JP2010200701A - Nucleic acid hybridization method, method for determination of single nucleotide polymorphism, and reaction vessel and determination apparatus for determination of single nucleotide polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hybridization method for a nucleic acid having a high specificity, and to provide a method for determination of single nucleotide polymorphism and a reaction vessel and a determination apparatus for determination of single nucleotide polymorphism which use the hybridization method and can be performed simply in medical practices or the like with high precision. <P>SOLUTION: The method hybridizes specifically a target nucleic acid and a probe nucleic acid by contacting a sample containing the target nucleic acid having a target nucleotide sequence (t), which is to be an object of hybridization, with the probe nucleic acid having a probe nucleotide sequence (p) complementary to the target nucleotide sequence (t). The target nucleic acid is brought into contact with a part or whole (a and/or b) of the nucleotide sequence in the target nucleic acid other than the target nucleotide sequence (t), and a block nucleic acid having hybridizable nucleotide sequence (a' and/or b'). Then, in the condition that the block nucleic acid is hybridized with the target nucleic acid, the target nucleic acid and the probe nucleic acid are hybridized specifically. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a nucleic acid hybridization method, a single nucleotide polymorphism determination method, a single nucleotide polymorphism determination reactor and a determination apparatus, and more particularly, a nucleic acid hybridization method having high specificity, and the use thereof The present invention relates to a single nucleotide polymorphism determination method, a single nucleotide polymorphism determination reactor, and a determination apparatus that can be easily implemented in a medical field or the like and have high determination accuracy.

There are individual differences in the whole base sequence of the genome. Among them, an individual difference of one base level (more precisely, one that appears at a frequency of 1% or more of the population) is a single nucleotide polymorphism (SNP). Call. In human DNA, SNPs are thought to exist at a frequency of about 1000 bases. The majority of SNPs are present in untranslated regions and do not cause changes in genetic characteristics, but SNPs in genes and control regions may cause genetic individual differences. Along with the improvement of gene therapy and diagnostic technology, various diseases such as drug susceptibility (resistance and presence of side effects, etc.), resistance to infectious diseases, environmental factors of lifestyle-related diseases, cancer, Alzheimer's disease, pet genetic diseases, etc. Many cases that can be explained by single nucleotide polymorphisms of genes have been discovered. From this, it is expected that the possibility of tailor-made medical care and predictive medical care for each individual will be expanded by the analysis of SNP.

In order to realize tailor-made medical care and predictive medical care, it is urgent to establish a reliable single nucleotide polymorphism determination (typing) method and system that can be implemented quickly, easily, and inexpensively in the medical field. The gene analysis market is steadily expanding, and many gene sequence analysis techniques have been developed. These techniques are broadly classified into those using hybridization of poly (oligo) nucleotides and those using the recognition efficiency of specific sequences by enzymes that perform nucleic acid elongation, ligation, cleavage, etc. (for example, non-patent literature) 1). In recent years, along with active research on SNP typing methods, many patent applications have been filed.

Patent Document 1 includes a sample having at least a sequence near the SNP of a specific gene and having four types of detection probes having different SNP base types, and at least a sequence near the SNP of the gene. A non-complementary base pair site of the double-stranded nucleic acid formed by the hybridization step using a hybridization step that hybridizes with the target nucleic acid, an enzyme that specifically cleaves the single-stranded nucleic acid site, etc. The mismatch site cleavage step to cleave, the nucleic acid denaturation step in which only the nucleic acid cleaved in the mismatch site cleavage step is denatured into single strands, and the base that detects the detection probe that retains the double strand state after the nucleic acid denaturation step An SNP discrimination method that includes a seed detection step and can be applied to a DNA chip or the like is disclosed.

In Patent Document 2, when a target nucleic acid in a specimen is hybridized with a plurality of types of probes on a solid phase carrier, the specimen is divided into a plurality of parts, thereby giving each probe type a plurality of stringencies and increasing the number of probes. In addition to hybridization, a probe having a base sequence that includes a mismatch with a probe consisting of a base sequence that is completely complementary to the target nucleic acid can be designed, and even a single base difference can be detected by comparing the hybridization results. A method for analyzing the base sequence of a nucleic acid that can be produced is disclosed.

In Patent Document 3, for each target nucleic acid, a probe group consisting of complete and / or incomplete match probes for at least a part of the target nucleic acid is prepared, and a sample containing a plurality of types of target nucleic acids and each probe group are appropriately combined. The hybrid signal intensity is measured corresponding to each of the perfect and / or incomplete match probes, and the first signal data corresponding to the perfect match probe and / or the second signal data corresponding to the incomplete match probe. From the first and / or second signal data of each probe group, data at the optimal reaction conditions associated with the probe group is extracted as first and / or second test data, Filter the second test data, determine the target nucleic acid sequence in the sample nucleic acid, and integrate the determination results for all target nucleic acids A column analysis method is disclosed.

JP 2007-175017 A JP 2007-174986 A JP 2006-6220 A

Mitsuo Sekine, “Science and Application of New DNA Chips”, Kodansha Scientific, July 30, 2007, ISBN 978-4-06-153864-1, p. 127-139

However, in the SNP typing method using nucleotide hybridization, it is necessary to strictly set the generation of background noise due to hybridization between mismatched polynucleotides (mishybridization) and hybridization conditions. Therefore, there is a problem that a great deal of labor is required for setting the conditions. In addition, SNP typing methods that utilize the recognition efficiency of specific sequences by enzymes often require special enzymes, and the conditions for the enzymes to work effectively are often special. There are problems such as requiring labor.

In the SNP discrimination method described in Patent Document 1, since it is necessary to hybridize even if there is a mismatch, it is necessary to prepare a long probe nucleic acid, and an expensive restriction enzyme is required. Since time is required, there is a problem that quick determination is difficult.

The nucleic acid base sequence analysis method described in Patent Document 2 requires a large amount of sample in order to perform reliable determination. In addition, since it is necessary to determine the binding to several probes under different conditions and to comprehensively determine it, the determination protocol after measurement becomes complicated. Furthermore, since it is necessary to measure the binding under different conditions, there is a problem that a great amount of measurement time is required.

In the nucleic acid sequence analysis method described in Patent Document 3, it is necessary to determine the hybridization for a plurality of probes under different conditions and to comprehensively determine the obtained results, so that a large amount of samples are required. In addition, the determination protocol after measurement becomes complicated. Further, since it is necessary to measure hybridization under different conditions, there are problems that a long measurement time is required and an expensive measurement device is required.

The present invention has been made in view of such circumstances, a nucleic acid hybridization method having high specificity, and a single nucleotide polymorphism determination method with high determination accuracy that can be easily performed in a medical field using the method, An object of the present invention is to provide a single nucleotide polymorphism determination reactor and determination apparatus.

In the first aspect of the present invention that meets the above-mentioned object, a sample containing a target nucleic acid having a target base sequence to be hybridized is brought into contact with a probe nucleic acid having a probe base sequence complementary to the target base sequence, In the method of specifically hybridizing the target nucleic acid and the probe nucleic acid in the sample, it hybridizes with a part or all of the base sequence in the target nucleic acid located at the 5 ′ end side of the target base sequence 5 'terminal block nucleic acid having a possible base sequence on the 5' end side and / or hybridizing with part or all of the base sequence in the target nucleic acid located on the 3 'end side of the target base sequence A 3 ′ terminal block nucleic acid having a base sequence on the 3 ′ terminal side is brought into contact with the target nucleic acid, and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid is contacted. Provided is a nucleic acid hybridization method, wherein the target nucleic acid and the probe nucleic acid are specifically hybridized in a state where the nucleic acid is hybridized with the hybridizable base sequence in the target nucleic acid. This solves the above problem.

In the present invention, “A and / or B” has the same meaning as one or both of A and B. For example, the expression “in the presence of A and / or B” includes (1) A All in the presence of (2) only B, and (3) both A and B are included.

By hybridizing the target nucleic acid and the probe nucleic acid in a state in which the probe nucleic acid is hybridized with the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid, the probe nucleic acid is inserted between the target base sequence and the probe base sequence. Hybridization of probe nucleic acid and target nucleic acid by reducing the efficiency of non-specific hybridization in the presence of mismatch and improving the hybridization efficiency of probe nucleic acid and target nucleic acid having complementary base sequences to each other The base sequence specificity in can be improved. Therefore, it is suitable for SNP typing that needs to detect the difference of only one base in the base sequence with high accuracy, and complicated procedures such as processing a plurality of experimental data obtained under a plurality of measurement conditions. Execution and the labor required for it can be reduced.

In addition, the efficiency of detection and separation of a nucleic acid having a specific base sequence can be improved as well as SNP typing using hybridization. In addition, in the base sequence portion other than the target base sequence, a portion where the target nucleic acid hybridizes with the 5 'terminal block nucleic acid and / or the 3' terminal block nucleic acid forms a double strand. Thereby, since the formation of the higher order structure of the target nucleic acid can be suppressed, the efficiency of hybridization with the probe nucleic acid can be improved.

In the method for hybridizing nucleic acids according to the first aspect of the present invention, the base sequence capable of hybridizing with the 3 ′ end side end of the target base sequence present in the target nucleic acid and the 3 ′ end side block nucleic acid Between the 5 ′ end side and / or the 5 ′ end side end of the base sequence complementary to the target base sequence and the 3 ′ end of the base sequence capable of hybridizing with the base sequence of the 5 ′ end side block nucleic acid There is preferably a gap of 1 to 15 bases between the sides.
By setting the gap length (number of bases) within the above range, the specificity of hybridization between the probe nucleic acid and the target nucleic acid can be improved without inhibiting the hybridization between the target nucleic acid and the probe nucleic acid.

In the nucleic acid hybridization method according to the first aspect of the present invention, the probe nucleic acid is preferably DNA.
By using a DNA that is more stable than RNA or the like as a probe nucleic acid, the durability of the probe nucleic acid can be increased and the reproducibility of hybridization can be improved.

In the nucleic acid hybridization method according to the first aspect of the present invention, the probe base sequence preferably has 10 to 20 bases.
By setting the number of bases in the probe base sequence within the above range, the base sequence specificity can be increased without increasing the background noise caused by the hybridization with the mismatched nucleic acid in the hybridization between the probe nucleic acid and the target nucleic acid. Can be improved.

According to a second aspect of the present invention, a target nucleic acid having a target base sequence to be determined is a probe base complementary to each of a part or all of single nucleotide polymorphisms (SNPs) present in the target base sequence. The target nucleic acid and the probe nucleic acid are brought into contact with a plurality of probe nucleic acids arranged in predetermined different regions on the inner wall surface of a single reactor. A step A of specifically hybridizing the target base sequence having the probe base sequence complementary to the target base sequence;
A step B for detecting the specific hybridization of the target nucleic acid and the probe nucleic acid having the probe base sequence complementary to the target base sequence in a position-specific manner. The above-described problems are solved by providing a polymorphism determination method.
In the present invention, the “reactor” means that a probe nucleic acid is arranged at a predetermined position on an inner wall surface (in contact with the solution) of a container filled with a solution containing a target nucleic acid or a flow channel to be circulated, A probe nucleic acid is configured to contact and hybridize.

Since all the probe nucleic acids are arranged in any one of the single container and the channel, the probe nucleic acids corresponding to all the single nucleotide polymorphisms can be hybridized under the same conditions. Therefore, it is possible to suppress the occurrence of background noise due to the difference in hybridization conditions, detect a difference of only one base with high accuracy, and reduce the labor for setting the conditions. Accordingly, it is possible to quickly and easily perform highly accurate SNP typing at a medical site or the like without requiring a great deal of labor and skill for setting complicated conditions.

In the method for determining a single nucleotide polymorphism according to the second aspect of the present invention, in the step A, it hybridizes with a part or all of the base sequence in the target nucleic acid located at the 5 ′ end side of the target base sequence. Can hybridize with a part or all of the base sequence in the target nucleic acid located at the 3 ′ end side of the 5 ′ end block nucleic acid and / or the target base sequence having a base sequence capable of soybean at the 5 ′ end side. A 3 ′ terminal block nucleic acid having a simple base sequence on the 3 ′ terminal side is brought into contact with the target nucleic acid, and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid is hybridized in the target nucleic acid. It is preferable to specifically hybridize the target nucleic acid and the probe nucleic acid in a state of being hybridized with a possible base sequence.

By hybridizing the target nucleic acid and the probe nucleic acid in a state in which the probe nucleic acid is hybridized with the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid, the probe nucleic acid is inserted between the target base sequence and the probe base sequence. Hybridization of probe nucleic acid and target nucleic acid by reducing the efficiency of non-specific hybridization in the presence of mismatch and improving the hybridization efficiency of probe nucleic acid and target nucleic acid having complementary base sequences to each other The base sequence specificity in can be improved. Therefore, it is possible to reduce the execution of complicated procedures such as processing a plurality of experimental data obtained under a plurality of measurement conditions and the labor required for the procedures. In addition, in the base sequence portion other than the target base sequence, a portion where the target nucleic acid hybridizes with the 5 'terminal block nucleic acid and / or the 3' terminal block nucleic acid forms a double strand. Thereby, since the formation of the higher order structure of the target nucleic acid can be suppressed, the efficiency of hybridization with the probe nucleic acid can be improved.

In the method for determining a single nucleotide polymorphism according to the second aspect of the present invention, a base capable of hybridizing with the 3 ′ end side end of the target base sequence present in the target nucleic acid and the 3 ′ end side block nucleic acid 1 to 15 bases between the 5 ′ end of the sequence and / or the 5 ′ end of the target base sequence and the 3 ′ end of the base sequence capable of hybridizing with the 5 ′ end block nucleic acid It is preferred that a minute gap exists.
By setting the gap length (number of bases) within the above range, the specificity of hybridization between the probe nucleic acid and the target nucleic acid can be improved without inhibiting the hybridization between the target nucleic acid and the probe nucleic acid.

In the single nucleotide polymorphism determination method according to the second aspect of the present invention, the probe nucleic acid is preferably DNA.
By using a DNA that is more stable than RNA or the like as a probe nucleic acid, the durability of the probe nucleic acid can be increased and the reproducibility of hybridization can be improved.

In the method for determining a single nucleotide polymorphism according to the second aspect of the present invention, the number of bases in the probe base sequence is preferably 10 or more and 20 or less.
By setting the number of bases in the probe base sequence within the above range, the base sequence specificity can be increased without increasing the background noise caused by the hybridization with the mismatched nucleic acid in the hybridization between the probe nucleic acid and the target nucleic acid. Can be improved.

In the method for determining a single nucleotide polymorphism according to the second aspect of the present invention, the positions of the bases corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence are both the probe base. It is preferably larger than 1/3 times the number of bases in the sequence.
When the base corresponding to the single nucleotide polymorphism is located at the center in the case where the probe base sequence is divided into three regions (specific examples will be described later) so that the number of bases in each region is (approximately) equal, Since the efficiency of hybridization with a nucleic acid in which a mismatch exists is lowered, the base sequence specificity in the hybridization between the probe nucleic acid and the target nucleic acid can be improved. The same applies to the position of the base corresponding to the single nucleotide polymorphism in the target base sequence complementary to the probe base sequence.

In the method for determining a single nucleotide polymorphism according to the second aspect of the present invention, specific hybridization between the target nucleic acid and the probe nucleic acid having a base sequence complementary to the target base sequence is performed on the surface. Detection may be performed by either plasmon resonance or abnormal reflection of gold.
According to these methods, since hybridization between the target nucleic acid and the probe nucleic acid can be detected in real time, the time required for SNP typing can be shortened and the throughput can be improved. In addition, since it is possible to detect position-specific hybridization with an inexpensive apparatus compared to a plate reader or the like, single nucleotide polymorphism typing can be easily performed in a medical field or the like.

The third aspect of the present invention has either or both of a container filled with a solution having a target base sequence to be determined and a flow path for circulating the solution, and is present in the target base sequence. Among a plurality of probe nucleic acids having probe base sequences complementary to part or all of single nucleotide polymorphisms (SNPs), those having different probe base sequences are either single containers or flow paths. The above-mentioned problems are solved by providing a single nucleotide polymorphism determination reactor characterized in that it is disposed at predetermined positions different from each other on one or both inner wall surfaces.
Since all the probe nucleic acids are arranged in any one of the single container and the channel, the probe nucleic acids corresponding to all the single nucleotide polymorphisms can be hybridized under the same conditions. Therefore, the generation of background noise due to the difference in hybridization conditions can be suppressed, and the labor for setting conditions can be reduced. Therefore, when the single nucleotide polymorphism determination reactor according to this embodiment is used, high-precision SNP typing can be performed quickly and easily in a medical field without requiring a great deal of labor and skill for setting complicated conditions. Can be done.

In the single nucleotide polymorphism determination reactor according to the third aspect of the present invention, the probe nucleic acid is preferably DNA.
By using a DNA that is more stable than RNA or the like as a probe nucleic acid, the durability of the probe nucleic acid can be increased and the reproducibility of hybridization can be improved.

In the single nucleotide polymorphism determination reactor according to the third aspect of the present invention, the number of bases in the probe base sequence is preferably 10 or more and 20 or less.
By setting the number of bases in the probe base sequence within the above range, the base sequence specificity can be increased without increasing the background noise caused by the hybridization with the mismatched nucleic acid in the hybridization between the probe nucleic acid and the target nucleic acid. Can be improved.

In the single nucleotide polymorphism determining reactor according to the third aspect of the present invention, the position of the base corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence is the probe. It is preferably larger than 1/3 times the number of bases in the base sequence.
When the base corresponding to the single nucleotide polymorphism is located in the center when the probe base sequence is divided into three regions so that the number of bases in each region is (approximately) equal, hybridization with a nucleic acid containing a mismatch Since the efficiency is lowered, the base sequence specificity in hybridization between the probe nucleic acid and the target nucleic acid can be improved. The same applies to the position of the base corresponding to the single nucleotide polymorphism in the target base sequence complementary to the probe base sequence.

The fourth aspect of the present invention has either or both of a container filled with a solution having a target base sequence to be judged and a flow path for circulating the solution, and is present in the target base sequence. A plurality of probe nucleic acids having a probe base sequence complementary to part or all of a single nucleotide polymorphism (SNP) having different probe base sequences are different from each other on the inner wall surface. A reactor arranged in a position;
A detection means for detecting position-specifically specific hybridization between a target nucleic acid having the target base sequence and a probe nucleic acid having the probe base sequence complementary to the target base sequence. The above-described problems are solved by providing a single nucleotide polymorphism determination device that is characterized.
Since all probe nucleic acids are arranged in a single reactor, probe nucleic acids corresponding to all single nucleotide polymorphisms can be hybridized under the same conditions. Therefore, the generation of background noise due to the difference in hybridization conditions can be suppressed, and the labor for setting conditions can be reduced.

In the single nucleotide polymorphism determining device according to the fourth aspect of the present invention, the single nucleotide polymorphism determining reactor according to the third aspect of the present invention may be used as the reactor.

In the single nucleotide polymorphism determination device according to the fourth aspect of the present invention, the detection means may detect specific hybridization by either surface plasmon resonance or abnormal reflection of gold.
According to these methods, since hybridization between the target nucleic acid and the probe nucleic acid can be detected in real time, the time required for SNP typing can be shortened and the throughput can be improved. In addition, since it is possible to detect position-specific hybridization with an inexpensive device compared to a plate reader or the like, single nucleotide polymorphism typing can be easily performed at a medical site, etc. Cost can be reduced.

According to the present invention, the base sequence specificity in the hybridization between the probe nucleic acid and the target nucleic acid is high, and the SNP typing or the specific base sequence that needs to detect the difference of only one base in the base sequence with high accuracy can be obtained. There is provided a nucleic acid hybridization method which can improve the efficiency and specificity of detection and separation of nucleic acids and can be suitably applied to them.
In addition, according to the present invention, a single nucleotide polymorphism (SNP) that can quickly and easily perform highly accurate SNP typing at a medical site or the like without requiring a great deal of labor and skill for setting complicated conditions. ) Determination method is provided.

In addition, according to the present invention, there are provided a single nucleotide polymorphism determining reactor that can be suitably used in the above-described single nucleotide polymorphism determining method and a single nucleotide polymorphism determining apparatus using the same.

The hybridization state of the target nucleic acid and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid in the nucleic acid hybridization method and single nucleotide polymorphism determination method according to the embodiment of the present invention is shown. It is a schematic diagram. It is a block diagram of the single nucleotide polymorphism judging device concerning a 4th embodiment of the present invention. 4 is a graph showing the relationship between the base length of a target base sequence and hybridization efficiency in Example 1. 2 is a graph showing the relationship between the base length of a target base sequence and the base sequence specificity of hybridization in Example 1. In Example 1, it is a graph which shows the relationship between the position of the mismatch in a target base sequence, and the base sequence specificity of hybridization. 3 is a graph showing the effect of 3 ′ terminal block nucleic acid on base sequence specificity of hybridization in Example 1. FIG. 3 is a view showing the base sequences of the target nucleic acid, probe nucleic acid, linker nucleic acid and 3 ′ end side block nucleic acid used for determination of single nucleotide polymorphism in Example 1. In the determination of the single nucleotide polymorphism in Example 1, it is a graph which shows the pattern of hybridization with the target nucleic acid and probe nucleic acid corresponding to each genotype. It is a figure which shows the algorithm for determining a genotype from the pattern of the hybridization. In Example 1, it is a graph which shows the pattern of the hybridization of the target nucleic acid and probe nucleic acid which originate in a test subject. In the determination of the single nucleotide polymorphism in Example 2, it is a graph which shows the pattern of hybridization with the target nucleic acid and probe nucleic acid corresponding to each genotype. In Example 2, it is a graph which shows the pattern and determination result of hybridization of the target nucleic acid and probe nucleic acid which originate in a test subject. In the determination of single nucleotide polymorphism in Example 3, it is a graph which shows the pattern of the hybridization of the target nucleic acid and probe nucleic acid corresponding to each genotype. In Example 3, it is a graph which shows the pattern and determination result of the hybridization of the target nucleic acid and probe nucleic acid which originate in a test subject.

Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.
(I) Nucleic Acid Hybridization Method A nucleic acid hybridization method according to the first embodiment of the present invention will be described with reference to FIG. In FIG. 1, base sequences that hybridize with other base sequences are represented by white or hatching, and other base sequences are represented by black. In the following description, the parenthesized alphabets attached after the base sequence names correspond to the alphabets attached to the respective base sequences in FIG.
In the nucleic acid hybridization method according to the present embodiment, a sample containing a target nucleic acid having a target base sequence to be hybridized is brought into contact with a probe nucleic acid having a probe base sequence complementary to the target base sequence, In which a target nucleic acid and a probe nucleic acid are specifically hybridized, as shown in FIG.
(A) A part or all of the base sequence in the target nucleic acid located 5 ′ end side from the target base sequence (t) (a: located 5 ′ end side from the target base sequence (t) in FIG. 1) The case where it is a part of the base sequence in the target nucleic acid is exemplified, but the base sequence (a ′) capable of hybridizing with the base sequence (a ′) up to the 5 ′ end may be used. 5 'terminal block nucleic acid on the side, and / or (B) part or all of the base sequence in the target nucleic acid located 3' end side from the target base sequence (t) (b: target base in Fig. 1) The case where it is a part of the base sequence in the target nucleic acid located on the 3 ′ end side from the sequence (t) is exemplified, but it may be the entire base sequence up to the 3 ′ end. A 3 ′ terminal block nucleic acid having a base sequence (b ′) capable of soybeans on the 3 ′ terminal side is contacted with a target nucleic acid. The target nucleic acid and the probe nucleic acid are specifically hybridized in a state where the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid is hybridized with the hybridizable base sequence in the target nucleic acid. Let

Since the target nucleic acid and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid hybridize to form a double strand, the base sequence in the target nucleic acid other than the target base sequence (t) is a probe. Nonspecific hybridization with the base sequence (p) can be suppressed. Moreover, since it can suppress that the base sequence which exists in a different position in a target nucleic acid hybridizes within a molecule | numerator, and forms a higher order structure, the hybridization efficiency of a target nucleic acid and a probe nucleic acid can be improved.

(1) A target nucleic acid having a target base sequence (t) to be subjected to target nucleic acid hybridization is an arbitrary polynucleotide comprising at least one deoxyribonucleotide, ribonucleotide and a derivative thereof. The “polynucleotide” means a compound in which an arbitrary number of two or more nucleotides are condensed, and so-called “oligonucleotide” is also included in the “polynucleotide” in the present invention. Specific examples of the polynucleotide include DNA, cDNA, mRNA (messenger RNA: either before or after splicing), tRNA (transfer RNA), or a fragment thereof. . In addition, the origin of the polynucleotide or the sample containing it is not particularly limited, and it is derived from a living body (meaning any living body such as animal, plant, bacteria, fungus, archaea, virus, etc.), derived from a biological sample Nucleic acids may be amplified by PCR and / or restriction enzymes, etc. (needed to select those that do not cleave the target base sequence in the middle) or those chemically synthesized. Of these, a mixture of two or more may be used.

The position of the target base sequence (t) contained in the target nucleic acid is not particularly limited, and may be near the center of the molecular chain of the nucleic acid, or near the 5 'end or 3' end. The length of the target base sequence (t), that is, the number of bases of the target base sequence (t) is equal to the number of bases of the probe base sequence (p) described later. The length of the base sequence other than the target nucleic acid base sequence is not particularly limited, and similarly, the total number of bases of the target nucleic acid is not particularly limited.

For the preparation of the sample containing the target nucleic acid, any solvent or buffer additive or preparation method usually used for the preparation of the sample containing the nucleic acid can be used. It can be appropriately selected according to hybridization conditions and the like.

For example, in a nucleic acid to be hybridized, if at least the base sequence near the target base sequence is known, a base sequence consisting of 30 to 1000 bases including the target base sequence can be selected and hybridized near both ends thereof Design primers. Any known method (for example, use of a primer design program such as Primer 3) can be used for designing the primer. The target nucleic acid is amplified by the PCR method using the primer thus designed. The target nucleic acid may be prepared as a single-stranded nucleic acid using an asymmetric PCR method.

(2) In order to improve the base sequence specificity in the hybridization between the 5 ′ terminal side and 3 ′ terminal block nucleic acid target nucleic acid and the probe nucleic acid, the 5 ′ terminal block nucleic acid shown in (A) below, and / or Alternatively, the 3 ′ terminal block nucleic acid shown in (B) below is brought into contact with the target nucleic acid, and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid can respectively hybridize in the target nucleic acid. The target nucleic acid and the probe nucleic acid are specifically hybridized in a state of being hybridized with (a and / or b).
(A) 5 ′ terminal block nucleic acid: a nucleic acid containing a base sequence (a ′) capable of hybridizing to the base sequence (a) in the target nucleic acid on the 5 ′ terminal side (B) 3 ′ terminal block nucleic acid: target nucleic acid Nucleic acid containing 3 'terminal side of base sequence (b') capable of hybridizing with base sequence (b) therein

In the following description, the 5 ′ terminal block nucleic acid and the 3 ′ terminal block nucleic acid may be collectively referred to as “block nucleic acid” in some cases.

As described above, the portion where the target nucleic acid is hybridized with the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid forms a double strand, and therefore, in the target nucleic acid other than the target base sequence (t). Can be prevented from hybridizing non-specifically with the probe base sequence (p). Moreover, since it can suppress that the base sequence which exists in a different position in a target nucleic acid hybridizes within a molecule | numerator, and forms a higher order structure, the hybridization efficiency of a target nucleic acid and a probe nucleic acid can be improved.

As shown in FIG. 1, the base sequence (a ′) in the 5 ′ terminal block nucleic acid that can hybridize with the base sequence (a) in the target nucleic acid is completely complementary to the base sequence (a). However, as long as it can hybridize with the base sequence (a) and can maintain the hybridized state even under the condition of hybridizing the target base sequence and the probe base sequence, it contains one or more mismatches. Also good. Similarly, the base sequence (b ′) in the 3 ′ terminal block nucleic acid capable of hybridizing with the base sequence (b) in the target nucleic acid is preferably completely complementary to the base sequence (b). As long as it can hybridize with the base sequence (b) and can maintain the hybridized state even under the condition of hybridizing the target base sequence and the probe base sequence, it may contain one or more mismatches.

There is no particular limitation on the number of base sequences (a ′ and / or b ′) in the target nucleic acid that can hybridize with the block nucleic acid, and of course, it may be the same as or different from the base number of the target base sequence. . Further, the number of bases in the base sequence (a and / or b) in the target nucleic acid capable of hybridizing with the block nucleic acid located 5 ′ end and 3 ′ end from the target base sequence may be the same. They may be different from each other.

The block nucleic acid may consist only of a base sequence (a ′ and / or b ′) that can hybridize with the base sequence in the target nucleic acid, and other than that as long as it does not inhibit the hybridization with the target nucleic acid. May be included.
The 5 ′ terminal block nucleic acid may further have one or more bases on the 5 ′ terminal side of the base sequence (a ′) capable of hybridizing with the base sequence (a). The base sequence (a ′) is located at the 5 ′ end and has no other base at the 5 ′ end.
Similarly, the 3 ′ terminal block nucleic acid may further have one or a plurality of bases on the 3 ′ terminal side of the base sequence (b ′) capable of hybridizing with the base sequence (b). The base sequence (b ′) is located at the 5 ′ end and has no other base at the 3 ′ end.

3 'end side of the target base sequence (t) present in the target nucleic acid, 5' end side of the base sequence (b) capable of hybridizing with the 3 'end side block nucleic acid, and / or the target base sequence (t ) Between the 5 ′ terminal end and the 3 ′ terminal side of the base sequence (a) capable of hybridizing with the 5 ′ terminal block nucleic acid, is 1 to 15 bases, preferably 1 to 6 bases There is a gap. If there is no gap, the distance between the double strand formed by hybridization of the target nucleic acid and the block nucleic acid and the target base sequence becomes too small, and the hybridization efficiency with the probe nucleic acid decreases due to steric hindrance. On the other hand, when the number of bases in the gap exceeds 15, the hybridization efficiency between the target nucleic acid and the probe nucleic acid decreases due to nonspecific hybridization between the base sequence in the target nucleic acid other than the target base sequence and the probe base sequence. To do. The number of bases in the gap present on the 5 'end side and 3' end side of the target base sequence may be the same or different from each other.

(3) Probe nucleic acid As the probe nucleic acid, any nucleic acid usually used in probe hybridization can be preferably used. Specific examples of nucleic acids that can be used include a polynucleotide comprising at least one or more of deoxyribonucleotide (DNA), ribonucleotide (RNA), and derivatives thereof having a probe base sequence complementary to a target base sequence. Can be mentioned.

The length of the probe base sequence (p), that is, the number of bases of the probe base sequence (p), depends on the base sequence and base length of the target nucleic acid to be hybridized, and therefore it is difficult to determine uniquely. However, it is generally 10 or more and 20 or less, preferably 11 or more and 15 or less. When the number of bases of the probe base sequence (p) is less than 10, the total number of statistically possible probe base sequences (for example, 4 ⁹ = 262144 when the base number of the probe base sequence is 9) is too small and specific The number of nucleic acids that can hybridize to the number decreases. On the other hand, when the number of bases in the probe base sequence (p) exceeds 20, only one base is mismatched with the double-stranded nucleic acid formed by hybridization of the target base sequence with no mismatch and the probe base sequence. The difference in thermal stability between the double-stranded nucleic acid formed by hybridization between the target base sequence and the probe base sequence is reduced, and the base sequence specificity in hybridization is reduced.

The probe base sequence (p) may be completely complementary to the target base sequence (t) to be hybridized, particularly when used for single nucleotide polymorphism (SNP) determination (typing). Although it is preferable, it may contain one or a plurality of mismatches if it has sufficient base sequence specificity in a specific target nucleic acid type or hybridization conditions.

The probe base sequence (p) may be present at any position in the probe nucleic acid, but is preferably present on either the 5 ′ end side or the 3 ′ end side. However, from the viewpoint of ease of hybridization (steric hindrance) and the like, it is more preferable that it exists on the end side opposite to the side fixed on the solid phase carrier.
The probe base sequence (p) may further have one or a plurality of bases on the 5 ′ end side or 3 ′ end side of the probe base sequence (p). Preferably, the probe base sequence (p) is 5 ′ end or 3 ′ end. And has no other base at the 5 'or 3' end.

As the probe nucleic acid, any of those prepared by PCR method, chemically synthesized, natural nucleic acids as they are or processed and used can be used. It is preferable to use a chemically synthesized product for ease of preparation. In this case, it is preferable to use polydeoxyribonucleotide (DNA), polyribonucleotide (RNA), PNA (peptide nucleic acid), etc. from the viewpoint of ease of synthesis, but from the viewpoint of synthesis and availability and stability, preferable.

Since the probe nucleic acid is used for hybridization on a solid phase carrier, the probe nucleic acid is modified with any functional group or molecule so long as it does not inhibit hybridization in order to continue binding and adsorption to the solid phase carrier before and after hybridization. May be. The functional group or molecule may be located on either the 5 'end or the 3' end. Therefore, the probe nucleic acid may be immobilized on the solid phase carrier at either the 5 'end side or the 3' end side. The functional group or molecule may be any functional group or molecule that binds to a functional group present on the surface of the solid phase carrier or a functional group or molecule bonded to the surface. Group, carboxyl group, aldehyde group, isocyanate group, isothiocyanate group, epoxy group, thiol group, sulfide group, vinyl group, biotin that specifically binds to avidin bound on solid support, bound on solid support A polynucleotide having a base sequence complementary to the polynucleotide (preferably having a base sequence that does not exist in either the target nucleic acid sequence or the probe base sequence, such as polydeoxyribonucleotide (DNA), polyribonucleotide (RNA) and PNA (peptide nucleic acid)).

When the probe nucleic acid is chemically synthesized, it may be synthesized using a solid phase synthesis method on a solid phase carrier, and the probe nucleic acid synthesized by any method such as a liquid phase method is immobilized on the solid phase carrier. May be.
Examples of solid phase carriers include: (1) a microtiter plate, cell, flow cell, microreactor made of a material such as glass, resin, ceramics, metal (can take any form such as bulk material, thin film, fine particles), Examples thereof include a tube and (2) a chromatography carrier made of a material such as silica or polystyrene. In order to avoid non-specific adsorption of the target nucleic acid, probe nucleic acid and block nucleic acid to the surface of the solid phase carrier, the surface of the solid phase carrier may be coated (blocked) with casein, bovine serum albumin (BSA) or the like. Good.

In the nucleic acid hybridization method according to the present embodiment, first, a 5′-end block nucleic acid and / or a 3′-end block nucleic acid is added to a sample containing a target nucleic acid, and these are brought into contact with the target nucleic acid. The side block nucleic acid and / or the 3 ′ end side block nucleic acid is hybridized with the hybridizable base sequence (a and / or b) in the target nucleic acid, respectively. Next, the target nucleic acid hybridized with the block nucleic acid is brought into contact with the probe nucleic acid, and these are hybridized. When the target nucleic acid and the probe nucleic acid are intended for detection of a base sequence-specific nucleic acid, the hybridization is detected using any method. In addition, when the hybridization between the target nucleic acid and the probe nucleic acid is intended to isolate a nucleic acid having a specific base sequence, it is not hybridized (does not have a base sequence complementary to the probe base sequence). After washing and removing the nucleic acid, the target nucleic acid is dissociated from the probe nucleic acid and the block nucleic acid, and separated from the block nucleic acid. Dissociation of the hybridized nucleic acid and separation of the block nucleic acid can be performed using any method.

(II) Single nucleotide polymorphism (SNP) determination (typing) method The single nucleotide polymorphism determination method according to the second embodiment of the present invention is a target having a target base sequence (t) to be determined. A nucleic acid comprising a probe base sequence (p) that is complementary to a part or all of a single nucleotide polymorphism (SNP) present in the target base sequence (t) and has a different probe base sequence (p) A probe base sequence complementary to the target base sequence (t) of the target nucleic acid and the probe nucleic acid is brought into contact with a plurality of probe nucleic acids arranged in different predetermined regions on the inner wall surface of the single reactor. A specific high of Step A for specifically hybridizing with (p) and a target nucleic acid and probe nucleic acid having a probe base sequence (p) complementary to the target base sequence (t) Hybridization置特 and a step B of different detectable.

In the present invention, the “reactor” means that a probe nucleic acid is arranged at a predetermined position on an inner wall surface (in contact with the solution) of a container filled with a solution containing a target nucleic acid or a flow channel to be circulated, The probe nucleic acid is configured to contact and hybridize with a probe nucleic acid, and may take any form such as a cell, a flow cell, a microreactor, a tube and the like. The material of the reactor is not particularly limited, and any material such as glass, resin, ceramics, and metal can be selected according to the method used for position-specific detection of hybridization.

A probe base sequence (p) that is complementary to part or all of the single nucleotide polymorphism (SNP) present in the target base sequence (t) is included on the inner wall surface of the container or flow path in the reactor. (Ie, only one base in the probe base sequence is different from each other) A plurality of types (bases at positions corresponding to single nucleotide polymorphisms in the probe base sequence (p) are adenine (A), thymine (T) (or uracil (U )), 2-4) probe nucleic acids of guanine (G) and cytosine (C) are arranged. Here, those having different probe base sequences (p) are arranged in different predetermined regions on the inner wall surface of a single reactor. The probe nucleic acid arranged on the inner wall surface of the reactor may be only 2 to 4 types corresponding to a single single nucleotide polymorphism, but the probe base sequences (p) are different from each other. As long as hybridization can be detected independently of each other, a combination of probe nucleic acids corresponding to a plurality of single nucleotide polymorphisms may be arranged on the inner wall surface of a single reactor.

In order to detect the hybridization between the target nucleic acid and the probe nucleic acid at a single molecule level, an expensive detection device is required. Usually, a plurality of probe nucleic acids corresponding to each single nucleotide polymorphism (SNP) are prepared. And placed on the inner wall of the reactor. In this case, in order to determine (typing) the SNP, it is necessary to detect the specific hybridization for each probe nucleic acid in a position-specific manner. Therefore, each of the probe nucleic acids having the same probe base sequence is placed in a predetermined region having a predetermined size and shape, and the regions are located at a predetermined distance from each other. The probe nucleic acid is fixed on the inner wall surface in a state where the probe nucleic acid is arranged. The method for immobilizing the probe nucleic acid on the inner wall surface is as described in the description of the nucleic acid hybridization method according to the first embodiment, and will not be described in detail. In addition, the shape and size of each region and the distance between the individual regions can be appropriately determined according to the size of the reactor, the spatial resolution of the means for detecting hybridization, and the like.

In order to improve the base sequence specificity in the hybridization of the target nucleic acid and the probe nucleic acid performed in the step A, as shown in the above (A), as in the nucleic acid hybridization method according to the first embodiment described above. The 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid shown in (B) above is brought into contact with the target nucleic acid, and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid is the target, respectively. It is preferable to specifically hybridize the target nucleic acid and the probe nucleic acid in a state of hybridization with the hybridizable base sequence (a and / or b) in the nucleic acid.

Since the target nucleic acid used in step A and the sample containing the same, the block nucleic acid and the probe nucleic acid, and the hybridization conditions are the same as those in the nucleic acid hybridization method according to the first embodiment described above, Detailed description is omitted. In the probe nucleic acid, the position of the base corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence (p) is 1/3 of the number of bases of the probe base sequence (p). It is preferably located at the center when the probe base sequence (p) is divided into three regions so that the number of bases in each region is (almost) equal. More preferably, the base corresponding to the mold is located in the center of the probe base sequence (p).
Note that “positioned at the center of the base sequence (base)” means that when the number of bases of the probe base sequence (p) is n,
(1) When n is an even number: (n / 2) th or (n / 2) + 1st from the 5 ′ end side (2) When n is an odd number: A base located at (n + 1) / 2th.

For example, when the number of bases of the probe base sequence (p) is 13, “the positions of the bases corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence (p) are both probe bases. The “position larger than 1/3 times the number of bases of the sequence (p)” refers to the 5th to 9th positions from the 5 ′ end side of the probe base sequence. In this case, the probe base sequence is divided into three regions having 4, 5, and 4 bases from the 5 'end toward the 3' end, respectively. In this case, the “center” of the probe base sequence (p) means the seventh position from the 5 ′ end side of the probe base sequence.

Similarly, when the number of bases of the probe base sequence (p) is 12, “the positions of the bases corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence (p) are both probes. The “position larger than 1/3 times the number of bases in the base sequence (p)” refers to the fifth to eighth positions from the 5 ′ end side of the probe base sequence (in this case, the probe base sequence is from the 5 ′ end side). That is, the region is divided into three regions of which the number of bases is all four toward the 3 ′ end.) In this case, the “center” of the probe base sequence (p) is 5 ′ of the probe base sequence. 6th or 7th from the end side.

Further, when the number of bases of the probe base sequence (p) is 14, “the positions of the bases corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence (p) are both probe bases. The “position larger than 1/3 times the number of bases of the sequence (p)” means the 5th to 10th positions from the 5 ′ end side of the probe base sequence (in this case, the probe base sequence is 3 from the 5 ′ end side 'Towards the end side, the number of bases is divided into three regions of 4, 6, and 4 respectively.) In this case, the "center" of the probe base sequence (p) is the probe base sequence. 7th or 8th from the 5 ′ end side.
That is, when the probe base sequence (p) is divided into three regions as described above, when the number of bases is a multiple of 3, the number of bases in all three regions is equal, and when divided by 3, one or two remainders are obtained. Sometimes it is divided so that the number of bases at the center is 1 or 2 more than that at both ends.

When the base corresponding to the single nucleotide polymorphism is located in the center of the probe base sequence (p), the efficiency of hybridization with a nucleic acid containing a mismatch decreases, so the base sequence specificity in the hybridization between the probe nucleic acid and the target nucleic acid And the efficiency and reliability of single nucleotide polymorphism (SNP) typing can be improved.

The length of the probe base sequence in step A and the presence / absence of use of the 3 ′ terminal block nucleic acid and / or the 5 ′ terminal block nucleic acid can be determined, for example, as follows.
(1) First, design and synthesis of a probe nucleic acid having a probe base sequence having an appropriate base length (for example, 13 bases) in which the base corresponding to the single nucleotide polymorphism is located at the center, and hybridization with the target nucleic acid Observe.
(2) For any probe nucleic acid, if the intensity of the resonance signal is large and sufficient base sequence specificity is not observed, the probe nucleic acid is reduced in length by reducing the base length of the probe base sequence and having a mismatch. When non-specific hybridization such as a higher hybridization efficiency is observed, the base length of the probe base sequence is increased.
(3) If the base sequence specificity is improved as a result of changing the base length of the probe base sequence as described above, but is still insufficient, the 3 ′ end side block nucleic acid and the 5 ′ end side block nucleic acid Add one of these to examine whether improvement is observed. If necessary, the other is added and examined in the same manner.

In the reactor that performs hybridization between the probe nucleic acid and the target nucleic acid, for example, a buffer such as a phosphate buffer, PBS (phosphate buffered saline), a HEPES buffer, or a buffer such as a Tris buffer. Add the liquid. By adjusting the concentration of the buffer salt (NaCl, MgCl _2, etc.), nonspecific adsorption of the probe nucleic acid and the target nucleic acid to the solid phase carrier or the like can be prevented. Further, when a salt-containing HEPES buffer or the like is used as a buffer, nonspecific adsorption of nucleic acids can be prevented.

A solution containing the target nucleic acid is dropped or supplied into the reactor containing the buffer as described above, and the probe nucleic acid and the target nucleic acid are hybridized. The temperature in the reactor is, for example, a temperature at which the lowest Tm value of double strands formed by hybridization of probe nucleic acid, block nucleic acid and target nucleic acid is not denatured into single-stranded nucleic acid after hybridization. To.

In step B, specific hybridization between the target nucleic acid and the probe nucleic acid having a probe base sequence (p) complementary to the target base sequence (t) is detected in a position-specific manner. For detection of hybridization, an arbitrary method having a spatial resolution smaller than the distance between individual regions where probe nucleic acids having the same probe base sequence (p) are arranged on the inner wall surface of the reactor is used. Can do. As a preferable detection method, a signal associated with hybridization can be detected in real time with high sensitivity, and either surface plasmon resonance (SPR) method or abnormal reflection of gold which does not require an expensive detection device can be mentioned. When these detection methods are used, a reactor in which the light incident surface side is transparent and a gold vapor deposition film is formed is used. In this case, the probe nucleic acid can also be immobilized on the surface via a gold-thiol bond.

Alternatively, the probe nucleic acid may be fixed to the surface of the gate electrode of the CMOS FET via an electrode such as a gold deposition film so that hybridization between the probe nucleic acid and the target nucleic acid can be electrically read out. Good. In this case, by integrating amplifiers and multiplexers, it is possible to perform signal processing such as signal amplification and single nucleotide polymorphism determination, thereby reducing the size and cost of the apparatus. This is advantageous from the viewpoints of easy operation and improved reliability.

(III) Reactor for judging single nucleotide polymorphism The reactor for judging single nucleotide polymorphism according to the third embodiment of the present invention is a container filled with a solution having a target base sequence to be judged and the solution. A plurality of probe nucleic acids having a probe base sequence complementary to each of a part or all of single nucleotide polymorphisms (SNPs) present in the target base sequence. Among them, those having different probe base sequences are arranged at predetermined positions different from each other on the inner wall surface.

Since the material and form of the reactor and the arrangement of the probe nucleic acid are as described in the description of the single nucleotide polymorphism determination method according to the second embodiment, detailed description thereof is omitted.

(IV) Single nucleotide polymorphism judging device As shown in FIG. 2, the single nucleotide polymorphism judging device 10 according to the fourth embodiment of the present invention is the single nucleotide polymorphism according to the second embodiment described above. Is a device for carrying out the determination method, and has either or both of a container filled with a solution having a target base sequence to be determined and a flow path through which the solution is circulated. Among a plurality of probe nucleic acids having probe base sequences complementary to some or all of existing single nucleotide polymorphisms (SNPs), those having different probe base sequences are different from each other on the inner wall surface. Specific hybridization between the reactor 12 arranged at a predetermined position, a target nucleic acid having a target base sequence, and a probe nucleic acid having a probe base sequence complementary to the target base sequence And a detecting means 13 for detecting the.

About the reactor 12 and the detection means 13, as described in the description regarding the single nucleotide polymorphism determining method according to the second embodiment and the single nucleotide polymorphism determining reactor according to the third embodiment. Therefore, for example, when surface plasmon resonance (SPR) is used for detection of hybridization between the target nucleic acid and the probe nucleic acid, the detection means 13 includes a light source (semiconductor laser or the like), an optical system, and the like. (Condensing lens, polarizer, prism, beam splitter, etc.), detector (CCD camera, photodiode array, etc.), electronic circuit (differential amplifier, A / D converter, etc.), etc.
Alternatively, the addition of target nucleic acid solution or hybridization in the reactor 12 may be performed manually.

The single nucleotide polymorphism determination apparatus 10 processes signals from the sample supply means (diaphragm pump, etc.) 11 and the detection means 13 for supplying a sample containing the target nucleic acid to the reactor 12, in addition to the reactor 12 and the detection means 13. In some cases, it has a data processing means 14 for determining a single nucleotide polymorphism, a sample supplying means 11, a detecting means 13 and a control means 15 for controlling the data processing means 14. The data processing means 14 and the control means 15 are, for example, personal computers, and a program stored in a storage device such as a hard disk drive or ROM is read into a central processing unit (CPU), whereby the data processing means 14 and the control means 15 15 functions. In addition, the single nucleotide polymorphism determination device 10 may have a heater or the like (not shown) for adjusting the temperature of the reactor 12.

Next, examples carried out for confirming the effects of the present invention will be described. These examples are merely illustrative examples, and the present invention is not limited to these examples. In this example, in order to make it easier to confirm the complementarity of base sequences and the presence or absence of mismatches, the base sequences of the probe nucleic acid, linker nucleic acid and block nucleic acid that hybridize to the target nucleic acid are changed from 3 ′ end to 5 ′ end. Write in the direction.

Example 1: Examination of hybridization detection conditions by model aldehyde dehydrogenase (ALDH2) ALDH2, which oxidizes acetaldehyde, an intermediate metabolite of ethanol ingested by drinking and converts it to acetic acid, has a gene polymorphism (single base) Polymorphism) and is divided into an ALDH2 * 1 type that is active and an ALDH2 * 2 type that is inactive. The 104th base (the 1035th position from the 5 ′ end) of the 12th exon of the ALDH2 gene is G (guanine) in the former (hereinafter referred to as “G type”), whereas A (adenine) in the latter. (Hereinafter referred to as “A type”). As a result, the 487th amino acid in ALDH2 is glutamic acid (GAA) in the former and lysine (AAA) in the latter. Persons with genotypes active homozygotes (G / G) have fast metabolism of ethanol and acetaldehyde, whereas those with heterozygotes (G / A) and inactive homozygotes (A / A) Because of the delayed metabolism of acetaldehyde, facial flushing and discomfort (flushing symptoms) appear in these people. The latter, in particular, is very weak against alcohol and is of a so-called “drinkable” type. In whites and blacks, the G / G ratio is almost 100%, while in Japanese, the G / G is 56%, G / A is 38%, and A / A is 4%. .

(1) Construction of hybridization detection system and detection of hybridization by surface resonance plasmon resonance (SPR) method As the target nucleic acid, the 104th base of exon 12 of G-type ALDH2 gene (shown in bold in the base sequence below) A nucleic acid having a base sequence represented by SEQ ID NO: 1 was synthesized.

As the probe nucleic acid having a probe base sequence having a mismatch of 0 to 3 bases, the underlined 20 bases in the base sequence represented by SEQ ID NO: 1 are represented by the following base sequences 2 to 9 Eight types of nucleic acids having the nucleotide sequences to be designed and synthesized.

In the base sequences represented by SEQ ID NOs: 2 to 9, the bases shown in bold are bases present at the positions corresponding to the nucleobases G shown in bold in SEQ ID NO: 1 (C is complementary, T is mismatch) And the underlined base is a base complementary to the corresponding base in the target base sequence. Further, the 15 base sequence on the 3 ′ end side shown in italics is complementary to the sequence of 15 bases on the 5 ′ end side of a linker nucleic acid (SEQ ID NO: 10) described later, and is applied to the surface of the SNP determination chip. It is a base sequence introduced for immobilization of these probe nucleic acids.

A sensor chip for surface plasmon resonance (SPR) was prepared, and avidin was fixed on the surface thereof. Next, a solution (linker nucleic acid concentration 2 μM, 0.3 M NaCl-containing HBS, 30 μL) containing a synthetic DNA (linker nucleic acid) having the base sequence represented by SEQ ID NO: 10 and bound to biotin at the 5′-end side was introduced. After the injection, Running Buffer (0.3 M NaCl-containing HBS) was allowed to flow for 15 minutes (flow rate: 5 μL / min), and immobilized on the surface of the sensor chip through specific binding between biotin and avidin. Thereafter, 8 μL of 50 mM NaOH (4 minutes) was flowed 4 times, and then 8 μL of 0.05% SDS was flowed once to wash the linker nucleic acid to obtain a single strand.

In the above base sequence, the base sequence shown in italics is complementary to the base sequence shown in italics in the base sequences represented by SEQ ID NOs: 2-9. Subsequently, a solution (probe nucleic acid concentration 2 μM, 1M NaCl-containing HBS) containing any of the probe nucleic acids having the base sequences represented by SEQ ID NOs: 2 to 9 was flowed for 1 minute (flow rate 5 μL / min). By this operation, a sensor chip was prepared in which any of these probe nucleic acids was bound and immobilized on the surface through hybridization with a complementary sequence in the base sequence represented by SEQ ID NO: 10 in the linker nucleic acid.

After injecting a solution containing the target nucleic acid (target nucleic acid concentration: 0.5 μM, 0 to 2 M NaCl-containing HBS) into the sensor chip having the probe nucleic acid immobilized on the surface as described above, running buffer (HBS, pH 7.4) , 0.05% Tween 20, 1 mM EDTA) was allowed to flow for 1 minute (flow rate: 5 μL / min), and hybridization between the target nucleic acid and the probe nucleic acid was monitored by surface plasmon resonance (SPR) measurement. From the intensity change of the sensorgram at equilibrium before and after hybridization, the magnitude of the change in resonance angle due to hybridization (resonance signal intensity: unit is represented by RU (resonance unit), and 1000 RU = 0.1 °. .) Subsequently, washing (5 μL / min, 1 minute) was performed with 50 mM NaOH to wash and remove the target nucleic acid and the probe nucleic acid. The above operation was performed for all the probe nucleic acids, and the resonance signal intensities of the probe nucleic acids having the base sequences represented by SEQ ID NOs: 2 to 9 were compared.

The intensity of the resonance signal observed in the probe nucleic acid (SEQ ID NO: 2) having a base sequence completely complementary to the target base sequence tended to increase as the NaCl concentration increased. On the other hand, the ratio of the signal intensity between the case where the probe base sequence is completely complementary to the target nucleic acid and the case where the probe base sequence has a mismatch is almost constant regardless of the NaCl concentration, and the one having a one base mismatch (SEQ ID NO: 3 It was only about 1.14 times that of 5) and only about 1.5 times that having a mismatch of 3 bases (SEQ ID NO: 9). As described above, when the base length of the probe base sequence is 20, high base sequence specificity was not observed in the hybridization between the target nucleic acid and the probe nucleic acid.

(2) From the result of (1) on the examination of the influence of the base length of the target base sequence on the hybridization efficiency, it was confirmed that the hybridization between the target nucleic acid and the probe nucleic acid can be detected by the SPR method. When the base length of the sequence is 20, since the base sequence specificity is not sufficient, the target nucleic acid having various base lengths is used, and the influence of the base length of the nucleic acid base sequence on the base sequence specificity in hybridization investigated. The probe nucleic acid and target nucleic acid used are as follows. Here, only probe nucleic acids having the base sequence represented by SEQ ID NO: 11 were used, and examination was performed using target nucleic acids (SEQ ID NOs: 12 to 20) having different base lengths.

The target nucleic acid having the base sequence represented by SEQ ID NOs: 12 to 20 has a nucleobase G complementary to the nucleobase C shown in bold in the probe base sequence having the base sequence represented by SEQ ID NO: 11 in the center. And has a base sequence of 3 to 20 bases complementary to the probe base sequence on both sides. Using these nucleic acids, the same operation as in (1) above was performed, and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined.

The results are shown in FIG. The horizontal axis represents the number of bases of the target nucleic acid, and the vertical axis represents the resonance signal intensity divided by the number of bases (resonance signal intensity per base). When the number of bases is 20, the resonance signal intensity per base is maximum, and it is considered that the change in hybridization efficiency due to the presence or absence of mismatching is less likely to appear.

Therefore, for target nucleic acids having a base length of 24 or less (SEQ ID NOs: 12 to 17), in addition to those having the base sequence represented by SEQ ID NO: 11, the following base sequences containing mismatches (SEQ ID NOs: 21 and 22) The probe nucleic acid having the same procedure as (1) above was examined, and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined.

In SEQ ID NOs: 21 and 22, the underlined base is a mismatch. The results are shown in FIG. When the base length of the target base sequence is 11 to 15, the ratio of signal intensity between the case where the probe base sequence is completely complementary to the target nucleic acid and the case where there is a mismatch depends on the NaCl concentration, and the maximum (NaCl concentration It was confirmed that it increased to 5 to 100 in the case of 1M. From the above results, it was confirmed that the base length of the target base sequence (probe base sequence) is preferably 11-15. In the following operations, the salt (NaCl) concentration was 1 M, which is the concentration at which the highest base sequence specificity was observed.

(3) Examination of influence of mismatch position on hybridization efficiency Probe nucleic acid having base sequence represented by SEQ ID NO: 11 (completely complementary) and 21 (having one base mismatch) Were subjected to the same operation as in the above (1) using each of the target nucleic acids having the following base sequence (the base length was 11), and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined. .

The results are shown in FIG. The bar graph shows the intensity of the resonance signal in the hybridization between the probe nucleic acid having the base sequence represented by SEQ ID NOs: 11 (completely complementary) and 21 (one base mismatch) and each target nucleic acid, and the line graph is The ratio of the latter to the former is expressed in%, and the smaller the value, the higher the nucleotide sequence specificity. As is clear from the line graph in FIG. 5, it was confirmed that the base sequence specificity was maximized when the mismatch base was in the center of the target base sequence (probe base sequence).

(4) Examination of influence of block nucleic acid on hybridization efficiency As a target nucleic acid, represented by any one of the following SEQ ID NO: 27 and SEQ ID NO: 28 comprising 50 bases including the 104th base of exon 12 of ALDH2 gene A nucleic acid having the base sequence to be synthesized was synthesized.

The nucleic acids represented by SEQ ID NOs: 27 and 28 correspond to G type and A type, respectively. In the base sequences represented by SEQ ID NOs: 1 and 2, the underlined portion includes a base corresponding to a single nucleotide polymorphism and 13 bases consisting of 6 bases before and after the base. Bases represented by the following SEQ ID NOs: 29 to 32 as probe nucleic acids having these bases or 11 bases excluding one base at both ends as a target base sequence and a complementary base sequence as a probe base sequence A nucleic acid having a sequence was designed and synthesized. Using these nucleic acids, the same operation as in (1) above was performed, and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined. The base lengths of the probe base sequences are SEQ ID NOs: 29 and 31, 13, 30 and 32. In SEQ ID NOs: 29 and 30, the probe base sequence is completely complementary to the target base sequence. The one base corresponding to the single nucleotide polymorphism is mismatched.

In addition, 5 ′ end side (SEQ ID NO: 33) and 3 ′ having base sequences complementary to the 5 ′ end and 3 ′ end side base sequences from the target base sequence in the target nucleic acid (SEQ ID NO: 27, 28). The terminal block nucleic acid (SEQ ID NO: 34) was synthesized, and the hybridization efficiency in a state where one or both of them were hybridized to the target nucleic acid was also examined. When conducting experiments in the presence of block nucleic acids, after injecting the target nucleic acid, inject the block nucleic acid at twice the concentration (1 μM), heat at 95 ° C. for 3 minutes, and then slowly cool to room temperature, then SPR Measurements were made.

The results when the base length of the probe base sequence is 11 (base sequences 30, 32) are shown in FIG. When any probe nucleic acid was used, it was confirmed that the mismatch / match ratio was minimized to 8 to 12% when the 3 ′ terminal block nucleic acid was added.
Similar results were obtained when the base length of the probe base sequence was 13 (base sequences 29 and 31).

(5) Examination of influence of addition amount of 3′-end side block nucleic acid on hybridization efficiency The influence of addition amount of 3′-end side block nucleic acid on hybridization efficiency was also examined. Although the results are not shown, the mismatch / match ratio decreased with increasing amount of 3 ′ terminal block nucleic acid (SEQ ID NO: 33) for any target nucleic acid (SEQ ID NO: 27 and 28). The effect of addition was saturated in the vicinity of double (molar ratio). Therefore, it was confirmed that the effect of sufficiently improving the base sequence specificity can be obtained when the 3 ′ terminal block nucleic acid is added at least twice (molar ratio) of the target nucleic acid.

(6) Examination of the influence of the length of the gap between the 5 ′ end side of the base sequence in the target nucleic acid capable of hybridizing to the 3 ′ end side block nucleic acid and the 3 ′ end side of the target base sequence on the hybridization efficiency A target nucleic acid having a base sequence represented by SEQ ID NOs: 27 and 28, a probe nucleic acid having a base sequence represented by SEQ ID NOs: 29 and 31, and a 3 ′ terminal block nucleic acid having the following SEQ ID NOs: 34 to 34 Having a base sequence represented by 40 (the numbers in parentheses are the 5 'end side of the base sequence in the target nucleic acid to which the 3' end block nucleic acid can hybridize and the 3 'end side of the target base sequence) (Hereinafter abbreviated as “gap length.”) The negative number is present on the 3 ′ end side of the 3 ′ end block nucleic acid, and overlaps with the target base sequence. Base Using represents a number.), In the same manner as the above (1) and (4) were examined hybridization efficiency between target nucleic acid and the probe nucleic acid.

When there was an overlap between the target base sequence and the base of the 3 'terminal block nucleic acid, the hybridization efficiency was lowered, but measurement by the SPR method was possible. When the gap length was 0, although the hybridization efficiency was high, considerable hybridization was observed even in the presence of mismatch, and a mismatch / match ratio of about 10% was observed at the minimum. When the gap length was 1 to 5, hybridization in the presence of a single base mismatch was greatly suppressed, and the mismatch / match ratio was 1% or less.

From the above results, in ALDH2, the S / N ratio (by SPR method) is determined by using the 3 ′ terminal block nucleic acid having a base length of the probe base sequence of 11 to 13 and a gap length of 1 to 5, by the SPR method. It was confirmed that single nucleotide polymorphisms can be determined with a match / mismatch ratio of 100 or more.

(7) Only a single nucleotide polymorphism determination target nucleic acid having the base sequence represented by SEQ ID NO: 27 (corresponding to G / G type), only a nucleic acid having the base sequence represented by SEQ ID NO: 28 (A / (Corresponding to type A), or a mixture of the two in a ratio of 1: 1 (corresponding to type G / A), having a nucleotide sequence represented by SEQ ID NOs: 29, 31 and 41 as a probe nucleic acid, 3 ′ end As the side block nucleic acids, those having the base sequence represented by SEQ ID NO: 34 were used (see FIG. 7: the black trapezoid represents avidin, and the pentagon with B written represents a biotin molecule), respectively ( The same operation as 1) and (4) was performed, and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined. The probe nucleic acids having the base sequences represented by SEQ ID NOs: 29, 31 and 41 have probe base sequences complementary to the target nucleic acids whose bases are G, A and C at the sites corresponding to the single nucleotide polymorphisms, respectively. Have.

The results are shown in FIG. For each target nucleic acid, each genotype of single nucleotide polymorphism is obtained from the resonance signal intensity pattern for each probe nucleic acid having the base sequence represented by SEQ ID NOs: 29, 31 and 41 using an algorithm as shown in FIG. It is considered that (G / G, A / A, G / A) can be determined.

(8) Preparation of single-stranded nucleic acid from DNA collected from hair by asymmetric PCR method DNA was extracted from human hair using DNA extraction kit ISOHAIR manufactured by Nippon Gene Co., Ltd. Using the extracted DNA as a template, a 430 bp PCR amplification product containing the 904th to 1332st bases from the 5 ′ end was obtained. The primer used was an oligodeoxyribonucleotide having a base sequence complementary to a base sequence of 25 bases from each end. Using the 430 bp amplification product thus obtained as a template, a 118 bp amplification product was obtained by asymmetric PCR. The conditions of the asymmetric PCR method are as follows.

430 bp amplification product: 200 fg
5′-end template (complementary to the 945th to 964th base sequences): 1 μM
3′-end template (complementary to the 1042 to 1062nd nucleotide sequences): 20 nM
Cycle: 95 ° C. 5 minutes → (94 ° C. 30 seconds−55 ° C. 30 seconds−72 ° C. 15 seconds) × 35 cycles

By the Southern hybridization method, it was confirmed that the obtained amplification product was a single-stranded DNA. It was confirmed that a single-stranded DNA that can be used for SNP determination was obtained by the above method.

(9) Preparation of target nucleic acid sample from DNA collected from hair by asymmetric PCR method DNA was extracted from hair collected from 13 volunteers by the same method as in (8) above to obtain a 430 bp PCR amplification product. The primer used was an oligodeoxyribonucleotide having a base sequence complementary to a base sequence of 25 bases from each end. Using the 430 bp amplification product thus obtained as a template, a 139 bp amplification product was obtained by asymmetric PCR. The conditions of the asymmetric PCR method are as follows.

430 bp amplification product: 200 fg
5′-end template (complementary to the nucleotide sequence of the 925th to 949th positions): 1 μM
3 ′ terminal template (complementary to the 1044 to 1063rd nucleotide sequence: SEQ ID NO: 34): 20 nM
Premix, MilliQ: 25 μL
Cycle: 95 ° C. for 5 minutes → (94 ° C. for 30 seconds−55 ° C. for 30 seconds−72 ° C. for 15 seconds) × 40 cycles

The target nucleic acid sample (139 bp amplification product) thus obtained was precipitated with ethanol, dissolved in 140 μL of MilliQ, and 70 μL of 3M Buffer was added. 1 μL of a 0.25 μM solution of 3 ′ terminal block nucleic acid having the base sequence represented by SEQ ID NO: 34 was added, and after heating, rapidly cooled and hybridized to the target nucleic acid.

A solution (probe nucleic acid concentration 200 nM, 1 M NaCl-containing HBS) containing any of the probe nucleic acids having the base sequences represented by SEQ ID NOs: 29, 31, and 41 on the sensor chip having the linker nucleic acid immobilized on the surface in (1) above 25 μL was allowed to flow for 5 minutes (flow rate 5 μL / min). By this operation, a sensor chip was prepared in which any of these probe nucleic acids was bound and immobilized on the surface through hybridization with a complementary sequence in the base sequence represented by SEQ ID NO: 10 in the linker nucleic acid. Subsequently, the solution containing the target nucleic acid prepared above was allowed to flow for 10 minutes (flow rate: 3 μL / min), and hybridization between the target nucleic acid and the probe nucleic acid was monitored by surface plasmon resonance (SPR) measurement. From the intensity change of the sensorgram at equilibrium before and after hybridization, the magnitude of the change in resonance angle due to hybridization (resonance signal intensity: unit is represented by RU (resonance unit), and 1000 RU = 0.1 °. .) Subsequently, washing (5 μL / min, 1 minute) was performed with 50 mM NaOH to wash and remove the target nucleic acid and the probe nucleic acid. The above operation was performed for all the probe nucleic acids, and the resonance signal intensities of the three types of probe nucleic acids were compared.

The results are shown in FIG. The genotype is determined using the algorithm shown in FIG. 9 from the ratio of the resonance signal intensities obtained using the three probe nucleic acids (SEQ ID NOs: 29, 31, 41), and obtained by the conventional method (DNA sequencing). Compared with the results.

As shown in Table 1, the genotype determined by this method completely matched the genotype determined by the conventional method.

Example 2: Determination of a single nucleotide polymorphism of methylenetetrahydrofolate reductase (MTHFR) MTHFR is one of the enzymes in the folate metabolic pathway and catalyzes the rate-limiting step of the process of converting homocystin to methionine. This enzyme abnormality resulting from mutation of the 677th C in the DNA encoding MTHFR to T causes folate deficiency and causes hyperhomocystinemia, which can be improved by administration of folic acid. However, folic acid deficiency is known to increase the risk of developing fetal neural tube closure disorders, and adequate intake of folic acid is necessary especially in the early stages of pregnancy. Therefore, if the diagnosis is TT homozygous, sufficient management of folic acid intake is necessary during pregnancy. Appearance frequency of each genotype in Japanese is C / C type 46%, C / T type 44%, T / T type 10%.

(1) Examination of optimum conditions using model system As a target nucleic acid, a base sequence represented by any one of the following SEQ ID NO: 42 and SEQ ID NO: 43 comprising 53 bases including the 677th base of the MTHFR gene Nucleic acid possessed was synthesized. A probe nucleic acid having a base sequence represented by SEQ ID NOs: 44 to 47 (having a base corresponding to a single nucleotide polymorphism in the center), and a 3 ′ terminal block nucleic acid represented by SEQ ID NO: 48 Each having a base sequence (the gap length is 1) was designed and synthesized. Using these nucleic acids, the same operations as in (1) and (4) of Example 1 were performed, and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined.

Even when 3 'terminal block nucleic acid was added, the mismatch / match ratio was as large as about 10 to 80%. This was thought to be because the base length of the probe base sequence was too large and the hybridization efficiency was too high, so that the bases represented by SEQ ID NOs: 49 to 52 were reduced to 11. A probe nucleic acid having a sequence was synthesized.

These probe nucleic acids, the target nucleic acids used above (SEQ ID NOs: 42 and 43, a 1: 1 mixture of both: corresponding to C / C type, T / T type and C / T type, respectively) and the 3 ′ end. A similar experiment was performed using the side block nucleic acid (SEQ ID NO: 48). The result is shown in FIG. From this result, it is considered that the genotype can be determined based on the following criteria for the ratio of the resonance signal intensities observed for the four probe nucleic acids.
(1) If SEQ ID NO: 50> SEQ ID NO: 49, T / T type (2) If SEQ ID NO: 49 / SEQ ID NO: 50> 3, C / C type (3) If 1 ≦ SEQ ID NO: 49 / SEQ ID NO: 50 ≦ 3 C / T type

(2) Determination of single nucleotide polymorphism using DNA collected from test subject's hair DNA was extracted from human hair using DNA extraction kit ISOHAIR manufactured by Nippon Gene Co., Ltd. Using the extracted DNA as a template, a primer having the nucleotide sequence represented by SEQ ID NOs: 53 and 54 below (the former is on the 5 ′ end side and the latter is on the 3 ′ end side), and 866 bp by an asymmetric PCR method. An amplification product was obtained. Using the obtained amplification product as a template, an asymmetric PCR method using primers having the nucleotide sequences represented by SEQ ID NOS: 55 and 56 below (the former is on the 5 ′ end side and the latter is on the 3 ′ end side) Gave a 72 bp amplification product. Using this as a target nucleic acid, an experiment similar to the above was performed.

The results are shown in FIG. The genotype was determined from the ratio of the resonance signal intensities obtained using the three probe nucleic acids (SEQ ID NOs: 29, 31, 41) using the above criteria, and obtained by the conventional method (DNA sequencing). When compared with the results, they all agreed.

Example 3: Determination of a single nucleotide polymorphism of apolipoprotein E (apo E) apo E is a protein that binds to lipid in plasma to become lipoprotein, and serves as a marker when lipoprotein is recognized by cells. is there. The apo E gene has three alleles (alleles) of ε2, ε3, and ε4 (isoforms of E2, E3, and E4 in protein). The difference between E2, E3, and E4 is the amino acid at positions 112 and 158. is there. In apo E3, the 112th is cysteine, but in apo E4, the 112th is arginine. Apo E4 is known as a risk factor for Alzheimer's disease, and “apo E4 homo” is 3 times more likely to be affected, and “apo E4 homo” and “ALDH2 is inactive” are 30 times more likely to suffer from Alzheimer's disease.

(1) Examination of optimum conditions using model system As target nucleic acids, the following SEQ ID NO: 57 and sequence comprising 57 bases including bases corresponding to the single nucleotide polymorphism in the 3 ′ → 5 ′ strand of the apo E gene A nucleic acid having the base sequence represented by any of No. 58 was synthesized. A probe nucleic acid having a base sequence represented by SEQ ID NOs: 59 to 62 (having a base corresponding to a single nucleotide polymorphism at the center), and a 3 ′ terminal block nucleic acid represented by SEQ ID NO: 63 Each having a base sequence (the gap length is 1) was designed and synthesized. Using these nucleic acids, the same operations as in (1) and (4) of Example 1 were performed, and the hybridization efficiency between the target nucleic acid and the probe nucleic acid was examined.

Any target nucleic acid hybridizes only to the probe nucleic acid having the base sequence represented by SEQ ID NO: 59 regardless of the presence or absence of the 3 ′ terminal block nucleic acid, and is significantly higher than the other probe nucleic acids. Non-base sequence hybridization that did not hybridize was observed. This is considered to be because the base length of the probe base sequence is too small, and thus the probe nucleic acid and the sequence having the base sequence represented by SEQ ID NOs: 64-67, in which the base length of the probe base sequence is increased to 15 A 3 ′ terminal block nucleic acid having the base sequence represented by No. 68 was synthesized. These probe nucleic acids, the target nucleic acids used above (SEQ ID NOs: 57 and 58, a 1: 1 mixture of both: corresponding to A / A type, G / G type and A / G type, respectively) and the 3 ′ end. A similar experiment was performed using a side block nucleic acid (SEQ ID NO: 68).

As a result of increasing the base length of the probe base sequence to 15, non-specific hybridization to the probe nucleic acid (SEQ ID NO: 64) whose base corresponding to the single nucleotide polymorphism is T was not observed. Regarding the target nucleic acid having the base sequence represented by No. 58, the result was observed that the hybridization efficiency with the probe nucleic acid having the base sequence represented by SEQ ID Nos. 65 to 67 hardly changed. Therefore, a 5 ′ terminal block nucleic acid having the base sequence represented by SEQ ID NO: 69 below was designed and synthesized, and the above target nucleic acid (SEQ ID NO: 57, 58), probe nucleic acid (SEQ ID NO: 64-67), and A similar experiment was performed using the 3 ′ terminal block nucleic acid (SEQ ID NO: 68).

The obtained result is shown in FIG. From this result, it is considered that the genotype can be determined based on the following criteria for the ratio of the resonance signal intensities observed for the four probe nucleic acids.
(1) A / A type if SEQ ID NO: 65> SEQ ID NO: 66 (2) G / G type if SEQ ID NO: 66 / SEQ ID NO: 65> 2 (3) If 1 ≦ SEQ ID NO: 66 / SEQ ID NO: 65 ≦ 2 A / G type

(2) Determination of single nucleotide polymorphism using DNA collected from test subject's hair DNA was extracted from human hair using DNA extraction kit ISOHAIR manufactured by Nippon Gene Co., Ltd. Using the extracted DNA as a template, a primer having the nucleotide sequence represented by SEQ ID NOs: 70 and 71 below (the former is on the 5 ′ end side and the latter is on the 3 ′ end side), and is 228 bp by asymmetric PCR. An amplification product was obtained. Using the obtained amplification product as a template, an asymmetric PCR method using primers having the nucleotide sequences represented by SEQ ID NOS: 72 and 73 below (the former is on the 5 ′ end side and the latter is on the 3 ′ end side) Gave a 125 bp amplification product. Using this as a target nucleic acid, an experiment similar to the above was performed.

The results are shown in FIG. Subjects 1 and 2 use three probe nucleic acids (SEQ ID NOs: 60, 61, and 62) showing the results of measuring twice for reproducibility confirmation of samples derived from the same subject. The genotype was determined from the ratio of resonance signal intensities obtained in the above manner using the above-mentioned criteria, and compared with the results obtained by the conventional method (DNA sequencing).

DESCRIPTION OF SYMBOLS 10 Single nucleotide polymorphism determination apparatus 11 Sample supply means 12 Reactor 13 Detection means 14 Data processing means 15 Control means

Claims (18)

A sample containing a target nucleic acid having a target base sequence to be hybridized is brought into contact with a probe nucleic acid having a probe base sequence complementary to the target base sequence, and the target nucleic acid and the probe nucleic acid in the sample are brought into contact with each other. In the method of specifically hybridizing,
5 'terminal block nucleic acid having a base sequence capable of hybridizing with part or all of the base sequence in the target nucleic acid located 5' end side of the target base sequence on the 5 'end side and / or the target A 3 'terminal block nucleic acid having a base sequence capable of hybridizing with a part or all of the base sequence in the target nucleic acid located 3' end side of the base sequence is contacted with the target nucleic acid. The target nucleic acid and the probe nucleic acid are specifically hybridized in a state where the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid is hybridized with the hybridizable base sequence in the target nucleic acid. A method for hybridizing nucleic acids, comprising making soybeans. Between the 3 ′ end of the target base sequence present in the target nucleic acid and the 5 ′ end of the base sequence capable of hybridizing with the 3 ′ end block nucleic acid and / or of the target base sequence 2. A gap of 1 to 15 bases exists between the 5 ′ terminal end and the 3 ′ terminal side of the base sequence capable of hybridizing with the 5 ′ terminal block nucleic acid. A method for hybridization of nucleic acids. The nucleic acid hybridization method according to any one of claims 1 and 2, wherein the probe nucleic acid is DNA. The nucleic acid hybridization method according to any one of claims 1 to 3, wherein the number of bases in the probe base sequence is 10 or more and 20 or less. A target nucleic acid having a target base sequence to be determined includes a probe base sequence complementary to each of a part or all of single nucleotide polymorphisms (SNPs) present in the target base sequence, and the different probe bases Those having sequences are brought into contact with a plurality of probe nucleic acids arranged in different predetermined regions on the inner wall surface of a single reactor, and complementary to the target base sequence of the target nucleic acid and the probe nucleic acid. A step A for specifically hybridizing with the probe having a probe base sequence;
A step B for detecting the specific hybridization of the target nucleic acid and the probe nucleic acid having the probe base sequence complementary to the target base sequence in a position-specific manner. How to determine polymorphism. 5 'terminal block nucleic acid having a base sequence on the 5' end side that can hybridize with part or all of the base sequence in the target nucleic acid located at the 5 'end side of the target base sequence in the step A And / or a 3 'terminal block nucleic acid having a base sequence on the 3' end side capable of hybridizing with part or all of the base sequence in the target nucleic acid located 3 'end side of the target base sequence The target nucleic acid and the probe nucleic acid are brought into contact with the target nucleic acid, and the 5 ′ terminal block nucleic acid and / or the 3 ′ terminal block nucleic acid is hybridized with the hybridizable base sequence in the target nucleic acid. 6. The method for determining a single nucleotide polymorphism according to claim 5, wherein The 3 ′ end side end of the target base sequence present in the target nucleic acid, the 5 ′ end side of the base sequence hybridizable with the 3 ′ end side block nucleic acid, and / or the 5 ′ end of the target base sequence The single base according to claim 6, wherein a gap of 1 to 15 bases exists between the side terminal and the 3 'terminal side of the base sequence capable of hybridizing with the 5' terminal block nucleic acid. How to determine polymorphism. The method for determining a single nucleotide polymorphism according to any one of claims 5 to 7, wherein the probe nucleic acid is DNA. The single nucleotide polymorphism determination method according to any one of claims 5 to 8, wherein the number of bases of the probe base sequence is 10 or more and 20 or less. In each of the probe nucleic acids, the position of the base corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence is more than 1/3 times the number of bases of the probe base sequence. The single nucleotide polymorphism determination method according to any one of claims 5 to 9, wherein the single nucleotide polymorphism is large. Specific hybridization between the target nucleic acid and the probe nucleic acid having a base sequence complementary to the target base sequence is detected by either surface plasmon resonance or abnormal reflection of gold. The method for determining a single nucleotide polymorphism according to any one of claims 5 to 10. One or both of a container filled with a solution having a target base sequence to be determined and a flow path for circulating the solution, and one single nucleotide polymorphism (SNP) present in the target base sequence A plurality of probe nucleic acids having a probe base sequence complementary to each of all or all of them have different probe base sequences on the inner wall surface of either one or both of the single container and the flow path A single nucleotide polymorphism determination reactor, which is disposed at predetermined positions different from each other. The single nucleotide polymorphism determination reactor according to claim 12, wherein the probe nucleic acid is DNA. The single nucleotide polymorphism determination reactor according to any one of claims 12 and 13, wherein the number of bases in the probe base sequence is 10 or more and 20 or less. In each of the probe nucleic acids, the position of the base corresponding to the single nucleotide polymorphism starting from the 5 ′ end and the 3 ′ end of the probe base sequence is more than 1/3 times the number of bases of the probe base sequence. The single nucleotide polymorphism determination reactor according to any one of claims 12 to 14, which is large. One or both of a container filled with a solution having a target base sequence to be determined and a flow path for circulating the solution, and one single nucleotide polymorphism (SNP) present in the target base sequence A plurality of probe nucleic acids having a probe base sequence complementary to each part or all of the nucleic acids having different probe base sequences arranged at different positions on the inner wall, respectively,
A detection means for detecting position-specifically specific hybridization between a target nucleic acid having the target base sequence and a probe nucleic acid having the probe base sequence complementary to the target base sequence. Characteristic single nucleotide polymorphism determination device. The single nucleotide polymorphism determining apparatus according to any one of claims 12 to 15, wherein the single nucleotide polymorphism determining reactor according to any one of claims 12 to 15 is used as the reactor. The single nucleotide polymorphism determination according to any one of claims 16 and 17, wherein the detection means detects specific hybridization by either surface plasmon resonance or abnormal reflection of gold. apparatus.