included in the identification target, The base sequence of the polymer is divided into a common region having the same sequence regardless of the type of biopolymer and a mutation region containing a mutation, and an auxiliary probe is separately provided for the common region and the mutation region. A method of designing a probe, comprising: a step of designing. 6. The method for designing a probe according to claim 5, wherein the step of separately designing an auxiliary probe for the common region and the mutation region covers the common region and overlaps each other. A step of selecting an auxiliary probe, a step of obtaining a sequence in the case of assuming that a mutation is not included in the mutated region, a step of covering a sequence corresponding to the common sequence, and selecting auxiliary probes overlapping each other A method of designing a probe, comprising: 7. The probe designing method according to claim 5, further comprising the step of selecting, for each mutated partial sequence in the mutation region, a plurality of auxiliary probes similar to the sequence. Probe design method. 8. The probe designing method according to claim 5, 6 or 7, wherein a part of the base sequence in the auxiliary probe selected is selected so that hybridization does not occur with the biopolymer to be identified. A method of designing a probe, comprising the step of changing.