JP2007174986A - Method for analyzing base sequence of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hybridization method not requiring setting of stringency optimum and common to a plurality of probe species in a method for analyzing point mutation of single nucleotide polymorphism or the like by using a DNA microarray carrying an oligonucleic acid probe. <P>SOLUTION: A plurality of the probe species are hybridized by giving a plurality of stringencies to each probe species by separating a specimen to two or more when hybridizing target nucleic acids in the specimen with the plurality of the probe species on a solid carrier. The detection of up to a single nucleotide polymorphism can be carried out by designing a probe comprising a completely complemental base sequence to the target nucleic acid, and a probe having a base sequence containing a mismatch in a probe sequence, and comparing the hybridization results. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for analyzing a base sequence of a nucleic acid useful for detecting a point mutation in a similar nucleic acid sequence, represented by a single nucleotide polymorphism (SNP). The present invention also relates to a method for determining the presence or absence of a target nucleic acid in a sample.

  As represented by the Human Genome Project, various analyzes have been made on the base sequences of various organisms such as genomic genes and mitochondrial genes. Furthermore, research on the relationship between the elucidated genes and the mechanisms of life activities, various diseases, diseases, genetic constitutions, etc. is also progressing, and research results have been reported one after another. From these research results, knowing the presence or absence of a specific gene, its nucleotide sequence variation, or the abundance (expression level) of its expression product, for example, more detailed features regarding factors such as various diseases and diseases It has been found that useful information can be obtained in the pasting and typing. In addition, when more detailed characterization and typing is performed on factors such as various diseases and diseases, it becomes easier to select an effective treatment method, and it is effectively used not only for diagnosis but also for treatment of the disease. It has also been verified that it is possible.

  As a method for detecting the presence or absence of a specific gene and the expression level thereof in a specimen, many methods have been proposed and used in practice. When the base sequence of the gene or nucleic acid molecule to be detected is known, the probe hybridization method is the most widely used method. In this technique, a characteristic partial base sequence is selected from the base sequence of a gene or nucleic acid molecule to be detected, and a nucleic acid probe having a complementary base sequence is used. This nucleic acid probe detects the presence or content of a nucleic acid chain containing such a characteristic partial base sequence. Specifically, a DNA strand for a probe having a complementary base sequence is prepared in advance, and a gene or nucleic acid molecule contained in a sample is made into a single-stranded nucleic acid, and a hybridization reaction is carried out with this DNA probe. . The presence or absence of the hybrid formation based on this reaction or the amount of formation is detected by some method.

  In this method of detecting a specific nucleic acid molecule by the probe hybridization method, the hybridization reaction itself can be performed either in the liquid phase or on the solid surface as long as the formed hybrid can be separated. Good. For example, when a hybridization reaction is performed on a solid phase surface, a DNA probe is previously immobilized on the solid phase surface by binding or adsorption, and the formed hybrid is immobilized on the solid phase. To separate. At that time, a labeled nucleic acid that has been labeled with a certain detectable labeling substance on a nucleic acid sample contained in a specimen, formed a hybrid with a DNA probe, and fixed and separated on a solid phase The presence / absence of a chain and the amount thereof are measured using a signal resulting from the labeling substance. Further, as a solid phase (base material) for fixing a DNA probe, a chip using a flat substrate surface such as glass or metal or a bead using a microparticle surface is a body surface.

  In the probe hybridization method, the first reason why a hybridization reaction using a DNA probe immobilized on a solid phase is preferred is that B / F separation is easy. In addition, since a DNA probe immobilized at a predetermined position on the solid phase is used, the detection region can be physically miniaturized, and as a result of specifying the detection region, highly sensitive measurement is possible. Become. At this time, multiple items can be detected at the same time by physically isolating the fixing positions of a plurality of types of DNA probes. Furthermore, if a form of a DNA microarray (or DNA chip) in which a predetermined amount of DNA probes are immobilized on a solid phase is selected in advance, there is an advantage that the handling and application thereof become easier.

  For example, Patent Document 1 discloses a technique in which a nucleic acid labeled with a fluorescent dye is allowed to act on oligo DNA synthesized on a flat substrate, and the resulting hybrid is detected by fluorescence derived from the fluorescent label. Is disclosed. By using a DNA array composed of oligo DNA synthesized on this substrate and a fluorescence detection method using a fluorescent dye label, it is possible to detect the presence and amount of a specific nucleic acid contained in a specimen.

Further, Patent Document 2 discloses that a DNA array is produced by applying a method of immobilizing a plurality of separately prepared DNA probes using an amino group previously introduced on the substrate surface. . In Patent Document 2, this DNA array is used to detect a labeled 22-mer single-stranded DNA.
US Pat. No. 6,410,229 JP 2001-128683 A

  A DNA microarray can simultaneously detect multiple items for the same specimen, and can be used as an effective means in determining the presence or absence of a specific gene and quantifying its expression.

  As a DNA microarray for quantifying the expression level of a gene to be examined, a cDNA array using the full length cDNA of the gene to be examined as a probe is known. However, this cDNA array often cannot detect single nucleotide polymorphisms (SNPs), mutations (including insertions and deletions), etc. with high accuracy. The reason for this is that the cDNA array generally has a length exceeding 100 bases for both the labeled DNA derived from the specimen and the probe DNA. With such a base length, the probe DNA and the labeled DNA are hybridized to the same extent regardless of the presence or absence of a single nucleotide polymorphism, making mutation discrimination impossible. Therefore, partial detection of single nucleotide polymorphisms has been analyzed by a technique capable of discriminating single nucleotide polymorphisms such as a sequence method, an RFLP method, an SSCP method, and a Tuckman method. However, in the case of humans, the number of single nucleotide polymorphisms and mutations in a gene is evenly distributed over the entire genome in excess of 100,000, so the entire gene can be analyzed using the above method. It was difficult to do.

  Therefore, in recent years, attempts have been made to comprehensively analyze single nucleotide polymorphisms using a DNA microarray using oligo DNA as probe DNA. If the 20-mer to 50-er single-stranded DNA is used as the probe DNA, the difference in Tm (melting temperature) can be made even if there is only a difference of about one base. Therefore, optimum stringency conditions including the hybridization temperature are set. This is because single nucleotide polymorphisms can be analyzed.

  However, in this case, it is a problem to adjust the sequence design between DNA probes having various base compositions and the number of bases, and to set common stringency conditions optimal for all the DNA probes in one DNA array. Become. The solution of this problem is very difficult compared to the cDNA array in the following points.

  First, in the optimal DNA probe sequence design, when a 20-mer to 50-mer single-stranded DNA is used as the probe DNA, the complementation with the base sequence other than the target region of the target sample is not or low. It is necessary to consider. In addition, in the expression analysis that examines the presence or absence of a gene, the entire sequence of the target gene is different from the target range of the probe sequence.When single nucleotide polymorphism is analyzed, the position to be detected in the sequence of the target gene The target range of the probe sequence is limited. For this reason, if priority is given to the setting of stringency conditions such as the hybridization temperature, the arrangement of probes becomes more difficult.

In setting the hybridization temperature, for example, it is necessary to detect a single nucleotide polymorphism in an extremely GC-rich or AT-rich sequence. For example, when designing a base sequence from a Tm value, it is common to use an average base composition and the number of bases corresponding to a desired Tm value. However, if an attempt is made to design a base sequence that is extremely GC-rich or AT-rich based on Tm set in advance in this way, there are cases where it becomes 20 mer or less, or 40 mer or more. In this case, if it is 20 mer or less, the possibility of hybridization other than the target region increases, and if it is 50 mer or more, the ability to distinguish single nucleotide polymorphisms decreases. Therefore, when the base sequence is designed with priority on the Tm value, there is a problem that only a probe DNA with low performance can be set in an extremely GC-rich or AT-rich sequence.

  Therefore, an object of the present invention is to provide a base sequence analysis method that does not require setting common stringency conditions between different probes in a method for comprehensively analyzing the complementarity of base sequences of a target nucleic acid and a probe. That is.

  Another object of the present invention is a method for comprehensively analyzing the complementarity of a base sequence between a target nucleic acid and a probe, and a base sequence analysis method capable of simultaneous detection of multiple items using a microarray provided with an oligonucleic acid probe. Is to provide. Another object of the present invention is to provide a base sequence analysis method capable of analyzing a point mutation such as a single nucleotide polymorphism in a method for analyzing complementarity between a nucleic acid base sequence and a probe base sequence.

  A further object of the present invention is useful for comprehensive analysis of the homology of a nucleic acid to be analyzed contained in a sample with respect to a reference nucleic acid, and determines whether the nucleic acid to be analyzed is a reference nucleic acid or a similar nucleic acid. It is an object of the present invention to provide a useful base sequence analysis method.

  Still another object of the present invention is to provide a method for determining the presence or absence of a target nucleic acid in a sample with high accuracy.

As a result of diligent investigations, the present inventors have come up with a base sequence analysis method for comprehensively analyzing the complementarity of a target nucleic acid and a probe that can detect even a single base difference as a method for solving the above problems.
That is, the first aspect of the base sequence analysis method of the present invention is a method for analyzing complementarity on a base sequence between a target nucleic acid and a plurality of types of probes on a solid phase carrier,
(1) preparing a probe carrier in which a plurality of types of probes predicted to hybridize to the target nucleic acid are immobilized on a solid phase carrier;
(2) dividing the sample containing the target nucleic acid;
(3) reacting the plurality of types of probes with each of the divided samples with different stringency for each divided sample;
(4) in the case where formation of a hybrid with the target nucleic acid occurs in each probe, a step of taking out a signal indicating the formation of the hybrid;
(5) for each probe species, comparing the signals obtained at multiple stringencies;
(6) for each stringency, comparing the signals obtained in different types of probes hybridized to the same region of the target nucleic acid;
(7) analyzing the complementarity on the base sequence between the target nucleic acid and a plurality of types of probes on the solid phase carrier based on the comparison result of the two types of signals;
A method for analyzing the complementarity of a target nucleic acid to a probe, characterized by comprising:

A second aspect of the base sequence analysis method of the present invention is a method for analyzing homology between a target nucleic acid obtained from a nucleic acid to be analyzed contained in a specimen and a reference nucleic acid,
(1) preparing a probe carrier in which a plurality of types of probes predicted to hybridize to the same region of the reference nucleic acid are immobilized on a solid phase carrier;
(2) dividing the sample containing the target nucleic acid obtained from the nucleic acid to be analyzed contained in the specimen;
(3) reacting the plurality of types of probes with each of the divided samples with different stringency for each divided sample;
(4) in the case where formation of a hybrid with the target nucleic acid occurs in each probe, a step of taking out a signal indicating the formation of the hybrid;
(5) for each probe species, comparing the signals obtained at multiple stringencies;
(6) for each stringency, comparing the signals obtained in different types of probes hybridized to the same region of the target nucleic acid;
(7) analyzing the homology on the base sequence between the target nucleic acid and the reference nucleic acid based on the comparison result of the two kinds of signals;
A method for analyzing homology of a nucleic acid to be analyzed with respect to a reference nucleic acid.

Furthermore, the third aspect of the base sequence analysis method of the present invention is a method for determining the presence or absence of a target nucleic acid in a sample,
(I) providing a probe carrier in which a first probe that is completely complementary to at least a predetermined part of the base sequence of the target nucleic acid and a second probe that is incompletely complementary are immobilized on a solid phase carrier;
(II) a step of subjecting each of the plurality of probe carriers and the specimen to a hybridization reaction with different stringencies;
(III) For each probe carrier that has undergone the step (II), the first signal indicating the formation of the hybrid derived from the first probe and the formation of the hybrid derived from the second probe at each stringency. Obtaining an intensity ratio (first signal intensity / second signal intensity) with the second signal to be shown;
(IV) A step of determining the presence or absence of the target nucleic acid in the sample based on whether or not the intensity ratio shows a predetermined value exceeding 1.0 at least one of the stringencies.
A method for determining the presence or absence of a target nucleic acid in a specimen.

  By using the nucleic acid base sequence analysis method according to the present invention, a point mutation in a similar nucleic acid sequence represented by a single nucleotide polymorphism (SNP) or the like can be easily detected. Moreover, according to the present invention, the presence / absence of the target nucleic acid in the sample can be determined with higher accuracy.

  Specific configuration examples will be described below, but the present invention is not limited to the following method.

As the probe in the present invention, any nucleic acid usually used in probe hybridization can be preferably used. Specifically, it is a polynucleotide comprising at least one or more deoxyribonucleotides, ribonucleotides and derivatives thereof, but in order to more preferably separate and analyze single nucleotide polymorphisms, it may be 10 mer to 100 mer. More preferably, it is preferably 15 mer to 80 mer.
As the probe, any probe prepared by PCR or the like, chemically synthesized, or a nucleic acid derived from nature as it is or after being processed can be used. It is preferable to use a chemically synthesized product for ease. In this case, it is preferable to use oligodeoxyribonucleotide, oligoribonucleotide, PNA (peptide nucleic acid) or the like because of easy synthesis. Since the probe is used for hybridization on the solid phase carrier, the probe may be modified with a functional group or the like as long as it does not inhibit the hybridization in order to continue binding and adsorption to the solid phase carrier before and after hybridization.

  The species in the probe is uniquely determined and distinguished by the base sequence of the probe. For example, the same type of probe refers to a probe having the same base sequence. The plural types of probes indicate that there are two or more probes having different base sequences.

  Complementarity in the present invention includes the case of complete complementation and the case of incomplete complementarity. Here, complete complement refers to a case where all bases are complementary (full match) between two base sequences, and incomplete complement refers to including one or more mismatch bases.

  Multiple types of probes that hybridize to the same region of the target nucleic acid are probes that are designed to form a hybrid with the same sequence region of the target nucleic acid among the multiple types of probes used for analysis. Refers to a combination. When analyzing point mutations such as single nucleotide polymorphisms, a combination of probes in which the difference in the base sequence of the different probes is only one base can be preferably used.

  The solid phase carrier in the present invention refers to a solid that can continue to carry a probe before and after hybridization. Any solid phase carrier can be used as long as it can identify the position of the probe in the detection after washing (B / F separation) after hybridization. Specifically, it is a flat solid or gel carrier made of inorganic materials such as silicon wafer, quartz and glass, organic materials such as polycarbonate, polystyrene, polyacrylic acid, polyacrylamide and carbon plate, or a combination thereof. is there. For the purpose of comprehensive analysis of single nucleotide polymorphisms, it is preferable to use a probe carrier such as a DNA microarray in which quartz, glass or resins are used as a solid phase carrier and the probe is covalently bound for ease of detection. It is done.

  The target nucleic acid in the present invention is a nucleic acid that is subjected to a reaction with these probes in order to determine complementarity with a plurality of types of probes immobilized on a solid phase carrier. From the results obtained by reacting the target nucleic acid with a plurality of types of probes under the conditions according to the present invention, it is possible to determine what complementarity the target nucleic acid has for each probe. When there is a probe having a completely complementary relationship with the target nucleic acid, the base sequence of the region hybridized with the probe of the target nucleic acid can be determined from the base sequence of the probe. Furthermore, according to the present invention, it can also be determined whether or not the target nucleic acid is in an incompletely complementary relationship (for example, a one-base mismatch relationship) with respect to a certain probe.

  On the other hand, a base sequence of a reference nucleic acid that serves as a base sequence analysis is set, a plurality of probes that can hybridize to this base sequence are prepared, and the above determination process is performed on the nucleic acid to be analyzed. Thus, the homology between the nucleic acid to be analyzed and the reference nucleic acid can be determined. When nucleic acid in a sample is to be analyzed, target nucleic acid is prepared from this target nucleic acid, this is reacted with multiple types of probes immobilized on a solid support under the conditions of the present invention, and the results are analyzed. To do. For example, if the homology between the probe whose complementarity to the reference nucleic acid is completely complementary and the target nucleic acid from this sample is completely identical (100%), the region hybridized with the probe of the target nucleic acid is the base of the reference nucleic acid. It can be determined that it consists of an array. If these homologies are different by one base, it can be determined that the region hybridized with the target nucleic acid probe is a similar sequence having a single base difference from the base sequence of the reference nucleic acid. Based on the base sequence of the target nucleic acid, the sequence of the nucleic acid to be analyzed that is the origin of the target nucleic acid can be analyzed.

  The sample in the present invention refers to a sample containing the nucleic acid to be analyzed. The target nucleic acid from the sample is obtained based on the nucleic acid to be analyzed contained in the sample. From the nucleic acid to be analyzed itself, a characteristic part of the nucleic acid to be analyzed, or a complementary sequence (complete complement) thereof. Can take any form of the nucleic acid.

  Accordingly, the target nucleic acid is a polynucleotide comprising at least one deoxyribonucleotide, ribonucleotide and derivatives thereof. Samples include, for example, animals such as humans and mice, plants such as rice and corn, whole and part of samples considered to contain nucleic acids such as microorganisms such as bacteria, fungi, archaea, and viruses, or combinations thereof. This is true.

  The design of a plurality of types of probes in the base sequence analysis method according to the first aspect of the present invention can be performed as follows. First, a probe (full match) consisting of a sequence predicted to be in a completely complementary relationship with the target nucleic acid is designed. Furthermore, a probe comprising a base sequence having a moderate base sequence difference (mismatch) that causes a significant difference in hybridization results with a full match probe under at least one stringency condition is designed. When analyzing point mutations such as single nucleotide polymorphisms, it is preferable to design sequences that differ only by one base in the mutation part. The complementarity of the probe sequence to the target nucleic acid can be confirmed with reference to various publicly available sequence information databases. For example, it is possible to verify the complementarity of the designed probe to the target nucleic acid by comparing the designed probe sequence with sequence information stored in various databases. As the database, NCBI (Nathional Center of Biotechnology Information) in the United States can be used.

  In the base sequence analysis method according to the first aspect of the present invention, a plurality of types of probes are designed in the same manner as described above. First, a probe (full match) having a completely complementary relationship with the base sequence of the reference nucleic acid is designed. A probe having a difference in the number of bases is designed.

  Analyzing complementarity to a probe includes analyzing the presence or absence of a target nucleic acid that is complementary to the probe, so that the sample prepared from the sample in the present invention does not contain the target nucleic acid. There is also a possibility.

  Further, in order to prevent the sample prepared from the sample from containing a substance that inhibits hybridization of the target nucleic acid, it is preferable to purify the sample by a commonly used purification method. As the target nucleic acid, DNA or RNA extracted from the sample from which it is derived can be used as it is, but those amplified by PCR or the like can be used depending on the application. For amplification of the target nucleic acid, any amplification method usually used for the target nucleic acid to be subjected to probe hybridization such as a DNA microarray can be preferably used. Specific examples include PCR (symmetric), PCR (asymmetric), RT-PCR, random primer method, and the like, and these may be used in combination.

  Plural types of probes are divided into various types on a solid phase carrier and fixed at preset positions. Formation of a hybrid with the target nucleic acid in the fixed region of each probe is taken out as a signal and used for analysis of the base sequence. Various methods can be used to extract this signal. For example, a labeling method using a label that enables signal transmission can be suitably used.

  The labeling method includes a direct method in which a target nucleic acid molecule is labeled in advance and an indirect method in which a label is added during or after hybridization. Either method can be used, but in order to obtain a good detection result, the direct method is used. More preferably, it is used. As a method of incorporating a labeling substance into the target nucleic acid, for example, there is a method of adding a labeled deoxynucleotide (for example, Cy3-dUTP) when performing PCR amplification. In addition, when PCR amplification is performed, ATCG4 types of deoxynucleotides (dATP, dCTP, dGTP, dTTP: generically dNTP) and labeled deoxynucleotides (for example, Cy3-dUTP) are prepared as substrates for the DNA polymerase enzyme. There is also a method in which labeled deoxynucleotides are incorporated into the prepared amplification product by adjusting the final concentration of each dNTP.

  In addition, a method for labeling a nucleic acid by performing a PCR reaction using a pre-labeled primer is known. When this pre-labeled primer is used, there is an advantage that the amount ratio of the labeling substance to be applied can be controlled per molecule of the target nucleic acid to be produced. Is preferred.

  The labeling substance preferably contains at least one of a fluorescent substance and biotin from the viewpoint of detection sensitivity and ease of labeling. Conventionally known fluorescent dyes can be used as the fluorescent substance, but FITC, FAM in terms of quantum yield, light resistance, gas resistance, chemical stability, and characteristics as a substrate of DNA polymerase. Cy3, Cy5, Cy5.5, Cy7, TAMRA, Dabcyl, ROX, TET, Rhodamine, Texas Red, HEX, Cyber Green and the like can be preferably used. In the case of biotin and digoxigenin, various biotin-bound labeled enzyme proteins are reacted, and detection is possible using the enzyme activity of the labeled enzyme protein as an index.

  Various fluorescent materials used as fluorescent labels enable highly sensitive detection by using an optical detection means for observing the fluorescence intensity derived from such fluorescent materials. In addition, the detection method using the enzyme activity of the labeled enzyme protein as an index can also use optical detection means by using various color development reactions, and highly sensitive detection is possible. And

  In the present invention, the sample containing the target nucleic acid is equally divided into at least two or more before hybridization, and a plurality of stringencies are given. Thereby, hybridization at a plurality of stringencies is performed for each probe species. In other words, even when the stringency for obtaining the optimal hybridization result varies greatly from probe to probe, multiple strings are given to the same sample, so that the optimal stringency is maintained for any probe. The result can be obtained. The optimal stringency also depends on the concentration of the target nucleic acid, so it is very difficult to estimate completely. In addition, it is necessary to consider the instability of hybridization and labeling of the target nucleic acid. Therefore, by comparing the signal detection results of the same type of probe with the detection results of different probes at two or more different stringencies, it is possible to perform complementary base sequence complementarity or homology analysis with high accuracy. In this case, the same type of probe and the different probe are both designed to be complementary to the same region of the same target nucleic acid. Further, in the analysis of point mutations such as single nucleotide polymorphisms, the sequences of the different probes are designed so that only one base is different in the same probe and the mutation part.

  Specifically, the case where the stringency to be changed is set as the hybridization temperature will be described. In a probe having a Tm of 60 ° C. with a completely complementary strand (full match), in order to make the signal different between a complementary strand (single base mismatch) that differs only in one base and a full match, usually 55 to 65 ° C. The hybridization temperature must be set within the range. This is because at 55 ° C. or less, there is no difference between the signals of the full match and the single base mismatch, and when it is higher than 65 ° C., the signal becomes weak in both. However, when a probe having a Tm of 75 ° C. with a completely complementary strand (full match) is on the same solid phase carrier, the hybridization temperature must usually be set at 70 to 80 ° C. for this probe. That is, it is not possible to set an optimal hybridization stringency for these two probes at the same time. Therefore, good results can be obtained by dividing the same sample and performing hybridization at at least two temperatures of 60 ° C and 75 ° C. Furthermore, in this case, the stringency is subdivided into two or more, and by comparing the signals of the full-match probe and the single-base mismatch probe, the reliability is higher than that of hybridization under stringency using a single probe. A stable result can be obtained. In other words, even when the optimal hybridization stringency varies from probe to probe, judgment is made using the value at the stringency that maximizes the signal intensity ratio between the full match and single-base mismatch among the multiple stringencies set. By performing this, a highly reliable value can be obtained. This method uses not only single-base mismatch typing but also the value at the stringency that maximizes the signal intensity ratio between similar sequences when separating multiple similar sequences that differ in two or more bases. Thus, it is possible to determine which sequence has the closest homology. Examples of stringency include hybridization temperature, hybridization time, washing temperature, washing time, washing flow rate, number of washings, hybridization solution composition, washing solution composition, and the like. Specific examples of the hybridization solution composition and the washing solution composition include ionic strength, denaturant concentration (one that antagonizes hydrogen bonding between bases such as formamide and DMSO), and the like.

  The plurality of stringencies in the present invention can be achieved by preparing a plurality of liquid chambers for hybridization reaction and setting one stringency for one liquid chamber. Specifically, stringency can be changed by providing a liquid chamber for each of a plurality of solid phase carriers each containing the same type of probe. Alternatively, hybridization can be performed by setting a plurality of liquid chambers each containing the same type of probe on one solid phase carrier and changing the stringency for each liquid chamber. Furthermore, when a stringency change is given to a plurality of types of probes, a microarray can be suitably used as the solid phase carrier.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the best mode according to the present invention, but the technical scope of the present invention is not limited to the modes shown in these examples.

Example 1
<Preparation of probe DNA>
This example shows an example of separation detection by typing using 16S rRNA genes of Staphylococcus aureus and Staphylococcus epidermidis, which belong to the same genus, as a use for detecting a point mutation similar to SNP (single nucleotide polymorphism). As a probe for selective detection of nucleic acid molecules derived from Staphylococcus aureus and Staphylococcus epidermidis, a probe DNA having the base sequence shown in Table 1 was designed.

  Specifically, based on the 16S rRNA sequences of Staphylococcus aureus and Staphylococcus epidermidis, the base sequences of the four types of probes shown in Table 1 were selected. The base sequences of these probes are selected so as to be effective for the separation and detection of the bacteria, but the optimum separation temperatures are greatly different. Probe pairs designed for regions homologous to the 16S rRNA sequences of Staphylococcus aureus and Staphylococcus epidermidis (Se-1 corresponding to Sa-1 and Se-2 corresponding to Sa-2 in the examples) It is assumed that the bacterial species having the stronger signal is separated and detected when all the signal intensity differences are significant (signal intensity ratio is greater than 1.0, preferably 4.0 or more).

For each probe shown in Table 1, after synthesis of the DNA strand, a thiol group was introduced at the 5 'end of the nucleic acid according to a standard method as a functional group for immobilization on the DNA microarray. After introduction of the functional group, it was purified and lyophilized. The lyophilized probe for internal standard was stored in a freezer at −30 ° C.

<Preparation of PCR Primer for target nucleic acid preparation>
A DNA primer having a nucleic acid sequence shown in Table 2 was designed as a PCR primer for amplification of 16s rRNA gene (target gene) derived from the target bacterium used for detection.
Specifically, a probe set that specifically amplifies a genomic portion encoding 16s rRNA derived from the standard strains of Staphylococcus aureus ATCC 12600 and Staphylococcus epidermidis ATCC 14990, that is, a 16s rRNA coding region having a length of about 1500 bases. Based on the base sequences at both ends, a plurality of primers having a base sequence complementary thereto and having substantially the same melting temperature when hybridized with the portions were designed.

Each Primer shown in Table 2 is purified by high performance liquid chromatography (HPLC) after synthesis of the DNA strand. This Forward Primer and Reverse Primer were mixed and dissolved in TE buffer so that each Primer concentration would be a final concentration of 10 pmol / μl.

<Extraction of DNA (model specimen)>
[Pretreatment of microorganism culture and genomic DNA extraction]
First, a Staphylococcus aureus standard strain was cultured according to a conventional method.
1.0 ml (OD ₆₀₀ = 0.7) of this microorganism culture solution was collected in a 1.5 ml capacity microtube, and the cells were separated by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the supernatant, 300 μl of Enzyme Buffer (50 mM Tris-HCl: pH 8.0, 25 mM EDTA) was added, and the cells were resuspended using a mixer. The bacterial cells were separated again from the resuspended bacterial cell solution by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the supernatant, 50 μl of each of the following two enzyme solutions was added to the collected cells and resuspended using a mixer.

Lysozyme 50 μl for lysis treatment (enzyme concentration: 20 mg / ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (enzyme concentration: 0.2 mg / ml in Enzyme Buffer)
Next, the bacterial cell solution resuspended in the buffer solution containing the enzyme was allowed to stand in an incubator at 37 ° C. for 30 minutes, and the cell membrane of the microorganism was dissolved.
(Genome extraction / purification)
After lysis treatment of the cell membrane, extraction and purification of Genome DNA derived from the microorganism was performed according to the following procedure using a nucleic acid purification kit (MagExtractor-Genome-: manufactured by TOYOBO).
(Step 1)
First, 750 μl of the lysis / adsorption solution of the kit and 40 μl of magnetic bead solution are added to the microorganism suspension subjected to the cell membrane dissolution treatment, and vigorously stirred for 10 minutes using a tube mixer. By this operation, the contained double-stranded DNA molecule is adsorbed on the surface of the magnetic particle (bead).
(Step 2)
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube. Thereafter, the supernatant is discarded while being set on the stand.
(Step 3)
Add 900 μl of the washing solution into the microtube and stir it for about 5 seconds with a mixer to resuspend it.
(Step 4)
Again, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect magnetic particles on the wall of the tube. Thereafter, the supernatant is discarded while being set on the stand.
(Step 5)
The washing operations in steps 3 and 4 are repeated to perform washing twice.
(Step 6)
After washing with the washing solution, 900 μl of 70% ethanol is added to the microtube, and the mixture is stirred for about 5 seconds with a mixer and re-suspended.
(Step 7)
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect magnetic particles on the wall surface of the tube. Thereafter, the supernatant is discarded while being set on the stand.
(Step 8)
By repeating the washing operations in steps 6 and 7, the second washing is performed with 70% ethanol in total. After washing twice with 70% ethanol, 100 μl of pure water is added into the microtube, and the liquid containing the collected magnetic particles is stirred for 10 minutes with a tube mixer. By this operation, the double-stranded DNA molecules adsorbed on the surface of the magnetic particles (beads) are re-eluted in pure water. Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect the magnetic particles on the wall surface of the tube. Finally, the supernatant containing the double-stranded DNA molecules is collected in a new tube while being set on the stand.
(Inspection of recovered Genome DNA)
The liquid containing purified nucleic acid molecules derived from the collected microorganisms (Staphylococcus aureus standard strain)
According to a standard method, agarose electrophoresis and absorbance measurement at 260/280 nm were performed, and the quality (contamination amount of low-molecular nucleic acid, the degree of degradation) and the recovery amount of Genome DNA contained in the solution were tested.

  In this example, about 10 μg of Genome DNA was recovered, and Genome DNA degradation and rRNA contamination were not observed.

<Production of DNA microarray>
In this experiment, four microarrays were prepared to give stringency of 4 conditions.

[1] Cleaning of glass substrate A synthetic quartz glass substrate (size: 25 mm (width) x 75 mm (length) x 1 mm (thickness), manufactured by Iiyama Special Glass Co., Ltd.) is placed in a heat and alkali resistant rack, It was immersed in a cleaning solution for ultrasonic cleaning adjusted to a concentration. After soaking in the cleaning solution overnight, ultrasonic cleaning was performed for 20 minutes. Subsequently, the substrate was taken out, rinsed lightly with pure water (rinse cleaning), and then ultrasonically cleaned in ultrapure water for 20 minutes. Next, the substrate was immersed in a 1N aqueous sodium hydroxide solution heated to 80 ° C. for 10 minutes. Thereafter, the substrate was taken out and rinsed with pure water and subjected to ultrasonic cleaning in ultrapure water. A quartz glass substrate for a DNA chip that has been washed was prepared by the above washing operation.

[2] A surface-treated silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone Co., Ltd.) was added to pure water so as to have a concentration of 1%, and stirred at room temperature for 2 hours to dissolve uniformly. Subsequently, the washed quartz glass substrate was immersed in an aqueous silane coupling agent solution and left at room temperature for 20 minutes. The quartz glass substrate was pulled up and lightly rinsed with pure water, and then dried by blowing nitrogen gas on both sides of the substrate. Next, the dried substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the binding treatment of the aminosilane coupling agent to the substrate surface. An amino group derived from the aminosilane coupling agent was introduced on the surface of the substrate.

  N-maleimidocaproyloxy succinimide (N- (6-Maleimidocaproyloxy) succinimido; hereinafter abbreviated as EMCS) manufactured by Dojindo Laboratories Ltd. in a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol has a final concentration of 0. An EMCS solution dissolved to 3 mg / ml was prepared. After completion of the baking treatment, the quartz glass substrate was allowed to cool to room temperature. Next, the quartz glass substrate having amino groups introduced on the surface was immersed in the EMCS solution at room temperature for 2 hours. During this immersion treatment, the amino group introduced to the substrate surface by the aminosilane coupling agent treatment and the succinimide group of EMCS reacted, and the maleimide group derived from EMCS was introduced to the quartz glass substrate surface. The quartz glass substrate pulled up from the EMCS solution was washed with a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol to remove unreacted EMCS. Further, after washing with ethanol, the surface-treated quartz glass substrate was dried in a nitrogen gas atmosphere.

[3] Probe DNA
The probe DNA for detection prepared in <Preparation of probe DNA> was dissolved in pure water, dispensed into micro vials so that the final concentration (when dissolved in ink) was 10 μM, and then lyophilized. The micro vials each containing the probe DNA excluding moisture were used for the preparation of a DNA solution for spotting by the bubble jet method according to the following procedure.

[4] Discharge of DNA solution by BJ printer and immobilization on substrate surface 7.0 wt%, ethylene glycol 5.0 wt%, hexanetriol 5.0 wt%, acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) 1.0 wt% % Aqueous solution was prepared. Subsequently, a predetermined amount of the above mixed solvent is added so that the final concentration (at the time of dissolving the ink) is 10 μM in the microvials each containing the four types of probe DNAs (Table 1) prepared above. Thus, a DNA solution (ink) was prepared. The obtained DNA solution was filled into an ink tank for a bubble jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.

  The bubble jet printer has been modified so that printing on a flat plate is possible for use in spotting a DNA solution on a substrate surface. In addition, the print head of this bubble jet printer can spot a DNA solution droplet of about 5 picoliters at a pitch of about 120 micrometers by inputting a print pattern according to a predetermined file creation method. Yes.

  Subsequently, using the modified bubble jet printer, a spotting operation of each DNA solution is performed according to a predetermined printing pattern (FIG. 1) on a quartz glass substrate having a maleimide group introduced on the surface thereof. An array was made. After confirming that the array-like spotting operation has been carried out reliably, it is allowed to stand in a humidified chamber for 30 minutes to react the maleimide group on the surface of the quartz glass substrate with the thiol group at the 5 ′ end of the probe DNA, thereby Was fixed. In the printed pattern of this experiment, four types of probes are physically separated from each other on a single microarray, so that four types of items can be detected simultaneously by a single hybridization.

[5] After the reaction for 30 minutes, the DNA solution remaining on the substrate surface was washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. A gene chip in which a plurality of types of single-stranded DNAs of interest were immobilized in an array on the surface of a quartz glass substrate was obtained.

<PCR amplification of sample DNA in genomic DNA extract>
PCR amplification of the 16s rRNA gene, which is the sample DNA, from the genomic DNA extract derived from the microorganism is performed according to the following procedures and conditions. First, PCR amplification reaction is performed using genomic DNA as a template, using a mixture of Forward Primer and Reverse Primer shown in Table 2 above. The PCR amplification reaction was performed using a commercially available PCR kit (TAKARA ExTaq), and the composition of the reaction solution is shown in Table 3.

  On the other hand, the temperature condition of the PCR reaction was performed according to the protocol shown in Table 4 using a commercially available thermal cycler.

After completion of this PCR amplification reaction, a commercially available PCR product purification column (QIAGEN QIAquick PCR
Purification Kit) was used to remove the primer and collect the PCR amplification product. At that time, the obtained PCR amplification product was quantified.

<Preparation of labeled DNA strand using PCR amplification product as template>
As a fluorescent label, a labeled DNA strand was prepared according to the following procedure and conditions using a labeled oligonucleotide in which Cy3 was bound to the 5 ′ end of the Reverse Primer. After the synthesis of the nucleotide chain, the fluorescently labeled compound Cy3 was covalently bound to the 5 ′ end of the nucleotide chain according to a conventional method. Thereafter, the labeled oligonucleotide was purified using HPLC.

Using the PCR amplification product obtained by the PCR amplification reaction as a template and a labeled primer having a base sequence shown in Table 5 as a reverse primer, a complementary DNA chain extension reaction was carried out according to the following procedures and conditions. Do.

  The extension reaction of the DNA strand was carried out in the form of a single-stranded PCR reaction using a commercially available PCR kit (TAKARA ExTaq). The reaction solution composition is shown in Table 6.

  On the other hand, the temperature condition of this extension reaction was carried out using a commercially available thermal cycler according to the protocol shown in Table 7 below.

After the completion of the DNA chain extension reaction, the primer is removed using a commercially available column for PCR product purification (QIAGEN QIAquick PCR Purification Kit), and a fluorescently labeled Cy3 derived from a labeled oligonucleotide is added to the 5 ′ end. The product was recovered. At that time, the obtained amplification product was quantified.

<Detection of sample DNA by hybridization method>
Using the gene chip prepared in <Preparation of DNA microarray> and the labeled sample DNA prepared in <Amplification and labeling of specimen (PCR amplification & incorporation of fluorescent label)>, a hybridization reaction is performed, and fluorescence labeling is performed. Was used to detect the formed hybrids.

(Gene chip blocking)
BSA (bovine serum albumin Fraction V: manufactured by Sigma) is added to 100 mM NaCl / 10 mM Phosphate Buffer to a concentration of 1 wt% to prepare a BSA solution. In this BSA solution, two gene chips prepared in <Preparation of DNA microarray> were immersed for 2 hours at room temperature, and the surface of the quartz glass substrate of the gene chip was subjected to blocking treatment with BSA. After completion of the blocking treatment, 2 × SSC solution (NaCl 300 mM containing 0.1 wt% SDS (sodium dodecyl sulfate), Sodium Citrate (trisodium citrate dihydrate , Na 3 C 6 H 5 O 7 · 2H 2 O) 30mM, pH 7.0 The BSA solution remaining on the quartz glass substrate surface of the gene chip was washed. Next, after rinsing with pure water, the gene chip surface was drained with a spin dryer.

(Hybridization reaction)
The gene chip subjected to the blocking treatment was set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station), and a hybridization reaction between the labeled sample DNA and the probe DNA was performed according to the following procedures and conditions.
The composition and conditions of the used hybridization solution are shown below.

[Hybridization solution]
<Sample amplification and labeling (PCR amplification & incorporation of fluorescent label)> The purified labeled sample DNA prepared in 6 × SSPE buffer solution containing 10% HCONH ₂ was dissolved into four solutions. Divided equally and used as hybridization solution.
6 × SSPE / 10% Form amide / Target (2 nd PCR Products total)
(6 × SSPE: NaCl 900 mM, NaH ₂ PO ₄ .H ₂ O 60 mM, EDTA 6 mM, pH 7.4)
[Hybridization conditions]
The hybridization reaction and the washing and drying operations after the reaction were performed under the four conditions shown in Table 8 below. One gene chip was used for each condition.

<Detection of hybrid body (fluorescence measurement)>
After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescence-labeled Cy3 is measured for each probe DNA spot on the spin-dried gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). It was.
Table 9 shows the fluorescence intensity observed at the spot points of each probe DNA.
As a standard for separating and detecting S. aureus and S. epidermidis, in this example, the two probes (Sa-1 / Se-1) and (Sa-2 / Se-2) in the target nucleic acid are used. It is assumed that a signal intensity ratio of 4 or more is confirmed. It is necessary to look at two or more signals to take into account the sequence mutations that bacteria have at the strain level and to increase the reliability of data for base incorporation errors in PCR.

Table 10 summarizes the hybridization results in Table 9 for each probe.
As this result shows, it is difficult to determine whether each probe is detected simply by comparing the fluorescence intensity between stringencies (conditions 1 to 4). Therefore, it cannot be determined that Staphylococcus aureus can be separated and detected only by comparing the fluorescence intensity of the same probe species between stringencies.

Table 11 summarizes the signal intensity ratios under each condition based on the hybridization results shown in Table 9. By comparing the fluorescence intensities of conditions 1 and 4, it can be detected that the sample nucleic acid is Staphylococcus aureus.

(Comparative Example 1)
Detection was performed in the same manner as in Example 1 except that the sample was not divided and hybridization was performed only under hybridization condition 1.

  After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescence-labeled Cy3 is measured for each probe DNA spot on the spin-dried gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). It was.

  Table 12 shows the fluorescence intensity observed at the spot points of each probe DNA. Table 13 shows the signal intensity ratio and the determination result obtained from this.

From this, the reference value could not be satisfied with the signal intensity ratio of (Sa-2 / Se-2), and thus determination was impossible.

(Comparative Example 2)
Detection was performed in the same manner as in Example 1 except that the sample was not divided and hybridization was performed only under the hybridization condition 4. After completion of the hybridization reaction, the fluorescence intensity derived from the fluorescence-labeled Cy3 is measured for each probe DNA spot on the spin-dried gene chip using a gene chip fluorescence detector (Axon, GenePix 4000B). It was. Table 14 shows the fluorescence intensities observed at the spot points of each probe DNA. Table 15 shows the signal intensity ratio and the determination result obtained from this.

From this, the reference value could not be satisfied with the signal intensity ratio of (Sa-1 / Se-1), and thus determination was impossible.

It is a printing pattern of the probe in an Example.

Claims (19)

A method for analyzing complementarity on a base sequence between a target nucleic acid and a plurality of types of probes on a solid phase carrier,
(1) preparing a probe carrier in which a plurality of types of probes predicted to hybridize to the same region of the target nucleic acid are immobilized on a solid phase carrier;
(2) dividing the sample containing the target nucleic acid;
(3) reacting the plurality of types of probes with each of the divided samples with different stringency for each divided sample;
(4) in the case where formation of a hybrid with the target nucleic acid occurs in each probe, a step of taking out a signal indicating the formation of the hybrid;
(5) for each probe species, comparing the signals obtained at multiple stringencies;
(6) for each stringency, comparing the signals obtained in different types of probes hybridized to the same region of the target nucleic acid;
(7) analyzing the complementarity on the base sequence between the target nucleic acid and a plurality of types of probes on the solid phase carrier based on the comparison result of the two types of signals;
A method for analyzing complementarity of a target nucleic acid to a probe, comprising:   The analysis according to claim 1, wherein the stringency is set based on any one or more of a hybridization temperature, a hybridization time, a hybridization solution composition, a washing temperature, a washing time, a washing solution composition, a washing flow rate, and the number of washings. Method .   The analysis method according to claim 1, wherein the solid phase carrier is a DNA microarray.   The analysis method according to claim 1, wherein the target nucleic acid is labeled.   The analysis method according to claim 4, wherein the label is made using one or more labels selected from a fluorescent substance and biotin.   The base sequence analysis method according to claim 1, wherein the target nucleic acid is amplified DNA or RNA.   The analysis method according to claim 6, wherein the amplification of the target nucleic acid is performed by PCR.   The analysis method according to any one of claims 1 to 7, wherein the difference in the base sequences of the different probes is only one base.   The analysis method according to any one of claims 1 to 7, wherein the probe has a base chain length of 10 mer to 100 mer. A method for analyzing homology between a target nucleic acid obtained from a target nucleic acid contained in a specimen and a reference nucleic acid,
(1) preparing a probe carrier in which a plurality of types of probes predicted to hybridize to the same region of the reference nucleic acid are immobilized on a solid phase carrier;
(2) dividing the sample containing the target nucleic acid obtained from the nucleic acid to be analyzed contained in the specimen;
(3) reacting the plurality of types of probes with each of the divided samples with different stringency for each divided sample;
(4) in the case where formation of a hybrid with the target nucleic acid occurs in each probe, a step of taking out a signal indicating the formation of the hybrid;
(5) for each probe species, comparing the signals obtained at multiple stringencies;
(6) for each stringency, comparing the signals obtained in different types of probes hybridized to the same region of the target nucleic acid;
(7) analyzing the homology on the base sequence between the target nucleic acid and the reference nucleic acid based on the comparison result of the two kinds of signals;
A method for analyzing the homology of a nucleic acid to be analyzed with respect to a reference nucleic acid, comprising:   The analysis according to claim 10, wherein the stringency is set based on any one or more of a hybridization temperature, a hybridization time, a hybridization solution composition, a washing temperature, a washing time, a washing solution composition, a washing flow rate, and the number of washings. Method .   The analysis method according to claim 10 or 11, wherein the solid phase carrier is a DNA microarray.   The analysis method according to claim 10, wherein the target nucleic acid is labeled.   The analysis method according to claim 13, wherein the label is made using one or more labels selected from a fluorescent substance and biotin.   The base sequence analysis method according to any one of claims 10 to 14, wherein the target nucleic acid is amplified DNA or RNA.   The analysis method according to claim 15, wherein the amplification of the target nucleic acid is performed by PCR. The analysis method according to any one of claims 10 to 16, wherein a difference in the base sequences of the different probes is only one base.   The analysis method according to any one of claims 10 to 16, wherein the probe has a base chain length of 10 mer to 100 mer. A method for determining the presence or absence of a target nucleic acid in a sample,
(I) providing a probe carrier in which a first probe that is completely complementary to at least a predetermined part of the base sequence of the target nucleic acid and a second probe that is incompletely complementary are immobilized on a solid phase carrier;
(II) a step of subjecting each of the plurality of probe carriers and the specimen to a hybridization reaction with different stringencies;
(III) For each probe carrier that has undergone the step (II), the first signal indicating the formation of the hybrid derived from the first probe and the formation of the hybrid derived from the second probe at each stringency. Obtaining an intensity ratio (first signal intensity / second signal intensity) with the second signal to be shown;
(IV) A step of determining the presence or absence of the target nucleic acid in the sample based on whether or not the intensity ratio shows a predetermined value exceeding 1.0 at least one of the stringencies.
A method for determining the presence or absence of a target nucleic acid in a sample, comprising:

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