JP2009240264A - Method for deciding base sequence of target nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for accurately and simply performing decision in deciding the base sequence of a target nucleic acid by using a hybridization method, irrespective of the base sequence of an oligonucleotide probe. <P>SOLUTION: The method for deciding the base sequence of a target nucleic acid includes (a) a step of hybridizing the probe with the target nucleic acid by bringing a test specimen containing the target nucleic acid into contact with a nucleic acid alley mounted with two or more kinds of oligonucleotide probes immobilized on a carrier, (b) a step of detecting a distribution amount of the hybridized target nucleic acid in the carrier for every carrier, and (c) a step of deciding the base sequence of the target nucleic acid based on the result obtained by the detection. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for determining a base sequence of a target nucleic acid based on a hybridization method using a nucleic acid array. Specifically, the present invention relates to a method for identifying which base sequence of a target nucleic acid is complementary to the base sequence of any oligonucleotide probe mounted on the nucleic acid array.

In recent years, many studies have been conducted on the causal relationship between genotypes (ie, gene polymorphisms) such as SNPs (single nucleotide polymorphisms) and diseases.
Methods for determining (detecting) genetic polymorphisms such as SNPs include sequencing methods that actually read base sequences, RFLP methods that use restriction enzymes as indicators, and bases that utilize the specificity of enzymes such as polymerases. In addition to the extension method and the PCR method, a probe hybridization method is also included.
In the probe hybridization method, the presence or absence of a gene polymorphism is simply determined using the ease of formation of hybridization between the probe nucleic acid and the target nucleic acid due to the difference in the base sequence as an index. Compared to the method described above, the accuracy is inferior. For this reason, when the probe hybridization method is used for the purpose of determining genetic polymorphisms such as SNPs, there are many cases where improvements are made to improve accuracy or performance other than accuracy.

In order to improve performance other than accuracy, for example, a technique is known in which a plurality of gene polymorphisms can be efficiently confirmed by immobilizing a probe on a carrier. In this case, the immobilized probe may be a microarray. When a gene polymorphism such as SNPs is determined using a microarray, since many polymorphic sites can be determined at a time, the efficiency of the analysis becomes very high.
In some cases, it is possible to strictly control hybridization and washing conditions by incorporating a microchannel or the like into an apparatus or an array.
There is also a method in which differences are made in hybridization and subsequent washing methods, and data under conditions more suitable for each probe is adopted (Patent Document 1).
Furthermore, a method has been devised that controls the hybridization to increase the accuracy of genetic polymorphism determination by using PNA, inosine, and other structures and characteristics that are different from normal ones. (Patent Document 2).

By using several of these techniques at the same time, the overall performance such as accuracy, efficiency, and operability may be further improved.
When a nucleotide sequence containing a gene polymorphism such as SNPs is determined by a hybridization method, it can be performed based on the amount of hybridization formed or the presence or absence of hybridization. In some cases, this method can be clearly determined between probes that tend to cause differences in hybridization efficiency.
On the other hand, depending on the probe setting conditions, the amount of hybridization formed between different probes may be close to each other, making it difficult to make a determination for determining the base sequence. As such a case, for example, a case where a probe consisting of about 20 bases and a target nucleic acid to be analyzed form hybridization is easy to think. However, even in this case, if there is a polymorphism to be detected in the base near the center of each probe, the polymorphism in the base sequence of the target nucleic acid can be determined by measuring and comparing the amount of hybridization formed. Sometimes the type can be determined.

However, when the base corresponding to the polymorphism is present in the base at the terminal portion of the probe, no significant difference is observed in the amount of hybridization, and it is extremely difficult to determine the polymorphism. In addition, when such a probe is used, even if the stringency of the hybridization or washing method is improved, there may be a case where no clear difference occurs in the amount of hybridization between the probes.
The determination of the presence or absence of genetic polymorphisms such as SNPs is directly linked to the individual's genotype, which may be pointed out to be related to disease, so it is as accurate as possible in both experimental animals and plants and humans. Should be done. However, when the hybridization method is used, the determination criteria are only the result of measuring the amount of the hybridized target nucleic acid, and as described above, it is difficult to accurately determine the base sequence of the target nucleic acid. was there.

JP 2007-174986 JP JP-A-8-70900

  Therefore, the problem to be solved by the present invention is to determine the base sequence of the oligonucleotide probe when determining the base sequence of the target nucleic acid using the hybridization method, particularly when detecting the gene polymorphism in the base sequence. Regardless of (for example, whether or not the position of the base corresponding to the gene polymorphism is at the end (or near the end) of the probe), the above determination (or detection) can be performed accurately and simply. Is to provide.

The present inventor has intensively studied to solve the above problems.
As a result, when determining the base sequence of the target nucleic acid using the hybridization method, the determination is not made based solely on the amount of hybridization between the probe and the target nucleic acid, but in each carrier to which the probe is immobilized. The inventors have found that the above-mentioned problems can be solved by making a determination based on the distribution of the amount of hybridization formed in (for example, the ratio of the distribution amount between the vicinity of the surface and the inside of the carrier), and the present invention has been completed.

That is, the present invention is as follows.
A method for determining a base sequence of a target nucleic acid, comprising:
(A) contacting a test sample containing the target nucleic acid with a nucleic acid array equipped with two or more kinds of oligonucleotide probes fixed to a carrier, and hybridizing the probe and the target nucleic acid;
(B) detecting a distribution amount of the hybridized target nucleic acid in the carrier for each carrier; and (c) determining a base sequence of the target nucleic acid based on a result obtained by the detection. Said method.
Examples of the method of the present invention include a method in which the carrier has a three-dimensional shape (for example, a cylindrical shape), and the detection of the distribution amount is performed in the vicinity of the surface and inside of the carrier.

In the method of the present invention, the determination in the step (c) is performed based on, for example, a ratio between the distribution amount near the surface of the carrier obtained by the detection and the distribution amount inside the carrier. Things. In the determination in the step (c), for example, it is specified whether the base sequence of the target nucleic acid is a base sequence complementary to the base sequence of the probe fixed to any of the carriers. Things.
In the method of the present invention, examples of the base sequences different from each other between the two or more types of oligonucleotide probes include those corresponding to gene polymorphic sites in the base sequence of the target nucleic acid. Here, examples of the gene polymorphism include at least one selected from the group consisting of single nucleotide polymorphisms (SNPs), insertion polymorphisms, deletion polymorphisms, and base repeat polymorphisms. The method of the present invention may be, for example, a method for detecting a gene polymorphism in the base sequence of the target nucleic acid.

According to the present invention, when determining the base sequence of a target nucleic acid using a hybridization method, particularly when detecting a gene polymorphism in the base sequence, the position of the base corresponding to the gene polymorphism is, for example, Regardless of the base sequence of the oligonucleotide probe used, such as whether it is the end of the probe, the determination (or detection) can be performed accurately and simply.
That is, by using the present invention, it is possible to determine the base sequence of the target nucleic acid more accurately than the determination based only on the amount of hybridization formed. In addition, the base sequence of the target nucleic acid can be determined very accurately even when there is a difference in the terminal base of each probe (for example, there is a base corresponding to a polymorphism), which was difficult to determine in the past. Is possible.

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

As described above, the method for determining the base sequence of the target nucleic acid of the present invention comprises the following steps (a) to (c).
(A) contacting a test sample containing the target nucleic acid with a nucleic acid array carrying two or more types of oligonucleotide probes immobilized on a carrier and hybridizing the probe and the target nucleic acid (hereinafter referred to as “high”). Called hybridization step).
(B) A step of detecting the distribution amount of the hybridized target nucleic acid in the carrier for each type of the carrier (hereinafter referred to as a detection step).
(C) A step of determining the base sequence of the target nucleic acid based on the result obtained by the detection (hereinafter referred to as a determination step).
Below, each process and use of the determination method of this invention are demonstrated concretely.

1. Hybridization process As described above, the hybridization process is performed by bringing a test sample containing a target nucleic acid into contact with a nucleic acid array equipped with two or more types of oligonucleotide probes (capture probes) immobilized on a carrier. This is a step of hybridizing with a nucleic acid.
The test sample used in the method of the present invention contains a target nucleic acid, and the target nucleic acid is a nucleic acid that is subjected to a hybridization reaction with an oligonucleotide probe described later for the purpose of determining (analyzing) the base sequence. is there. The target nucleic acid is not particularly limited in the type and origin of the nucleic acid such as DNA or RNA, and may be prepared using PCR, reverse transcription reaction, transcription reaction or the like. It is preferable that the target nucleic acid is labeled by adding an appropriate labeling substance directly or indirectly so that the amount bound to the probe (hybridized amount) can be detected as a signal after the hybridization reaction. . Examples of the signal include fluorescence, luminescence, radioisotope, color development, and the like, and fluorescence is preferable from the viewpoint of simplicity of detection. When the signal is fluorescent, fluorescent substances that serve as labeling substances include various reporter dyes such as Cy5, Cy3, VIC, FAM, HEX, TET, fluorescein, FITC, TAMRA, Texas red, Yakima Yellow, ULYSIS, ARES, etc. Can be used.

A test sample containing a target nucleic acid is usually prepared and used as a target nucleic acid solution, that is, a hybridization solution. The hybridization solution can be appropriately prepared according to a conventional method using a buffer such as SDS or SSC.
The nucleic acid array used in the method of the present invention has two or more kinds of oligonucleotide probes mounted thereon.
The oligonucleotide probe refers to a nucleic acid that is subjected to hybridization with a target nucleic acid, and the type thereof may be DNA, RNA, PNA, or a mixture thereof, and is not particularly limited. The probe may be chemically modified as long as it does not inhibit the hybridization reaction.

  The length (base length) of the probe is not limited, but is, for example, 100 bases or less from the viewpoint of effectively determining the base sequence of the target nucleic acid (particularly, the base sequence difference from other target nucleic acids). It is preferably 10 bases to 100 bases, more preferably 10 bases to 30 bases. In addition, examples of the difference in the base sequence include polymorphisms such as SNPs, point mutations, and combinations thereof, and it is clear that variations in the difference are clarified at the stage of producing the probe. Desirable in design. In the probe, the position corresponding to the difference in the base sequence of the target nucleic acid to be determined is not limited and may be plural, but it is one in terms of efficiency and ease of determination. It is preferable. Moreover, it is preferable that the number of bases of the said location is 10% or less of the total base length of a probe.

  The two or more types of oligonucleotide probes have different base sequence portions between the probes, and the base sequences correspond to, for example, gene polymorphic sites in the base sequence of the target nucleic acid. It is preferable. Examples of gene polymorphism include, but are not limited to, single nucleotide polymorphisms (SNPs), insertion polymorphisms, deletion polymorphisms, and base repeat polymorphisms. For example, when there are 4 types of SNPs (A, T, G, C), it is preferable to design and use 4 types of probes corresponding to each type. In this case, each probe corresponds to a polymorphic site. It is preferable that all the bases including the chain length (base length) are the same except for the different bases. In addition, when a plurality of bases corresponding to SNPs exist in one probe, for example, it is desirable to design a probe that covers as many SNPs as the number of bases. Specifically, for example, when confirming two types of four types of SNPs at the same probe design position, it is desirable to prepare all 16 types of probes represented by 4 × 4.

In the present invention, each of the two or more kinds of oligonucleotide probes described above is mounted on the nucleic acid array in a state of being immobilized on different carriers. Specifically, if each oligonucleotide probe is not mixed in the nucleic acid array, the experimental operation such as hybridization reaction and subsequent washing becomes very simple. It is preferably modified with a group and fixed to different carriers by covalent bonds.
The carrier preferably has a three-dimensional shape, that is, a shape in which the probe can be immobilized three-dimensionally. The three-dimensional shape (particularly the shape of the carrier region on which the probe is immobilized) is not limited. For example, a cylindrical shape is preferable. Further, a target nucleic acid (solution) is provided on the side surface of the cylindrical carrier during the hybridization reaction. It is preferable that the structure does not contact. When the probe is immobilized on a carrier having a three-dimensional shape, the carrier is made of a material that can detect not only the vicinity of the surface but also the signal emitted by the target nucleic acid hybridized to the probe immobilized therein. It is preferred that The carrier is preferably made of a structure and material that allows the target nucleic acid to reach the inside of the carrier. Examples of such a material include porous resins and gels. The type of gel is not particularly limited as long as it can confirm the distribution pattern of the target nucleic acid inside the carrier when detecting the signal intensity of the target nucleic acid. For example, an agarose gel or an acrylamide gel is preferable, and heating is performed. An acrylamide gel which is difficult to dissolve by is more preferable.

Specific examples of the nucleic acid array used in the method of the present invention as described above include a fiber type microarray (product name: Genopal ^™ ; published website: http://www.mrc.co.jp/genome/top. html; see Japanese Patent No. 3488456, Japanese Patent No. 3510882, etc.). A fiber-type microarray uses a hollow fiber or other tubular body in which a gel-like material is held as a carrier for fixing a probe. It is a microarray obtained by fixing to fibers, converging and fixing all hollow fibers, and then repeating cutting in the longitudinal direction of the fibers. This fiber type microarray can also be described as a type in which a nucleic acid is immobilized on a through-hole substrate.

Hereinafter, the fiber type microarray will be described in detail. This microarray can be manufactured through the following steps (i) to (iv), for example.
(i) A step of producing an array by arranging a plurality of hollow fibers in three dimensions so that the longitudinal directions of the hollow fibers are the same direction
(ii) a step of embedding the array and producing a block
(iii) A step of introducing a gel precursor polymerizable solution containing an oligonucleotide probe into the hollow part of each hollow fiber of the block body to carry out a polymerization reaction and holding the gel-like substance containing the probe in the hollow part
(iv) A step of cutting the block body into pieces by cutting in a direction crossing the longitudinal direction of the hollow fiber

The material used for the hollow fiber is not limited, but for example, materials described in JP-A No. 2004-163211 and the like are preferable.
The hollow fibers are arranged three-dimensionally so that the lengths in the longitudinal direction are the same (step (i)). As an arrangement method, for example, a method of arranging a plurality of hollow fibers in parallel with a predetermined interval on a sheet-like material such as an adhesive sheet, forming a sheet, and then winding the sheet in a spiral manner (Japanese Patent Laid-Open No. 11-1990). No. 108928), or two perforated plates in which a plurality of holes are provided at a predetermined interval are overlapped so that the holes coincide with each other, and hollow fibers are passed through these holes, and then the two perforated plates And a method of temporarily fixing the gap between the two perforated plates and filling the periphery of the hollow fiber with a curable resin raw material to cure (see JP 2001-133453 A).

The manufactured array is embedded so that the array is not disturbed (step (ii)). Examples of the embedding method include a method in which a polyurethane resin, an epoxy resin, or the like is poured into a gap between fibers, and a method in which fibers are bonded together by heat fusion.
The embedded array is filled with a gel precursor polymerizable solution (gel forming solution) containing an oligonucleotide probe in the hollow part of each hollow fiber, and a polymerization reaction is performed in the hollow part (step (iii)). . Thereby, the gel-like thing with which the probe was fixed can be hold | maintained in the hollow part of each hollow fiber.
The gel precursor polymerizable solution is a solution containing a reactive substance such as a gel-forming polymerizable monomer, and the solution can be converted into a gel by polymerizing and crosslinking the monomer. Say. Examples of such monomers include acrylamide, dimethylacrylamide, vinyl pyrrolidone, methylene bisacrylamide and the like. In this case, the solution may contain a polymerization initiator and the like.

After fixing the probe in the hollow fiber, the block body is cut in a direction intersecting with the longitudinal direction of the hollow fiber (preferably in a direction perpendicular to the hollow fiber) to make a thin piece (step (iv)). The flakes thus obtained can be used as a DNA microarray. The thickness of the array is preferably about 0.01 to 1 mm. The block body can be cut by, for example, a microtome and a laser.
The contact between the hybridization solution containing the target nucleic acid and the nucleic acid array, that is, the hybridization reaction between the target nucleic acid and the oligonucleotide probe is carried out under conditions suitable for substantially forming a double strand between the target nucleic acid and the probe. The test sample containing the target nucleic acid is brought into contact with the nucleic acid array described above. Conditions suitable for the hybridization reaction vary depending on the base length and GC content of the probe, but it is usually easy for those skilled in the art to uniquely set them with reference to a known Tm calculation formula. Specifically, the reaction conditions (buffer type, pH, temperature, etc.) are appropriately set so that the target nucleic acid in the hybridization solution can hybridize with the probes mounted on the nucleic acid array under stringent conditions. Can be done. After the hybridization reaction, it is preferable to wash excess target nucleic acid among the target nucleic acids subjected to hybridization. The above-mentioned “stringent conditions” means the conditions for washing the nucleic acid array after the hybridization reaction, and those skilled in the art uniquely set the washing conditions with reference to a known Tm calculation formula. However, for example, a condition where the salt (sodium) concentration is 48 to 780 mM and the temperature is 37 to 80 ° C. is preferable, and a condition where the salt concentration is 97.5 to 390 mM and the temperature is 50 to 75 ° C. is more preferable. .

2. Detection Step The detection step is a step of detecting the distribution amount of the hybridized target nucleic acid in the carrier for each carrier as described above after washing the nucleic acid array.
Detection of the amount of the target nucleic acid hybridized to the probe immobilized on each carrier can be performed by imaging and / or digitizing the signal intensity derived from the target nucleic acid. When the signal derived from the target nucleic acid is fluorescence, an excitation light source, a filter set, a CCD camera, and a microscope as necessary are combined in accordance with the fluorescence wavelength to be detected. When the carrier has a three-dimensional shape (for example, a cylindrical shape), confocal light using a predetermined excitation light source or filter set is used in order to easily and three-dimensionally capture the signal emitted by the target nucleic acid in the carrier. It is desirable to use a microscope. Since the signal intensity corresponds to the amount of a substance that emits a signal, the intensity of the signal intensity indicates the amount of the hybridized target nucleic acid.

  In the method of the present invention, the detection step preferably detects the amount (distribution amount) of the hybridized target nucleic acid in the vicinity and inside of the surface of a carrier having a three-dimensional shape (for example, a cylindrical shape). Here, in the vicinity of the surface of the carrier, in a carrier having a uniform structure, the length from the contact boundary between the target nucleic acid solution and the carrier to the same boundary in the opposite direction through the center of the carrier is 3 The portion of the carrier within one-fifth means the inside of the carrier means the remaining portion excluding the portion defined as the vicinity of the surface in the carrier. For example, when the carrier is cylindrical, it is within a predetermined distance (that is, a third of the distance between the upper and lower surfaces) from two upper and lower surfaces corresponding to the cross section (circular cross section) of the column. The part is in the vicinity of the surface of the carrier, and the other part, that is, the part within the range exceeding the predetermined distance from the two upper and lower surfaces is the inside of the carrier. Therefore, the inside of the carrier can be said to be the central portion (center of gravity) of the carrier and the vicinity thereof. The detection of the signal intensity in the vicinity of the carrier surface and in the inside of the carrier may be performed for an arbitrary part or the entire region, and is not limited. In addition, when a hybridization reaction is performed with a plurality of carriers using the same type of probe, an average value of a plurality of detected values may be adopted as the signal intensity detected in the vicinity of the carrier surface and inside the carrier. The maximum value or the minimum value may be adopted and is not limited.

  In the conventional detection method using various nucleic acid arrays, the signal of the hybridized target nucleic acid is such that one average value from the entire carrier is detected for each carrier. On the other hand, the method of the present invention detects a signal for each predetermined part of the carrier (that is, detects a plurality of values from one carrier) and comprehensively analyzes them, so that the conventional method can make a determination. Since it is possible to determine the type of base in the target nucleic acid that has been difficult, it has a very remarkable effect.

3. Determination Step The determination step is a step of determining the base sequence of the target nucleic acid based on the result obtained by the detection step as described above.
To determine the base sequence that constitutes the target nucleic acid is known to have variations in the base sequence depending on the individual or species in a certain base sequence in the chromosomal DNA, but the individual or species to be investigated, etc. If the variation is unknown in, it is to clarify it. Variations in the base sequence are mainly called polymorphisms such as SNPs or point mutations, but there may be a plurality of bases corresponding to the base to be determined in one type of probe. .

  In the determination step in the method of the present invention, specifically, based on the ratio of the distribution amount of the target nucleic acid in the vicinity of the carrier surface obtained by the detection step and the distribution amount of the target nucleic acid inside the carrier, the target nucleic acid The specific base sequence variations (SNPs, etc.) are determined. That is, the ratio of the signal intensity value derived from the target nucleic acid in the vicinity of the carrier surface and in the inside of the carrier (specifically, “near the carrier surface”) The signal intensity value / the signal intensity value inside the carrier ") is compared. Then, the complementary base sequence of the probe fixed to the carrier having the largest ratio value is determined as the base sequence to be determined in the target nucleic acid. Therefore, in the determination step, the base sequence of the target nucleic acid (especially the base sequence to be determined in the target nucleic acid) is identified as a base sequence complementary to the base sequence of the probe fixed to which carrier. Can do.

  Further, in the determination step of the present invention, even if the ratio of the signal intensity values is not specifically determined by numerical value as described above, the base of the target nucleic acid is qualitatively determined by relative comparison of the detection images of the respective carriers. It is also possible to determine the sequence. In this case, in a carrier including a probe having a base sequence complementary to the base sequence of the target nucleic acid, the distribution pattern of the hybridized target nucleic acid is relatively higher than the signal intensity inside the carrier. Judgment is possible because it is large. Here, the distribution pattern of the target nucleic acid is the difference in the signal intensity derived from the target nucleic acid at different positions (near the carrier surface and inside the carrier) in the carrier region where one kind of probe is immobilized and the characteristics of the spots. That means.

  The determination method of the present invention is particularly useful when the oligonucleotide probe used is designed so that the base sequence corresponding to the base sequence to be determined in the target nucleic acid is at or near the end of the probe. It is. In the conventional method, when the probe is designed in this way, it is extremely difficult to determine the target base sequence simply by detecting the signal intensity from the entire carrier. However, if the method of the present invention is used, The base sequence of interest can be easily determined by comprehensively judging the signal intensity of each part of the carrier.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

<Preparation of target nucleic acid>
As target nucleic acids, oligonucleotides having the sequences shown in Table 1 were synthesized.
Using 2 μg of the obtained oligonucleotide, fluorescent labeling was performed using ULYSIS Alexa Fluor 546 (Invitrogen). For labeling, 2 μg of oligonucleotide was dissolved in 20 μl of labeling buffer attached to the kit, then 5 μl of ULYSIS Alexa Fluor 546 DMF lysate was added, stirred well, and then heated at 85 ° C. for 30 minutes. . The labeled oligonucleotide was purified using MicroSpin G-25 Columns (manufactured by GE Healthcare Bioscience). The concentration of the fluorescently labeled oligonucleotide after purification was measured, and water was added to prepare a concentration of 1 ng / μl.

  With the solution composition shown in Table 2, 150 μl (× 5 samples) of a hybridization solution was prepared.

<Synthesis of oligonucleotide probe>
Oligonucleotides having the sequences shown in Table 3 were synthesized as probes (capture probes) according to the following method.
First, an oligonucleotide serving as a probe was synthesized by an automatic DNA synthesizer. In the final stage of synthesis, aminolink TM (manufactured by PE Biosystems) was reacted with the oligonucleotide and then subjected to deprotection, whereby an aminohexyl group was introduced at the end of each oligonucleotide. -O-aminohexyl oligonucleotide was prepared. Next, the oligonucleotide was reacted with methacrylic anhydride to prepare a 5 ′ terminal vinylated oligonucleotide.

<Manufacture of nucleic acid array>
A hollow fiber bundle was manufactured using the array fixture shown in FIG. In the figure, x, y, and z are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.
First, two perforated plates (21) each having a thickness of 0.32 mm and having a total distance of 228 in 12 rows and 19 rows were prepared with the distance between the centers of the holes being 0.42 mm. These perforated plates were overlapped, and polycarbonate hollow fibers (31) (manufactured by Mitsubishi Engineering Plastics Co., Ltd., 1% by mass of carbon black) were passed through each of the holes one by one.
The position of the two perforated plates was moved in a state where 0.1 N tension was applied to each fiber in the X-axis direction, and fixed at two positions, 20 mm and 100 mm from one end of the hollow fiber. That is, the interval between the two perforated plates was 80 mm.

Next, the three surrounding surfaces of the space between the perforated plates were surrounded by a plate-like object (41). In this way, a container having an open top only was obtained. The resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the total weight of a polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd. NIPPORAN 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle.
Next, a gel precursor polymerizable solution containing a monomer and an initiator mixed at a mass ratio shown in Table 4 was prepared for each type of probe mounted on the nucleic acid array.

Next, in order to fill the hollow portion of the corresponding hollow fiber of the hollow fiber bundle with the gel precursor polymerizable solution containing each nucleic acid probe prepared above so as to have the probe arrangement shown in FIG. The container containing the functional solution and the hollow fiber bundle prepared above were placed in a desiccator. After the inside of the desiccator was decompressed, the unsealed end of each yarn of the hollow fiber bundle was immersed in a container containing the predetermined polymerizable solution. Nitrogen gas was sealed in a desiccator, and a gel precursor polymerizable solution containing a probe was introduced into the hollow portion of the hollow fiber. Next, the inside of the container was brought to 70 ° C., and a polymerization reaction was carried out over 3 hours.
In this way, a hollow fiber bundle was obtained in which the probe was held in the hollow part of the hollow fiber via the gel-like material.
Next, the obtained hollow fiber bundle was sliced in a direction orthogonal to the longitudinal direction of the fiber using a microtome to obtain 50 thin sheet (nucleic acid array) having a thickness of 0.25 mm.

<Hybridization reaction>
Five of the obtained nucleic acid arrays are sealed in five independent plastic containers, and then the hybridization solution containing the target nucleic acid is placed in each plastic container to contact the nucleic acid array with the target nucleic acid. I let you.
The plastic container was sealed with a plastic lid, and hybridization was formed under temperature conditions of 35 ° C., 40 ° C., 45 ° C., 50 ° C., and 65 ° C., respectively.
After completion of the hybridization reaction, each nucleic acid array was washed independently under the following conditions. First, the nucleic acid array was immersed in 10 ml of a washing solution composed of 0.12M Tris-HCl, 0.12M NaCl, 0.05% Tween20. During immersion, each nucleic acid array was maintained for 20 minutes at the same temperature as during the hybridization reaction. Thereafter, the same operation was repeated, and then the nucleic acid array was immersed in 10 ml of a washing solution composed of 0.12M Tris-HCl and 0.12M NaCl. During the immersion, each nucleic acid array was maintained at the same temperature as the hybridization reaction for 10 minutes, and the washing was completed.

<Target nucleic acid signal detection and nucleotide sequence determination>
For detection operation, using a cooled CCD camera type nucleic acid array automatic detection device (Mitsubishi Rayon Co., Ltd.), immerse the array in 0.12M Tris-HCl, 0.12M NaCl, cover with a cover glass, and then label nucleic acid sample The fluorescence signal intensity of the molecule was measured.
The probes of probe numbers 1 and 5 are complementary strands that are completely complementary to the base sequence of the target nucleic acid. The probes of probe numbers 2, 3, and 4 are sequences in which the base sequence of the target nucleic acid and the terminal single base are not complementary. Probes with probe numbers 6, 7, and 8 are sequences in which the base sequence of the target nucleic acid is non-complementary to one base at the non-terminal (center portion of the probe).
The detected signal intensity is shown in FIGS. The signal intensity indicates an average value of four carriers on which the same probe mounted on each nucleic acid array is immobilized.

  As shown in FIG. 4, the signal intensity of the carrier on which the probe numbers 6, 7, and 8 having non-complementary bases in the central part are fixed is slightly lower than that of the probe number 5, and the washing temperature is increased. Under conditions where hybridization was difficult to form, it was possible to detect a single base difference only with signal intensity. On the other hand, as shown in FIG. 3, the signal intensity of the carrier on which probe numbers 2, 3, and 4 having non-complementary bases at the terminal portion are immobilized is almost the same as that of probe number 1, as compared with probe number 1. Even when the washing temperature was raised, the signal intensity as a whole only decreased, and the difference was not clarified. That is, it was difficult to detect the difference between the terminal single bases only by the hybridization signal intensity.

  Next, FIGS. 5 and 6 show detection images on a carrier on which probe numbers 1, 2, 3, and 4 are immobilized during hybridization under a temperature condition of 50 ° C. using a confocal microscope. It was. FIG. 5 is an image of signal intensity when the carrier to which the probe is fixed is viewed from directly above. FIG. 6 is a white line portion in the image shown in FIG. This is the distribution of signal intensity when viewed. As shown in FIG. 3 and FIG. 5, the average signal intensity is almost the same in any of the carriers on which the probe numbers 1, 2, 3, and 4 are fixed. However, as shown in FIG. 6, the distribution of signal intensity in each carrier (that is, the distribution amount of the hybridized target nucleic acid) is as follows. 3 and 4 were found to be greatly different from the fixed carrier. That is, the signal intensity in the vicinity of the carrier surface is relatively high in the carrier on which probe number 1 is immobilized, and the signal intensity inside the carrier is immobilized on probe numbers 2, 3 and 4 whose terminal single bases are non-complementary. It was found to be relatively high in other carriers.

FIG. 7 is a graph showing the distribution of signal intensity in the vertical direction based on the image shown in FIG. As can be seen from FIG. 7, it was confirmed that there was a maximum value of the signal intensity near the surface of the carrier, and there was a minimum value inside the carrier sandwiched between the maximum values. Then, it was found that the local maximum value was larger and the local minimum value was smaller in the carrier on which the probe number 1 was fixed than in the carrier on which other probes were fixed. Therefore, it was considered that the ratio between the larger one of the two maximum values and the minimum value was calculated, and the one with the largest ratio value could be judged as having a base sequence complementary to the target nucleic acid. Note that the ratio between the maximum value and the minimum value referred to here was calculated as a value of “maximum value / minimum value”.
Table 5 shows the maximum value and the minimum value of the signal intensity distribution and the value of the ratio between the maximum value and the minimum value in each carrier to which the probe numbers 1, 2, 3, and 4 are fixed.

It was found that the carrier with the probe number 1 immobilized had a clearly larger value of the ratio between the signal intensity near the carrier surface and the signal intensity inside the carrier than the carrier with other probes immobilized. That is, among the probe numbers 1 to 4, it was possible to clearly determine that the base sequence of probe number 1 was a base sequence complementary to the target nucleic acid.
By using the determination method of the present invention, it is possible to clearly determine the base sequence of the target nucleic acid even when using a probe whose terminal single base is non-complementary, which is difficult to discriminate only by the average signal intensity. became.

It is the schematic which shows the arrangement | sequence fixing jig for manufacture of a hollow fiber bundle (hollow fiber array body). It is a figure which shows the design of a nucleic acid array. Each numerical value corresponds to the sequence number of the probe shown in Table 3. It is a graph which shows the detection result of the signal intensity | strength (average value) of the target nucleic acid hybridized to each probe (probe number 1-4). The vertical axis shows the signal intensity. It is a graph which shows the detection result of the signal intensity | strength (average value) of the target nucleic acid hybridized to each probe (probe number 5-8). The vertical axis shows the signal intensity. FIG. 5 is a photograph of an image showing the signal intensity when the carrier on which each probe (probe numbers 1 to 4) is fixed is viewed from directly above. FIG. 6 is a photograph of an image showing a signal intensity distribution when the carrier on which each probe (probe numbers 1 to 4) is fixed is viewed from the side in the white line portion in the image shown in FIG. 5. It is the graph which showed distribution of the signal strength in the vertical direction of the support | carrier to which each probe (probe number 1-4) was fixed based on the image shown in FIG. The vertical axis shows the signal intensity.

Explanation of symbols

11 hole 21 perforated plate 31 hollow fiber 41 plate

SEQ ID NO: 1 synthetic DNA
Sequence number 2: Synthetic DNA
Sequence number 3: Synthetic DNA
Sequence number 4: Synthetic DNA
Sequence number 5: Synthetic DNA
Sequence number 6: Synthetic DNA
Sequence number 7: Synthetic DNA
Sequence number 8: Synthetic DNA
Sequence number 9: Synthetic DNA

Claims (8)

A method for determining a base sequence of a target nucleic acid, comprising:
(A) contacting a test sample containing the target nucleic acid with a nucleic acid array equipped with two or more kinds of oligonucleotide probes fixed to a carrier, and hybridizing the probe and the target nucleic acid;
(B) detecting a distribution amount of the hybridized target nucleic acid in the carrier for each carrier; and (c) determining a base sequence of the target nucleic acid based on a result obtained by the detection. Said method.   The method according to claim 1, wherein the carrier has a three-dimensional shape, and the distribution amount is detected in the vicinity of the surface and the inside of the carrier.   The method according to claim 2, wherein the three-dimensional shape is cylindrical.   The determination in the step (c) is performed based on a ratio between the distribution amount in the vicinity of the surface of the carrier obtained by the detection and the distribution amount in the carrier. 3. The method according to 3.   The determination in the step (c) specifies whether the base sequence of the target nucleic acid is a base sequence complementary to the base sequence of the probe fixed to any of the carriers. Item 5. The method according to any one of Items 1 to 4.   The method according to any one of claims 1 to 5, wherein the nucleotide sequences different from each other between the two or more types of oligonucleotide probes correspond to gene polymorphic sites in the nucleotide sequence of the target nucleic acid.   The method according to any one of claims 1 to 6, which is a method for detecting a gene polymorphism in the base sequence of the target nucleic acid.   The method according to claim 6 or 7, wherein the gene polymorphism is at least one selected from the group consisting of a single nucleotide polymorphism, an insertion polymorphism, a deletion polymorphism, and a base repeat polymorphism.