JP2010284159A - Probe having high specificity, nucleic acid array using the same and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an efficient blocking nucleic acid to improve specificity of hybridization. <P>SOLUTION: The nucleic acid composition is a mixture of a nucleic acid A containing linked base sequences A1 and A2 and a nucleic acid B containing linked base sequences A1' and A2' which are complementary to the base sequences A1 and A2, wherein a 5' terminal of the base sequence A2 is linked to a 3' terminal of the base sequence A1, and a 3' terminal of the base sequence A1' is linked to a 5' terminal of the base sequence A2'. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a nucleic acid detection probe for more accurately detecting a target nucleic acid to be detected. The present invention also relates to a nucleic acid array for simultaneously detecting a plurality of target nucleic acids more accurately and a method for producing the nucleic acid array.

Various methods have been developed to date for detecting and identifying nucleic acids. As a basic technique for that purpose, a hybridization method is widely used.

Hybridization means that nucleic acid molecules complementarily form a complex (double strand). This is due to the continuation of hydrogen bonding between complementary bases.
In general, the longer the base length of the nucleic acid constituting the double strand, the higher the affinity for hybridization, and the shorter the base length, the lower the affinity for hybridization. The affinity of hybridization also depends on the bases constituting the nucleic acid. When there are many G and C in the nucleic acid which comprises a double strand, the affinity of hybridization becomes high, and when there are many A and T, the affinity of hybridization becomes low. This is due to the difference in the number of hydrogen bonds formed by the base (three for the former and two for the latter).

  As a method for detecting a specific nucleic acid using hybridization, there are Southern hybridization and Northern hybridization. In this method, the target nucleic acid is detected by immobilizing the nucleic acid to be detected (target nucleic acid) on a carrier, bringing the complementary strand of the nucleic acid (probe nucleic acid) into contact therewith, and detecting the formed hybridization. It is a method to do. With these techniques, the number of nucleic acids that can be detected at one time is small.

  On the other hand, there is a nucleic acid array (DNA chip) as a method for simultaneously detecting many kinds of nucleic acids. In recent years, nucleic acid arrays in which many types of probe nucleic acids are arranged at high density have been actively developed. The basic detection principle of the nucleic acid array is the same as that described above. By using a nucleic acid array, it has become possible to comprehensively evaluate the movement of the entire gene.

However, in hybridization, not only a completely complementary sequence forms a double strand, but a similar base sequence may form a double strand. For this reason, detection of nucleic acids by a hybridization method may cause a problem of so-called “cross-hybridization” (also referred to as “cross-hybridization” in this specification), which detects nucleic acids that are not for detection purposes. It was.
Many methods have been devised so far to eliminate cross-hybridization as much as possible and improve the specificity of hybridization between the probe nucleic acid and the target nucleic acid.

  For example, optimization of hybridization conditions can be mentioned. The first condition is the hybridization temperature. Nucleic acid hybridization is due to hydrogen bonding. When the temperature is raised, hybridization becomes difficult to occur. Therefore, by raising the temperature, it is possible to dissociate only the cross-hybridization while maintaining the target hybridization as much as possible.

  The condition is secondly the salt concentration of the hybridization solution. Since nucleic acids have negative charges, the nucleic acids repel each other electrically. When the salt concentration of the solution is increased, the negative charge of the nucleic acid is offset, so that the repulsion between the nucleic acids is weakened and hybridization is likely to occur. Therefore, by adjusting the salt concentration of the solution, it is possible to set conditions that prevent cross hybridization as much as possible while maintaining the target hybridization.

  It is also possible to improve specificity by adding any additive to the hybridization solution. By adding a surfactant, betaine, Denhardt's solution, formamide, etc., it is possible to make it difficult to take a three-dimensional structure in the molecule. This makes it possible to improve the specificity of hybridization.

  When a relatively long chain cDNA is used as the probe nucleic acid, cross-hybridization derived from repetitive sequences can be prevented by adding Cot-1 or salmon sperm DNA to the hybridization solution. . Thereby, it is possible to improve the specificity of hybridization.

  Furthermore, with the recent accumulation of a large amount of genome information and the development of information processing technology, programs for designing probe nucleic acids and the like have been developed so as to minimize cross hybridization. By using these, it is possible to improve the specificity of hybridization.

Under such circumstances, a method for improving the specificity of hybridization using “blocking nucleic acid” has also been developed.
A blocking nucleic acid is a nucleic acid composed of a partially complementary strand of a probe nucleic acid. A blocking nucleic acid has a higher affinity for a probe nucleic acid than a nucleic acid that causes cross-hybridization (cross-hybrid nucleic acid), and an affinity for a probe nucleic acid compared to the target nucleic acid that is the original detection target. Low nucleic acids are used.

  This blocking nucleic acid is previously hybridized to the probe nucleic acid. Then, it is possible to inhibit the cross hybrid nucleic acid from hybridizing to the probe nucleic acid. On the other hand, the target nucleic acid hybridizes to the probe nucleic acid. As a result, the specificity of hybridization is improved.

  A method using a blocking nucleic acid is known for detection of a single nucleotide polymorphism using a probe nucleic acid. In the probe nucleic acid, a blocking nucleic acid is hybridized to an invariable base sequence portion that is not a single nucleotide polymorphism portion, and this portion is blocked. On the other hand, the single nucleotide polymorphism part is exposed. Then, the nucleic acid having the target polymorphic sequence hybridizes to the probe nucleic acid, but the nucleic acid having a different polymorphic sequence does not hybridize to the probe nucleic acid. In this way, it is possible to detect a minute difference of a single base with high sensitivity using a blocking nucleic acid (Patent Document 1).

  As long as the blocking nucleic acid satisfies the above conditions, the base sequence and sequence length can be designed freely.

However, when the affinity for hybridization between the blocking nucleic acid and the probe nucleic acid is low (for example, when the sequence length of the blocking nucleic acid is set short), the blocking nucleic acid cannot sufficiently inhibit cross-hybridization and has high specificity. Sexuality may not be obtained. On the other hand, when the affinity between the blocking nucleic acid and the probe nucleic acid is high (for example, when the sequence length of the blocking nucleic acid is set long), the hybridization between the target nucleic acid and the probe nucleic acid is also inhibited, resulting in detection. There may be a significant decrease in sensitivity. That is, it is difficult to achieve both sensitivity and specificity in detecting a nucleic acid using a blocking nucleic acid.
Therefore, the method for improving the specificity of hybridization using a blocking nucleic acid is mainly applicable when a large amount of target nucleic acid is present, such as polymorphism analysis. In normal mRNA expression analysis, the absolute amount of the target nucleic acid is often small. In such a case, there was a concern that the target nucleic acid could not be detected due to a decrease in detection sensitivity.

WO2005 / 012571

  Therefore, an efficient blocking nucleic acid that improves the specificity of hybridization without inhibiting the hybridization between the probe nucleic acid and the target nucleic acid has been desired.

In order to solve the above-mentioned problems, the present inventors have paid attention to a method for designing a blocking nucleic acid and have conducted intensive studies. As a result, designing a blocking nucleic acid so that a portion of the blocking nucleic acid hybridizes to a portion of the probe nucleic acid and another portion of the blocking nucleic acid to a portion of another probe nucleic acid effectively hybridizes. The inventors have found that the specificity of can be improved, and have completed the present invention.
That is, the present invention is as follows.
(1) Nucleic acid obtained by mixing nucleic acid A in which base sequences A1 and A2 are linked, and nucleic acid B in which base sequences A1 ′ and A2 ′ are complementary to base sequences A1 and A2, respectively. In the composition, the 5 ′ end of the base sequence A2 is linked to the 3 ′ end of the base sequence A1, and the 3 ′ end of the base sequence A1 ′ is linked to the 5 ′ end of the base sequence A2 ′. A nucleic acid composition.
(2) A nucleic acid array in which the nucleic acid composition according to (1) above is arranged on a carrier in an addressable manner, wherein either one of nucleic acids A and B is immobilized on the carrier.
(3) One of nucleic acid A formed by linking base sequences A1 and A2 and nucleic acid B formed by linking base sequences A1 ′ and A2 ′ that are complementary to base sequences A1 and A2, respectively. A method for producing a nucleic acid array comprising the step of immobilizing a nucleic acid on a carrier in an addressable manner and then mixing the other nucleic acid, wherein the 5 ′ end of the base sequence A2 is located at the 3 ′ end of the base sequence A1. A method for producing a nucleic acid array, wherein the 3 ′ end of the base sequence A1 ′ is linked to the 5 ′ end of the base sequence A2 ′.
(4) A method for detecting a target nucleic acid, comprising a step of contacting the nucleic acid composition according to (1) or the nucleic acid array according to (2) and a target nucleic acid, and detecting a signal of the target nucleic acid.

Conventional blocking nucleic acids reduce the signal strength of hybridization when trying to ensure sufficient specificity for the probe nucleic acid. In addition, when trying to maintain the signal intensity, it is difficult to ensure sufficient specificity for the probe nucleic acid. Therefore, it has been difficult to apply this to expression analysis.
However, by applying the present invention, it is possible to prevent a reduction in hybridization efficiency as occurs with conventional blocking nucleic acids and to improve the specificity of hybridization efficiently. As a result, it has become possible to use the method for improving specificity using a blocking nucleic acid not only for polymorphism analysis but also for expression analysis.

It is a schematic diagram of the probe nucleic acid and blocking nucleic acid of the present invention. It is a schematic diagram of the probe nucleic acid and blocking nucleic acid of the present invention. It is a figure which shows one aspect | mode of the nucleic acid array of this invention. It is a figure which shows one aspect | mode of the detection method using the nucleic acid array of this invention. It is a figure of the arrangement | sequence fixing jig which manufactures a fiber array. It is a figure which shows the design of a nucleic acid array. The numbers are SEQ ID NOs, and B is a diagram showing spots where no probe nucleic acid is mounted. It is a figure which shows the signal intensity | strength of the model sample in Example 1, and its ratio. It is a figure which shows the signal intensity | strength of the model sample in Comparative Examples 1-4, and its ratio. It is a figure which shows the signal intensity | strength of the model sample in Comparative Examples 5-6, and its ratio. It is the figure which put together the result in the probe X and the probe Y among FIGS. It is a figure which shows the signal intensity | strength of the model sample in Example 2, and its ratio. It is a figure which shows the signal intensity | strength of the model sample in Comparative Examples 7-8, and its ratio. It is the figure which put together the result in the probe A and the probe B among FIG. It is a figure which shows the signal intensity | strength of the model sample in Example 3, and its ratio. It is a figure which shows the signal intensity | strength of the model sample in Comparative Examples 9-11, and its ratio. It is the figure which put together the result in the probe C and the probe D among FIG.

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.
The first of the present invention is a mixture of nucleic acid A in which base sequences A1 and A2 are linked, and nucleic acid B in which base sequences A1 ′ and A2 ′ complementary to the base sequences A1 and A2 are linked, respectively. Nucleic acid composition. The nucleic acid A has a form in which the 5 ′ end of the base sequence A2 is linked to the 3 ′ end of the base sequence A1, and the nucleic acid B has a form in which the 3 ′ end of A1 ′ is linked to the 5 ′ end of A2 ′ ( FIG. 1).
Here, the base sequence A1 and the base sequence A2 are arbitrary nucleic acid base sequences.
The base sequence A1 ′ is a base sequence composed of a complementary sequence of the base sequence A1.
The base sequence A2 ′ is a base sequence composed of a complementary sequence of the base sequence A2.

Therefore, when one molecule of the nucleic acid A and the nucleic acid B is present, a form in which the sequence on the 3 ′ side of the nucleic acid B (A2 ′ which is a complementary sequence of A2) is hybridized to the A2 on the 3 ′ side of the nucleic acid A (Fig. 1, upper panel). Alternatively, the 5 ′ side (A1) of the nucleic acid A can be hybridized with the 5 ′ side of the nucleic acid B (A1 ′ which is a complementary sequence of A1). When a plurality of nucleic acids A and B are mixed, the nucleic acids A and B hybridize alternately (lower panel in FIG. 1). In the present invention, one of the nucleic acid compositions containing such nucleic acids A and B is used as a probe nucleic acid, and the other is used as a blocking nucleic acid.
Nucleic acids are RNA, DNA, analogs such as PNA and LNA, and those that are mixed together, and can form double strands by hybridization with complementary strands As long as it is, it may be anything.
The base sequence may be completely random, or may be the same sequence in whole or in part as that gene in order to detect any gene, and the gene and all or part of it may be a complementary sequence. There may be. The base sequence may have any length and configuration as long as the sequence can be distinguished from other base sequences. However, in order for the linked nucleic acids A and B to function as probe nucleic acids, the sequence length of the linked nucleic acids A and B is preferably 10 to 100 bases, and preferably 20 to 70 bases. Further preferred.
Regarding the relationship between the base sequences A1 and A2 (the relationship between A1 ′ and A2 ′), the base sequence lengths are preferably similar to each other. Specifically, it is desirable that the difference in sequence length between the base sequences A1 and A2 (A1 ′ and A2 ′) is within 5 bases, and the difference is within 2 bases.
In addition, it is desirable that the base sequences A1 and A2 have the same degree of ease in forming a double strand with their complementary sequences (A1 ′ and A2 ′). Specifically, among the bases forming the base sequences A1 and A2, A and T (or U) are set to 1, G and C are set to 2, and the values of all bases constituting each sequence are added. In this case, the difference between the values of the base sequences A1 and A2 is preferably 0 to 5, and more preferably 0 to 3.
A complementary sequence is a sequence that is paired with a certain sequence. Nucleic acids form a pair by pairing A and T (or U) and G and C, respectively, by hydrogen bonding. Thus, for example, the complementary sequence of ATGC is GCAT, and the complementary sequence of TACG is CGTA. However, the complementary sequence is not limited only to the above base pair. In a base sequence having a predetermined length, even if some bases in the sequence do not form the above base pair, the sequence and its pair As long as it can hybridize with the sequence forming the sequence, it is included in the “complementary sequence”.

Ligation means the hydroxyl group at the 3 ′ end in the base sequence A1 and the phosphate group at the 5 ′ end in the base sequence A2, or the hydroxyl group at the 3 ′ end in the base sequence A1 ′ and the 5 ′ end in the base sequence A2 ′. It shows that the phosphate group is phosphodiester linked.
The 3 ′ end is the 3 ′ end of the nucleotide, which has a hydroxyl group. The 5 ′ end is the 5 ′ end of the nucleotide, and has a phosphate group. A nucleic acid can have a chain-like molecular structure by phosphodiester bonding between the hydroxyl group at the 3 ′ end of a certain nucleotide and the phosphate group at the 5 ′ end of a different nucleotide. The 5 ′ end and 3 ′ end indicate the direction of the chain nucleic acid.
The nucleic acid A is a nucleic acid having a base sequence formed by linking the 5 ′ end of the base sequence A2 to the 3 ′ end of the base sequence A1.

  The nucleic acid B is a nucleic acid having a base sequence formed by linking the 3 'end of the base sequence A1' to the 5 'end of the base sequence A2'.

“Mixed” means that the mixed materials are in contact with each other or in close contact with each other. By mixing the nucleic acid A and the nucleic acid B described above, the nucleic acid A and the nucleic acid B are converted into a 3 ′ side region (A2) of the nucleic acid A and a 3 ′ side region (A2 ′) of the nucleic acid B, or a nucleic acid. Hybridization is formed in the 5 ′ region (A1) of A and the 5 ′ region (A1 ′) of nucleic acid B. At this time, if the nucleic acid A and the nucleic acid B are one molecule, it is considered that the portion not subjected to hybridization remains a single strand (A1 and A1 ′ in the upper panel of FIG. 1).
However, when nucleic acid A and nucleic acid B are present in large quantities, it is presumed that hybridization occurs continuously and takes a double-stranded structure composed of a plurality of nucleic acids A and a plurality of nucleic acids B (bottom of FIG. 1). panel).
The target nucleic acid is a target nucleic acid to be detected.

A probe nucleic acid is a nucleic acid for capturing a target nucleic acid by hybridization. That is, the probe nucleic acid is a complementary strand of the target nucleic acid.
A cross-hybrid nucleic acid is a nucleic acid that hybridizes to a probe nucleic acid other than the target nucleic acid (also referred to as a cross-hybridized nucleic acid).
A blocking nucleic acid is a nucleic acid composed of a partially complementary strand of a probe nucleic acid. The blocking nucleic acid prevents the cross-hybrid nucleic acid from hybridizing to the probe nucleic acid.

Here, consider a case where nucleic acid A is used as a probe nucleic acid and nucleic acid B is used as a blocking nucleic acid. The target nucleic acid is a complementary strand of nucleic acid A. A nucleic acid that hybridizes to nucleic acid A other than the target nucleic acid is a cross-hybridized nucleic acid.
Since the base sequence A2 of the probe nucleic acid A and the base sequence A2 ′ of the blocking nucleic acid B hybridize, the hybridization of the cross-hybrid nucleic acid having a sequence complementary to a part of the base sequence A2 is inhibited by the blocking nucleic acid. Is done. Further, since the base sequence A1 of the probe nucleic acid A hybridizes with a base sequence A1 ′ of a blocking nucleic acid B different from the above, a cross-hybrid nucleic acid having a sequence complementary to a partial sequence of the base sequence A1 is also available. Hybridization is inhibited by the blocking nucleic acid. On the other hand, in the state where the probe nucleic acid and the blocking nucleic acid are continuously hybridized in this way, the hybridization between the target nucleic acid and the probe nucleic acid is hardly inhibited. That is, according to this method, the target nucleic acid can hybridize with the probe nucleic acid while suppressing the hybridization by suppressing the hybridization of the cross-hybrid nucleic acid to the probe nucleic acid, and the detection sensitivity (specificity of the target nucleic acid) Property) can be improved (FIG. 2).
The second aspect of the present invention is a nucleic acid array in which the nucleic acid composition described above is arranged on a carrier in an addressable manner, wherein either one of the nucleic acids A and B is immobilized on the carrier. It is a nucleic acid array.

That is, the second aspect of the present invention is a nucleic acid array in which the nucleic acid composition is arranged on a carrier and the arrangement position thereof is defined.
A third aspect of the present invention is a method for producing the nucleic acid array, wherein either one of the nucleic acids A and B is immobilized in advance so as to be addressable to a carrier, and then the other nucleic acid is prepared. Mixing.
Here, addressable means that the relative or absolute position can be discriminated. Specifically, when a plurality of nucleic acid compositions are arranged, it has been clarified or can be clarified which nucleic acid composition is arranged at which position on the carrier. Refers to that.

For example, by fixing one end of the probe nucleic acid at a predetermined position on the array and regularly arranging it, it is possible to know which probe nucleic acid is fixed at which position. FIG. 3 is a diagram showing a state in which one end of the nucleic acid A is fixed on a carrier.
The carrier is a substance that immobilizes the probe nucleic acid and a substance on which the nucleic acid composition is arranged. Various substances such as glass, silicon, resin, gel, and beads can be used as the carrier.

  The nucleic acid array is formed by immobilizing probe nucleic acids on a carrier so that a plurality of addresses can be addressed. In addition, the nucleic acid array referred to in the present invention is one in which a blocking nucleic acid is further hybridized to a probe nucleic acid immobilized on a carrier so that a plurality of addresses can be addressed.

Immobilization refers to a process that prevents the probe nucleic acid from leaving the carrier. For example, it is possible to immobilize the probe nucleic acid on the carrier by modifying the probe nucleic acid or the carrier itself with some functional group and chemically bonding it. Also, immobilization is possible by electrostatic bonding or hydrophobic bonding.
The nucleic acid array of the present invention can be produced by immobilizing only one of nucleic acid A and nucleic acid B on a carrier and hybridizing the other to the immobilized nucleic acid. In this case, the nucleic acid immobilized first becomes the probe nucleic acid, and the nucleic acid hybridized later becomes the blocking nucleic acid.
The nucleic acid array of the present invention can also be produced by mixing nucleic acid A and nucleic acid B in advance and placing the mixture on a carrier.
Furthermore, the fourth aspect of the present invention is a method for detecting a nucleic acid, comprising a step of contacting a target nucleic acid with the nucleic acid array or nucleic acid composition and detecting a signal of the target nucleic acid.
Contact means that the nucleic acid array or nucleic acid composition and the target nucleic acid are present in the same reaction system. More specifically, adding a target nucleic acid on a nucleic acid array, mixing a nucleic acid composition and a target nucleic acid, etc. are mentioned.
Consider a case where nucleic acid A is used as a probe nucleic acid (FIG. 2). Hybridization between the probe nucleic acid and the cross-hybrid nucleic acid is suppressed by the blocking nucleic acid. By contacting the target nucleic acid with the nucleic acid composition or the nucleic acid array in this state, the target nucleic acid can be hybridized with the probe nucleic acid while preventing hybridization of the cross-hybrid nucleic acid to the probe nucleic acid (FIG. 2).

  FIG. 4 is a diagram showing an aspect of detection of a target nucleic acid in a nucleic acid array when a probe nucleic acid is immobilized on a carrier in an addressable manner. Hybridization between the probe nucleic acid A immobilized on the carrier and the cross-hybrid nucleic acid is suppressed by the blocking nucleic acid. By bringing the target nucleic acid into contact with the nucleic acid array in this state, the probe nucleic acid and the target nucleic acid are hybridized. If the target nucleic acid does not exist, the base sequence A2 'of the blocking nucleic acid B is hybridized to the base sequence A2 of the probe nucleic acid A. Further, the base sequence A1 of the probe nucleic acid A is hybridized with the base sequence A1 'of the blocking nucleic acid B that is hybridized to another probe nucleic acid A (FIG. 3). When the target nucleic acid is present, the hybridization between the base sequence A1 and the base sequence A1 'and the base sequence A2 and the base sequence A2' that have been previously hybridized is released. As a result, the target nucleic acid can hybridize with the probe nucleic acid while preventing the hybridization of the cross-hybrid nucleic acid (FIG. 4).

The target nucleic acid can be detected by labeling the target nucleic acid with a fluorescent label, a radiolabel or the like and detecting the signal with a detection device. By quantifying or qualifying the hybridization between the probe nucleic acid and the target nucleic acid using a signal derived from a substance previously labeled with the target nucleic acid, such as fluorescence, chemiluminescence, or radioisotope, the target nucleic acid corresponding to the probe nucleic acid The existence can be confirmed. Further, the detection method is not limited to the above. For example, it is possible to label the blocking nucleic acid and confirm the loss of the blocking nucleic acid due to the hybridization of the target nucleic acid with a decrease in signal.
The above “labeling” method is not particularly limited as long as hybridization of nucleic acid can be detected. Examples of the labeling substance used for labeling include fluorescent substances such as Cy3, Cy5, and Alexa Fluor, enzymes or proteins for utilizing chemiluminescence accompanying substrate degradation using alkaline phosphatase and horseradish peroxidase, γ-32P, A radioisotope such as α-32P can be used, but it is preferable to use a fluorescent substance because it is simple.

  When incorporating these labeling substances into nucleic acid molecules, direct, indirect, physical, or chemical binding is possible as long as the labeling method is stable and does not inhibit specific hybridization with the probe nucleic acid. Regardless, any method can be used. As chemical modification methods, a base previously labeled with a fluorescent substance is incorporated during the reaction, or an analog base modified with biotin or an aminoallyl group is incorporated during the reaction, and the labeled substance is incorporated via the base. A method is mentioned. In addition, a method of intercalating a labeling substance to a complementary nucleic acid molecule using SYBR Green, acridine orange, SYBR Gold, etc., a method of binding a labeling substance to a complementary nucleic acid molecule via platinum, such as ULYSIS, etc. You can also

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
Example 1
Preparation of Nucleic Acid Array In order to prepare a nucleic acid array, oligonucleotides (60 base length) represented by SEQ ID NO: 1 and 2 shown in Table 1 were synthesized.

  In the final stage of synthesis, aminolink TM (manufactured by PE Biosystems) was reacted with the oligonucleotide, followed by deprotection, whereby an aminohexyl group was introduced at the end of each oligonucleotide. -O-aminohexyl oligonucleotide was prepared.

  Subsequently, these oligonucleotides were reacted with methacrylic anhydride to prepare 5'-terminal vinylated oligonucleotides, which were used as probe nucleic acids.

  The 1st to 30th and 31st to 60th bases of the probe X correspond to the base sequence A1 and the base sequence A2 in FIG. 1, respectively. The same applies to the probe Y.

  Next, a hollow fiber bundle was manufactured using the array fixing device shown in FIG. Note that x, y, and z in FIG. 5 are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.

  First, a perforated plate (21) having a thickness of 0.12 provided with a total of 228 holes (11) having a diameter of 0.32 mm and having a center-to-center distance of 0.42 mm in 12 rows and 19 rows each. Two sheets were prepared. These perforated plates were overlapped, and polycarbonate hollow fibers (31) (added by 1% by mass of carbon black manufactured by Mitsubishi Engineering Plastics) were passed through all of the holes one by one.

  The position of the two perforated plates was moved in a state in which a tension of 0.1 N was applied to each fiber in the X-axis direction, and was fixed at two positions of 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. Nippon Run 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 2 was prepared for each probe nucleic acid to be immobilized on the 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 prepared above so as to have the arrangement shown in FIG. And the hollow fiber bundle produced above was installed in a desiccator. After reducing the pressure in the desiccator, 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 the desiccator, and a gel precursor polymerizable solution containing the probe nucleic acid was introduced into the hollow part of the hollow fiber. Subsequently, the inside of a container was 70 degreeC and the polymerization reaction was performed over 3 hours.

  In this way, a hollow fiber bundle in which the probe nucleic acid was held in the hollow portion of the hollow fiber via a gel-like material was obtained.

  Next, the obtained hollow fiber bundle was sliced in a direction orthogonal to the longitudinal direction of the fiber using a microtome to obtain 300 thin sheet (nucleic acid array) having a thickness of 0.25 mm.

The nucleic acid array was prepared at the probe position shown in FIG. The numbers 1 and 2 in FIG. 6 indicate the probe nucleic acid sequence numbers (SEQ ID NO: 1 and SEQ ID NO: 2 respectively), and probe nucleic acids having the base sequences indicated by the respective sequence numbers are arranged in the nucleic acid array. ing. B shows a spot without probe nucleic acid.
Preparation of Blocking Nucleic Acid Mixed Nucleic Acid Array Next, an oligonucleotide that is a blocking nucleic acid was designed and mixed with the nucleic acid array prepared above to prepare a blocking nucleic acid mixed nucleic acid array.

  First, oligonucleotides represented by SEQ ID NO: 3 and SEQ ID NO: 4 shown in Table 3 were synthesized and used as blocking nucleic acids.

  The oligonucleotide represented by SEQ ID NO: 3 is composed of a structure in which 30 bases on the 5 terminal side and 30 bases on the 3 terminal side are replaced in the completely complementary strand (60 bases) of SEQ ID NO: 1. In addition, the oligonucleotide represented by SEQ ID NO: 4 has a structure in which 30 bases on the 5 terminal side and 30 bases on the 3 terminal side are replaced in the completely complementary strand (60 bases) of SEQ ID NO: 2.

  SEQ ID NO: 3 is a blocking nucleic acid for the probe nucleic acid of SEQ ID NO: 1, and SEQ ID NO: 4 is a blocking nucleic acid for the probe nucleic acid of SEQ ID NO: 2. That is, in X-60T and Y-60T, the 1st to 30th bases correspond to the base sequence A1 ′ when the nucleic acid A of FIG. 1 is a probe nucleic acid and the nucleic acid B is a blocking nucleic acid, and the 31st to 60th bases. This base corresponds to the base sequence A2 ′. In order to mix the prepared blocking nucleic acid with the probe nucleic acid already immobilized on the nucleic acid array, the solutions shown in Table 4 were prepared and contacted at 60 ° C. for 16 hours in a sealed state with the nucleic acid array.

  The nucleic acid array after contact was prepared with 0.12 M TNT buffer (0.12 M Tris-HCl, 0.12 M NaCl, 0.05% Tween 20 aqueous solution), 0.12 M TN buffer (0.12 M Tris-HCl, 0.12 M NaCl). Each solution was washed with an aqueous solution, and then stored in a light-shielded state at 4 ° C. while immersed in a 0.12 M TN buffer.

Thus, a “blocking nucleic acid mixed nucleic acid array” equipped with a “blocking nucleic acid mixed probe nucleic acid” obtained by mixing a blocking nucleic acid with a predetermined probe nucleic acid was completed.
Model specimen preparation and hybridization A model specimen used for evaluating the specificity of a nucleic acid array mixed with blocking nucleic acids was prepared.

  As the probe nucleic acid detection target (target model sample), oligonucleotides of SEQ ID NO: 5 and SEQ ID NO: 6 described in Table 5, which are completely complementary sequences to two types of probe nucleic acids, were used.

  In addition, as a probe nucleic acid non-detection target (cross-hybrid model sample), the 60-base target model sample represented by SEQ ID NO: 5 and SEQ ID NO: 6 was divided into 20 equal bases and divided into 3 equal parts, as shown in Table 5. (SEQ ID NOs: 7 to 12) were used.

  For the target model sample (SEQ ID NOs: 5 and 6), labeling is performed using ULYSIS-Alexa Fluor 647 (manufactured by Invitrogen), and for the cross-hybrid model sample (SEQ ID NOs: 7 to 12), ULYSIS-Alexa Fluor 546 is used. (Invitrogen) was used for labeling. That is, even when the target model sample and the cross-hybrid model sample are labeled with different labeling substances and the mixture of the target model sample and the cross-hybrid model sample is hybridized to the nucleic acid array, each is independently independent at the same probe nucleic acid position. Detectable.

  For each target model specimen and cross-hybrid model specimen that had been labeled, a hybridization solution was prepared so as to have the composition shown in Table 6. This hybridization solution was hybridized to the blocking nucleic acid mixed nucleic acid array prepared above. Hybridization was performed by contacting the nucleic acid array and the hybridization solution in a sealed state at 60 ° C. for 16 hours.

  The detection operation was performed by immersing the nucleic acid array in 0.12M TN buffer using a cooled CCD camera type nucleic acid array automatic detection device, and covering the cover glass, and then measuring the fluorescence signal intensity of the labeled nucleic acid sample molecule. .

  The data is obtained by averaging the fluorescent signal derived from AlexaFluor 647 and the fluorescent signal derived from AlexaFluor 546 detected at 10 spots mounted on the nucleic acid array for both the signals of probe X and probe Y. Indicated.

  The result is shown in FIG. For probe X, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 20590, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 4, and the target / cross hybrid ratio is 5147. there were.

For probe Y, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 8408, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 4, and the target / cross-hybrid ratio was 2102.
Comparative Example 1
The procedure was the same as in Example 1 except that a nucleic acid array not mixed with blocking nucleic acid was used.

  The result is shown in FIG. 8A. For probe X, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 35818, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 9074, and the target / cross hybrid ratio is 4. there were.

  For probe Y, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 16853, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 3871, and the target / cross hybrid ratio is 4. there were.

The signal intensity of the target model sample was higher than that of Example 1, but the signal intensity derived from the cross-hybrid model sample was higher at a higher rate, and the target / cross-hybrid ratio was extremely higher than that of Example. The value was low. This indicates that in the state where no blocking nucleic acid is used, many cross-hybrid nucleic acids form hybridization with probe nucleic acids, and high specificity cannot be obtained.
Comparative Example 2
As the blocking nucleic acid, one having 60 bases consisting of the complete complementary sequence of the probe nucleic acid represented by SEQ ID NO: 5 and SEQ ID NO: 6 shown in Table 5 was used. Other than that was carried out in the same procedure as in Example 1.

  The result is shown in FIG. 8B. For probe X, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 3585, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 4, and the target / cross hybrid ratio is 896. there were.

For probe Y, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 1488, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 4, and the target / cross hybrid ratio was 372.

In this comparative example, since the length of the blocking nucleic acid used is the same as that of the probe nucleic acid, the efficiency of hybridization is not different from that of the target model sample. Therefore, the blocking nucleic acid also competitively inhibits the formation of hybridization between the target model sample and the probe nucleic acid, and the signal intensity derived from the target model sample becomes extremely low. Although the signal intensity derived from the cross-hybrid model specimen was hardly detected, the target / cross-hybrid ratio was lower than that of Example 1 because the signal intensity of the target model specimen was significantly reduced.
Comparative Example 3
As the blocking nucleic acid, one having a length of 50 bases (SEQ ID NOs: 13 and 14) shown in Table 7 was used. These sequences are composed of sequences from the 6th base to the 55th base of SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The same procedure as in Example 1 was performed except for the blocking nucleic acid.

  The result is shown in FIG. 8C. For probe X, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 10932, the signal intensity of the fluorescence derived from the cross-hybrid model sample (Alexa Fluor 546) is 4, and the target / cross hybrid ratio is 2733 there were.

For probe Y, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 3474, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 4, and the target / cross hybrid ratio was 869.

  In this comparative example, since the length of the blocking nucleic acid used was 10 bases shorter than the probe nucleic acid (the lengths of the blocking nucleic acid and the probe nucleic acid are not equal), the target model sample and Hybridization with a probe nucleic acid is easy. However, compared with the blocking nucleic acid used in Example 1, the signal intensity derived from the target model sample was low, and thus the target / cross-hybrid ratio was also low.

Comparative Example 4
As the blocking nucleic acid, those having a length of 40 bases (SEQ ID NOs: 15 and 16) shown in Table 8 were used. These sequences are composed of sequences from the 11th base to the 50th base of each of SEQ ID NO: 5 and SEQ ID NO: 6. The same procedure as in Example 1 was performed except for the blocking nucleic acid.

  The result is shown in FIG. 8D. For probe X, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 25202, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 263, and the target / cross hybrid ratio is 96. there were.

  For probe Y, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 12416, the signal intensity of the fluorescence derived from the cross-hybrid model sample (Alexa Fluor 546) is 26, and the target / cross hybrid ratio is 471. there were.

  The chain length of the blocking nucleic acid used is 40 bases. However, under this condition, a signal derived from a cross-hybrid sample is detected, so that the value of the target / cross-hybrid ratio decreases.

Comparative Example 5
As the blocking nucleic acid, those having a length of 30 bases (SEQ ID NOs: 17 and 18) described in Table 9 were used. These sequences are composed of sequences from the 16th base to the 45th base of each of SEQ ID NO: 5 and SEQ ID NO: 6. The same procedure as in Example 1 was performed except for the blocking nucleic acid.

  The result is shown in FIG. 9A. For probe X, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 35037, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 3459, and the target / cross hybrid ratio is 10. there were.

  For probe Y, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 16641, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 946, and the target / cross hybrid ratio is 18. there were.

  As in the case of Comparative Example 4, since the signal derived from the cross-hybrid sample is detected more in comparison with the signal intensity derived from the target model sample, the target / cross-hybrid ratio value decreases.

Comparative Example 6
As the blocking nucleic acid, those having a length of 30 bases (SEQ ID NOs: 19, 20, 21, and 22) shown in Table 10 were used. These sequences are composed of sequences from the first base to the 30th base and from the 31st base to the 60th base of each of SEQ ID NO: 5 and SEQ ID NO: 6. Except for the blocking nucleic acid used, the procedure was the same as in Example 1.

  The result is shown in FIG. 9B. For probe X, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 25712, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 179, and the target / cross hybrid ratio is 144. there were.

  For probe Y, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 11439, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 44, and the target / cross hybrid ratio is 260. there were.

  The blocking nucleic acid used here uses two 30-base long blocking nucleic acids for one 60-base long probe nucleic acid to block the entire sequence of the probe nucleic acid. Although it is the same as Example 1 and Comparative Example 2 in that the entire sequence of the probe nucleic acid can be blocked, the target / cross-hybrid ratio was lower than that of Example 1.

Summary of Example 1 and Comparative Examples 2 to 6 In FIG. 10, Examples and Comparative Examples are plotted on the same graph. In FIG. 10, panel A is a summary of the results for probe X, and panel B is the summary for probe Y. As a result, the target / cross-hybrid ratio in the example was very high compared to any other comparative examples. From the above, it was proved that the blocking nucleic acid in this example improves the specificity of the probe nucleic acid as compared with the blocking nucleic acid used in the comparative example.

Example 2
Preparation of Nucleic Acid Array In order to prepare a nucleic acid array, oligonucleotides (30 base length) represented by SEQ ID NO: 23 and 24 shown in Table 11 were synthesized.

  In the same manner as in Example 1, a 5 'terminal vinylated oligonucleotide was prepared and used as a probe nucleic acid.

  The 1st to 15th and 16th to 30th bases of probe A correspond to base sequence A1 and base sequence A2 in FIG. 1, respectively. The same applies to the probe B.

Other than that, Example 1
In the same manner as described above, a nucleic acid array was prepared.

Preparation of Blocking Nucleic Acid Mixed Nucleic Acid Array Next, an oligonucleotide that is a blocking nucleic acid was designed and mixed with the nucleic acid array prepared above to prepare a blocking nucleic acid mixed nucleic acid array.

  First, oligonucleotides represented by SEQ ID NO: 27 and SEQ ID NO: 28 shown in Table 11 were synthesized and used as blocking nucleic acids.

  The oligonucleotide represented by SEQ ID NO: 27 is composed of a structure in which 15 bases on the 5 terminal side and 15 bases on the 3 terminal side are replaced in the completely complementary strand (30 bases) of SEQ ID NO: 23. In addition, the oligonucleotide represented by SEQ ID NO: 28 has a structure in which 15 bases on the 5 terminal side and 15 bases on the 3 terminal side are replaced in the completely complementary strand (30 bases) of SEQ ID NO: 24.

  SEQ ID NO: 27 is a blocking nucleic acid for the probe nucleic acid of SEQ ID NO: 23, and SEQ ID NO: 28 is a blocking nucleic acid for the probe nucleic acid of SEQ ID NO: 24. That is, in A-30 and B-30, the 1st to 15th bases correspond to the base sequence A1 ′ when the nucleic acid A of FIG. 1 is a probe nucleic acid and the nucleic acid B is a blocking nucleic acid, and the 16th to 30th bases. This base corresponds to the base sequence A2 ′. In order to mix the prepared blocking nucleic acid with the probe nucleic acid already immobilized on the nucleic acid array, the solutions shown in Table 12 were prepared and contacted at 60 ° C. for 16 hours in a sealed state with the nucleic acid array.

The nucleic acid array after the contact was prepared using 0.24 M TNT buffer (0.24 M Tris-HCl, 0.24 M NaCl, 0.05% Tween 20 aqueous solution), 0.24 M TN buffer (0.24 M Tris-HCl, 0.24 M NaCl). Each solution was washed with an aqueous solution and then stored in a light-shielded state at 4 ° C. in a state immersed in a 0.24 M TN buffer.
Thus, a “blocking nucleic acid mixed nucleic acid array” on which a “blocking nucleic acid mixed probe nucleic acid” obtained by mixing a blocking nucleic acid with a predetermined probe nucleic acid was completed.

Model specimen preparation and hybridization A model specimen used for evaluating the specificity of a nucleic acid array mixed with blocking nucleic acids was prepared.

  As the probe nucleic acid detection target (target model sample), oligonucleotides of SEQ ID NO: 25 and SEQ ID NO: 26 shown in Table 11, which are completely complementary sequences to two types of probe nucleic acids, were used.

In addition, as a probe nucleic acid non-detection target (cross-hybrid model sample), oligonucleotides having 15 bases represented by SEQ ID NO: 29 and SEQ ID NO: 30 were used.

For the target model samples (SEQ ID NOs: 25 and 26), labeling is performed using ULYSIS-Alexa Fluor 647 (manufactured by Invitrogen), and for the cross-hybrid model samples (SEQ ID NOs: 29 and 30), ULYSIS-Alexa Fluor 546 is used. (Invitrogen) was used for labeling. That is, even when the target model sample and the cross-hybrid model sample are labeled with different labeling substances and the mixture of the target model sample and the cross-hybrid model sample is hybridized to the nucleic acid array, each is independently independent at the same probe nucleic acid position. Detectable.

  For each target model specimen and cross-hybrid model specimen that had been labeled, a hybridization solution was prepared so as to have the composition shown in Table 13. This hybridization solution was hybridized to the blocking nucleic acid mixed nucleic acid array prepared above. Hybridization was performed by contacting the nucleic acid array and the hybridization solution in a sealed state at 60 ° C. for 16 hours. Washing was performed by the same method as that after contact with the blocking nucleic acid.

  The detection operation was performed by immersing the nucleic acid array in 0.24M TN buffer using a cooled CCD camera type nucleic acid array automatic detection apparatus, and covering the cover glass, and then measuring the fluorescence signal intensity of the labeled nucleic acid sample molecule. .

  The data showed the value of the fluorescent signal derived from AlexaFluor 647 and the fluorescent signal derived from AlexaFluor 546, which was detected at the spot mounted on the nucleic acid array, together with the signals of probe A and probe B.

  The result is shown in FIG. For probe A, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 166516, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 2964, and the target / cross hybrid ratio is 56. there were.

For probe B, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 89108, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 2054, and the target / cross hybrid ratio was 43.

Comparative Example 7
The procedure was the same as in Example 2 except that a nucleic acid array not mixed with blocking nucleic acid was used.

  The result is shown in FIG. 12A. For probe A, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 159204, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 10850, and the target / cross hybrid ratio is 15. there were.

For probe B, fluorescence from the target model specimen (Alexa Fluor
647) had a signal intensity of 85560, the signal intensity of fluorescence derived from the cross-hybrid model sample (Alexa Fluor 546) was 8484, and the target / cross-hybrid ratio was 10.

The signal intensity of the target model sample was about the same as that of Example 2, but the signal intensity derived from the cross-hybrid model sample was high at a higher rate, and the target / cross-hybrid ratio was much higher than that of Example. The value was low. This indicates that in the state where no blocking nucleic acid is used, many cross-hybrid nucleic acids form hybridization with probe nucleic acids, and high specificity cannot be obtained.

Comparative Example 8
As the blocking nucleic acid, a 15-base nucleic acid consisting of the complete complementary sequence of the probe nucleic acid shown in SEQ ID NO: 29 and SEQ ID NO: 30 shown in Table 11 was used. Other than that was carried out in the same procedure as in Example 2.

  The result is shown in FIG. 12B. For probe A, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 166700, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 3476, and the target / cross hybrid ratio is 48. there were.

For probe B, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 94244, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 2900, and the target / cross hybrid ratio was 32.

The chain length of the blocking nucleic acid used was 15 bases. Under these conditions, a signal derived from the cross-hybrid specimen was detected higher than that in Example 2.

Summary of Example 2, Comparative Example 7, and Comparative Example 8 The results of Example 2, Comparative Example 7, and Comparative Example 8 are summarized in FIG. In FIG. 13, panel A is a summary of the results for probe A, and panel B is the summary for probe B. Even in the case of using probes A and B having a length of 30 bases, it was revealed that the method described in the examples can obtain highly specific data as compared with the method described in the comparative example.

Example 3
Preparation of Nucleic Acid Array In order to prepare a nucleic acid array, oligonucleotides (20 base length) represented by SEQ ID NOs: 31 and 32 shown in Table 14 were synthesized.

  In the same manner as in Example 1, a 5 'terminal vinylated oligonucleotide was prepared and used as a probe nucleic acid.

  The 1st to 10th and 11th to 20th bases of the probe C correspond to the base sequence A1 and the base sequence A2 in FIG. 1, respectively. The same applies to the probe D.

Other than that was carried out similarly to Example 1, and produced the nucleic acid array.

Preparation of Blocking Nucleic Acid Mixed Nucleic Acid Array Next, an oligonucleotide that is a blocking nucleic acid was designed and mixed with the nucleic acid array prepared above to prepare a blocking nucleic acid mixed nucleic acid array.

  First, oligonucleotides represented by SEQ ID NO: 33 and SEQ ID NO: 34 shown in Table 14 were synthesized and used as blocking nucleic acids.

The oligonucleotide represented by SEQ ID NO: 33 has a structure in which 10 bases on the 5 terminal side and 10 bases on the 3 terminal side are replaced in the completely complementary strand (20 bases) of SEQ ID NO: 31. In addition, the oligonucleotide represented by SEQ ID NO: 34 has a structure in which 10 bases on the 5 terminal side and 10 bases on the 3 terminal side are replaced in the completely complementary strand (20 bases) of SEQ ID NO: 32.

SEQ ID NO: 33 is a blocking nucleic acid for the probe nucleic acid of SEQ ID NO: 31, and SEQ ID NO: 34 is a blocking nucleic acid for the probe nucleic acid of SEQ ID NO: 32. That is, in C-20T and D-20T, the 1st to 10th bases correspond to the base sequence A1 ′ when the nucleic acid A of FIG. 1 is a probe nucleic acid and the nucleic acid B is a blocking nucleic acid, and the 11th to 20th bases. This base corresponds to the base sequence A2 ′. In order to mix the prepared blocking nucleic acid with the probe nucleic acid already immobilized on the nucleic acid array, the solutions described in Table 12 were prepared and contacted at 50 ° C. for 16 hours in a sealed state with the nucleic acid array.

The nucleic acid array after the contact was prepared using 0.24 M TNT buffer (0.24 M Tris-HCl, 0.24 M NaCl, 0.05% Tween 20 aqueous solution), 0.24 M TN buffer (0.24 M Tris-HCl, 0.24 M NaCl). Each solution was washed with an aqueous solution and then stored in a light-shielded state at 4 ° C. in a state immersed in a 0.24 M TN buffer.

Thus, a “blocking nucleic acid mixed nucleic acid array” on which a “blocking nucleic acid mixed probe nucleic acid” obtained by mixing a blocking nucleic acid with a predetermined probe nucleic acid was completed.

Model specimen preparation and hybridization A model specimen used for evaluating the specificity of a nucleic acid array mixed with blocking nucleic acids was prepared.

  As the probe nucleic acid detection target (target model specimen), oligonucleotides of SEQ ID NO: 35 and SEQ ID NO: 36 described in Table 14, which are completely complementary sequences to two types of probe nucleic acids, were used.

In addition, as a probe nucleic acid non-detection target (cross-hybrid model sample), a 10-base oligonucleotide represented by SEQ ID NO: 37 and SEQ ID NO: 38 was used.

The target model sample (SEQ ID NOs: 35 and 36) is labeled using ULYSIS-Alexa Fluor 647 (manufactured by Invitrogen), and the cross-hybrid model sample (SEQ ID NOs: 37 and 38) is labeled with ULYSIS-Alexa Fluor 546. (Invitrogen) was used for labeling. That is, even when the target model sample and the cross-hybrid model sample are labeled with different labeling substances and the mixture of the target model sample and the cross-hybrid model sample is hybridized to the nucleic acid array, each is independently independent at the same probe nucleic acid position. Detectable.

For each target model specimen and cross-hybrid model specimen that had been labeled, a hybridization solution was prepared so as to have the composition shown in Table 13. This hybridization solution was hybridized to the blocking nucleic acid mixed nucleic acid array prepared above. Hybridization was carried out by contacting the nucleic acid array and the hybridization solution in a sealed state at 50 ° C. for 16 hours. Washing was performed by the same method as that after contact with the blocking nucleic acid.

The detection operation was performed by immersing the nucleic acid array in 0.24M TN buffer using a cooled CCD camera type nucleic acid array automatic detection apparatus, and covering the cover glass, and then measuring the fluorescence signal intensity of the labeled nucleic acid sample molecule. .

  The data showed the value of the fluorescent signal derived from Alexa Fluor 647 and the fluorescent signal derived from Alexa Fluor 546, which was detected at the spot mounted on the nucleic acid array, together with the signals of probe C and probe D.

  The result is shown in FIG. For probe C, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 23456, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 558, and the target / cross hybrid ratio is 42. there were.

For probe D, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 1728, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 156 and the target / cross hybrid ratio was 11.

Comparative Example 9
The procedure was the same as in Example 2 except that a nucleic acid array not mixed with blocking nucleic acid was used.

  The result is shown in FIG. 15A. For probe C, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 21712, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 6751, and the target / cross hybrid ratio is 3. there were.

  For probe D, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 1432, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 1045, and the target / cross hybrid ratio is 1. there were.

The signal intensity of the target model sample was about the same as that of Example 3, but the signal intensity derived from the cross-hybrid model sample was higher than that of Example 3, so that the target / cross-hybrid ratio was as in Example 3. The value was extremely low compared.

Comparative Example 10
As the blocking nucleic acid, a 10-base nucleic acid consisting of a complementary sequence of the probe nucleic acid shown in SEQ ID NO: 37 and SEQ ID NO: 38 shown in Table 14 was used. Other than that was carried out in the same procedure as in Example 2.

  The result is shown in FIG. 15B. For probe C, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 19480, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 521, and the target / cross hybrid ratio is 37. there were.

For probe D, the signal intensity of the target model specimen-derived fluorescence (Alexa Fluor 647) is 1380, and the cross-hybrid model specimen-derived fluorescence (Alexa
The signal intensity of Fluor 546) was 213 and the target / cross hybrid ratio was 6.

The blocking nucleic acid used has a chain length of 10 bases, but under these conditions, a signal derived from a cross-hybrid specimen is detected, so that the value of the target / cross-hybrid ratio decreases.

Comparative Example 11
As the blocking nucleic acid, a 20-base nucleic acid consisting of a completely complementary sequence of the probe nucleic acid represented by SEQ ID NO: 35 and SEQ ID NO: 36 shown in Table 14 was used. Other than that was carried out in the same procedure as in Example 2.

  The result is shown in FIG. 15C. For probe C, the signal intensity of the fluorescence derived from the target model specimen (Alexa Fluor 647) is 3656, the signal intensity of the fluorescence derived from the cross-hybrid model specimen (Alexa Fluor 546) is 386, and the target / cross hybrid ratio is 9. there were.

For probe D, the signal intensity of the fluorescence derived from the target model sample (Alexa Fluor 647) is 752, and the fluorescence derived from the cross-hybrid model sample (Alexa
The signal intensity of Fluor 546) was 122 and the target / cross hybrid ratio was 6.

The chain length of the blocking nucleic acid used is 20 bases. However, under this condition, the chain length of the target model sample is the same as that of the target model sample, so that the efficiency of hybridization of the target model sample is significantly reduced and the signal intensity is greatly reduced. To do. As a result, the value of the target / cross hybrid ratio decreases.

Summary of Example 3, Comparative Example 9, Comparative Example 10, and Comparative Example 11 FIG. 16 summarizes the results of Example 3, Comparative Example 9, Comparative Example 10, and Comparative Example 11. In FIG. 16, panel A is a summary of the results for probe C, and panel B is the summary for probe D. Even in the case of using probes C and D having a length of 20 bases, it was revealed that the method described in the examples can obtain highly specific data as compared with the method described in the comparative example.

  The present invention provides a nucleic acid composition and a nucleic acid array that can be used not only for polymorphism analysis but also for expression analysis, preventing reduction in hybridization efficiency.

11... Hole 21... Perforated plate 31... Hollow fiber 41.

  SEQ ID NOs: 1-38: Synthetic DNA

Claims (4)

A nucleic acid composition in which nucleic acid A formed by linking base sequences A1 and A2 and nucleic acid B formed by linking base sequences A1 ′ and A2 ′ that are complementary to base sequences A1 and A2, respectively, are mixed. A nucleic acid in which the 5 ′ end of the base sequence A2 is linked to the 3 ′ end of the base sequence A1, and the 3 ′ end of the base sequence A1 ′ is linked to the 5 ′ end of the base sequence A2 ′. Composition. A nucleic acid array in which the nucleic acid composition according to claim 1 is arranged on a carrier in an addressable manner, wherein either one of nucleic acids A and B is immobilized on the carrier. A nucleic acid A formed by linking base sequences A1 and A2 and a nucleic acid B formed by linking base sequences A1 ′ and A2 ′ that are complementary to the base sequences A1 and A2, respectively. A method for producing a nucleic acid array, comprising the step of immobilizing the nucleotide sequence on the other nucleic acid and then mixing the other nucleic acid, wherein the 5 ′ end of the base sequence A2 is linked to the 3 ′ end of the base sequence A1, A method for producing a nucleic acid array, wherein the 3 ′ end of the base sequence A1 ′ is linked to the 5 ′ end of the base sequence A2 ′. A method for detecting a target nucleic acid, comprising the step of bringing the nucleic acid composition according to claim 1 or the nucleic acid array according to claim 2 into contact with a target nucleic acid, and detecting a signal of the target nucleic acid.