JP2008125439A - Method for detecting recurring base sequence, and detection kit for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting the number of recurrence of recurring sequences in many target nucleic acids, in a quick and simple manner, and to provide a detection kit for the same. <P>SOLUTION: This method for detecting recurring base sequences comprises a first process of amplifying the target nucleic acid, having the recurring base sequences by using a primer; a second process of hybridizing a complementary base chain with the recurring base sequences of the amplified nucleic acid sequences to form a recurring base sequence-complementary base chain complex; a third process of making the target nucleic acid hybridize with an immobilizing carrier, to immobilize the recurring base sequence-complementary base chain complex; and a fourth process of determining the amount of the hybridized complementary base chain from the immobilized recurring base sequence-complementary base chain complex. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting a repetitive base sequence.

  Differences in nucleic acid base sequences are caused by nucleic acid substitution, insertion, deletion or recombination. Among the differences in the nucleotide sequence of the nucleic acid, a mutation present at a frequency of 1% or more in a certain group is particularly called a gene polymorphism. Known genetic polymorphisms include SNPs in which one base is replaced with another base, VNTRs with several to several tens of repeat units, and microsatellite polymorphisms with 2 to 4 repeat units It has been.

  Genetic polymorphism is spread within a population with a certain frequency. For this reason, it has been attracting attention as a trait that influences the traits that can be said to be a constitution, not a trait change at all, or a trait that is not disadvantageous for survival (reproduction). For example, it is known that gene susceptibility to susceptibility to lifestyle diseases such as diabetes, hypertension and obesity, immune diseases such as rheumatism and allergies, and diseases such as cancer. In addition, drug metabolism (how the drug works) and human leukocyte histocompatibility antigens are considered to be governed by genetic polymorphism. Furthermore, since the repetition frequency of the repeating unit varies from person to person, an individual can be identified.

  In the vicinity of the insulin gene that controls the blood glucose level, there are VNTR sequences (repeating units: several types such as 5'-ACAggggTgTgggg-3 ') having a repeating unit of 14 bases. It is known that when the number of repeating units is large, the amount of insulin secreted is larger than when the number of repeating units is small. That is, the expression level of insulin is regulated by the number of repeating units of VTNR (see, for example, Non-Patent Document 1).

  In Huntington's disease, the number of repeats of the microsatellite polymorphism (repetition unit: CAg) present in the first gene of the short arm of chromosome 4 is abnormally large, 36 or more. This sequence encodes glutamine. This produces a long chain of glutamine, which inhibits the transcription mechanism and is thought to cause Huntington's disease.

  When detecting a certain type of nucleic acid (target nucleic acid) such as genetic diagnosis, identification of pathogenic bacteria, detection of genetic polymorphism, etc., a DNA chip or DNA microarray in which a large number of probe nucleic acids are immobilized on a substrate is used. Since oligo DNA can be easily synthesized by a DNA synthesizer, oligo DNA is mainly used as a probe nucleic acid. The probe nucleic acid immobilized on the substrate is mixed with the target nucleic acid and hybridized.

  Whether or not the probe nucleic acid and the target nucleic acid are hybridized is determined, for example, by detecting the target nucleic acid using a label or the like. As the label, a fluorescent label or RI (radioactive isotopic) is used. Known detection methods that do not use a labeling body include methods using a crystal resonator method (QCM), a surface plasmon resonance method (SPR method), and an ellipsometer (see, for example, Patent Documents 1 to 3). In these methods, the target nucleic acid immobilized on the substrate is quantified.

  In addition, an attempt has been made to measure SNPs by measuring the redox potential difference before and after the target nucleic acid is hybridized to the probe by an electrochemical technique (see, for example, Non-Patent Document 2).

On the other hand, as a method for detecting a repetitive base sequence, a method is known in which a repetitive base sequence in a genome is detected using a labeled probe (see, for example, Patent Documents 4 and 5). In Patent Document 4, a telomere size is measured by measuring a label signal of a hybridized DNA probe. Further, in Patent Document 5, the repetitive sequence length is detected by irradiating the labeled probe hybridized with the target nucleic acid with excitation light and detecting the fluctuation of the fluorescence of the labeled probe.
John Dee. H. St. D. H. Stead, et al., "Structural analysis of insulin minisatellite allele reveals unusually large differences between Africans and non-Africans (Structural Analysis of Insulin Minisellelite) Alles Reveals Unusual Large Differences in Diversity between Africans and Non-Africans, American Journal of Human Genetics (American Journal of Human Genetics, American Journal of Human Genetics) Human Gene ics), 2002 years, the first Vol. 71, p. 1273-1284 Hea Yon Lee, et al., “SNPs Feasibility of Nonlabeled Oligonucleids on US, ElectroSensics, Electrochemical Sensing” Willy-VCH, 2004, Vol. 16, No. 23, p. 1999-2002 JP-A-7-72060 JP 2002-51774 A JP 2002-189028 A JP 2001-95586 A JP 2005-237334 A

  In Patent Documents 1 to 3 and Non-Patent Document 2, the amount of hybridization between the probe nucleic acid and the target nucleic acid is measured. Therefore, even if the methods described in Patent Documents 1 to 3 and Non-Patent Document 2 are directly used, it is not possible to detect a repeated base sequence.

  On the other hand, in the inventions described in Patent Documents 4 and 5, the fluctuation of the labeled molecule is measured in the solution. That is, since one sample is measured in one reaction system, it is not easy to measure many samples simultaneously. In addition, currently used methods for detecting repeated polymorphisms are mainly detected using Southern hybridization after amplification of the repeated sites by PCR or the like. This method requires a time of 1 to 2 days and has a problem that a quick diagnosis cannot be performed.

  Furthermore, it is not easy to examine a large number of genetic polymorphisms simultaneously with conventional detection methods. In particular, SNPs and repeated polymorphisms are closely related. Accordingly, simultaneous diagnosis of these is required for more accurate genetic diagnosis.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a method and a detection kit for quickly and easily detecting the number of repetitive base sequences in a large number of target nucleic acids.

  Another object of the present invention is to provide a method and a detection kit for simultaneously detecting SNPs and repeated polymorphisms.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have amplified a repetitive base sequence using a primer and combined a complementary base chain that complementarily binds to this repetitive base sequence (composite) And the number of repeats of the repetitive base sequence can be detected quickly and easily from a large number of target nucleic acids, thereby completing the present invention. That is, the present invention is as follows.

  In the method for detecting a repetitive base sequence of the present invention, a first step of amplifying a target nucleic acid having a repetitive base sequence using a primer and a base chain complementary to the repetitive base sequence of the amplified target nucleic acid are hybridized. And a second step of forming a repetitive base sequence-complementary base chain complex, the tag sequence and an immobilization carrier complementary to the tag sequence are hybridized, and the repetitive base sequence-complementary base A third step of immobilizing the chain complex, and a fourth step of quantifying the amount of complementary base chain hybridized from the immobilized repetitive base sequence-complementary base chain complex.

  The primer may have a tag sequence. In addition, it is preferable that the complementary base chain includes a label, and the amount of the complex is measured by measuring the signal intensity of the label.

  In the method, it is possible to simultaneously detect a repeated nucleotide sequence and a single nucleotide polymorphism.

  The immobilization carrier may be a non-natural nucleic acid.

  The repetitive base sequence detection kit of the present invention comprises a primer for amplifying a target nucleic acid having a repetitive base sequence, a complementary base chain that complementarily binds to the repetitive base sequence, and immobilization complementary to the tag sequence. A carrier.

  The primer may have a tag sequence. The immobilization carrier may be fixed on a substrate. The detection kit of the present invention preferably further contains a label.

  In the present invention, the number of repetitive base sequences is detected by immobilizing a compound having a complementary base chain bonded to a repetitive site and quantifying the combined complementary base chain. As a result, it is possible to provide a method and a detection kit for detecting quickly and easily.

  The method for detecting a repetitive base sequence of the present invention comprises (1) a target nucleic acid having a repetitive base sequence is amplified using a primer, and (2) a base chain complementary to the repetitive base sequence of the amplified target nucleic acid is hybridized. (3) hybridizing the target nucleic acid to an immobilization carrier and immobilizing the repetitive base sequence-complementary base chain complex (4) ) The amount of the complementary base chain hybridized from the immobilized repetitive base sequence-complementary base chain complex is quantified. This will be specifically described below.

[Repetitive nucleotide sequence]
The repetitive base sequences to be detected in the present invention include VNTR having a repetitive unit of several to tens of bases, microsatellite polymorphism having a repetitive unit of 2 to 4 bases, and the like existing in the genome.

[Primer]
As shown in FIG. 1, the target nucleic acid having the repetitive base sequence is amplified from the target site 2 in the PCR product using the primer 1 (base extension portion 3). The target nucleic acid (nucleic acid 4 with extended base) is usually amplified using the polymerase chain reaction (PCR). The primer 1 to be used is not particularly limited as long as it can amplify a repetitive base sequence to be detected. As described below, a primer having a tag sequence 5 can also be used. Further, a primer having a tag sequence may be used as a forward primer, and another primer may be used as a reverse primer.

[Complementary base strand]
The complementary base chain used in the present invention refers to a nucleic acid aggregate that can hybridize with a repetitive base sequence in a target nucleic acid to form a double strand. As shown in FIG. 2, the complementary base chain 8 binds to the repetitive base sequence 7 in the target nucleic acid 6 to form a repetitive base sequence-complementary base chain complex. The complementary base chain may be any one that hybridizes with all or part of the repetitive base sequence. The length of the complementary base chain is not particularly limited, but is preferably a nucleic acid aggregate having 5 or more bases. This is because the duplex stability is improved. The nucleic acid assembly may be an assembly of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), or the like.

[Immobilization of repetitive base sequence-complementary base chain complex]
The immobilization method is not particularly limited, and may be immobilized using an immobilization carrier on which a part of either the target nucleic acid or the complementary base chain is hybridized. Immobilization can be performed using, for example, a sequence between repeated base sequences of the amplified target nucleic acid or a sequence at the end of the target nucleic acid. Alternatively, a complementary base chain shorter than the repetitive base sequence may be used to form a duplex, and immobilization may be performed using a portion that did not form a duplex. For example, a primer having a tag sequence as shown in FIG. 1 is used. In the example shown in FIG. 3, a tag sequence of a primer having a tag sequence in which a complementary base chain 8 is bound to a repetitive base sequence 7 of a target nucleic acid 6 and an immobilization carrier 9 are hybridized. In the example of this figure, a label 10 is added to the complementary base chain 8.

  The tag sequence of the primer having the tag sequence hybridizes with the immobilization carrier. The tag sequence may be included in a part of the primer. The tag sequence may be added to the primer in advance, or may be added after amplifying the target nucleic acid. Thus, when the primer which has a tag sequence is used, the amplified target nucleic acid can be fix | immobilized easily.

  The immobilization carrier only needs to be immobilized on a base material such as a substrate or beads. When a repetitive base sequence-complementary base chain complex is immobilized on a substrate, a plurality of complexes can be selectively immobilized. As a result, it is possible to process a large number of samples, which is preferable.

  The immobilization carrier may be a nucleic acid. This is because a nucleic acid can complementarily bind to a part (including a tag sequence) of either the target nucleic acid or the complementary base strand. The immobilization carrier and part of the target nucleic acid or complementary base strand (including the tag sequence) may be a natural nucleic acid or a non-natural nucleic acid. A natural nucleic acid refers to a nucleic acid in which all the sugars contained in the nucleic acid are in D form. Non-natural nucleic acid means a nucleic acid that does not exist in nature, and refers to an L-type nucleic acid in which at least one of the sugars contained in the nucleic acid is L-form. When a non-natural nucleic acid is used, highly selective hybridization is possible.

  When a nucleic acid having a natural D-type sugar is used in an immobilization carrier and a sequence (including a tag sequence) that complementarily binds to the immobilization carrier, a portion other than the sequence to be examined causes hybridization (cross-hybridization). There is. For this reason, when analysis is performed using a DNA chip or the like, great care has been required to determine the sequence of the immobilized nucleic acid. The D-form sugar and the L-form sugar are in an enantiomeric relationship with each other. For this reason, a D-type nucleic acid having a D-form sugar and an L-type nucleic acid having an L-form sugar do not form a double strand by hybridization even when the complementarity of the base sequences is high. Therefore, when an L-type nucleic acid is used for the immobilization carrier and a sequence (including a tag sequence) that complementarily binds to the immobilization carrier, the target nucleic acid generated in the genome or by PCR other than the sequence portion that complementarily binds to the immobilization carrier The D-type part of does not hybridize. On the other hand, an immobilization carrier that is an L-type nucleic acid and a sequence (including a tag sequence) that complementarily binds to the immobilization carrier are hybridized. As described above, when an L-type nucleic acid is used as an immobilization carrier and a sequence (including a tag sequence) that complementarily binds to the immobilization carrier, sequencing of the immobilized target nucleic acid becomes very easy.

  The introduction of the L-type nucleic acid is performed, for example, by amplifying a target portion in the genome using a primer having a tag sequence of the L-type nucleic acid.

  The method for supporting the immobilization carrier on the substrate is not particularly limited, and for example, the following known methods can be used. For example, DNA is fixed at a high density by spotting DNA on a nylon sheet or the like using a device called a spotter. Alternatively, as described in JP-A-1-505144, oligonucleic acid can be spotted on a flat substrate such as a slide glass using a device called a spotter. Further, as described in Japanese Patent Application Laid-Open No. 2000-508542 and the like, oligonucleic acid may be synthesized on a chip based on a technique used for semiconductor production called photolithography. Furthermore, as described in JP-A-2005-233703, etc., a functional group is generated on the surface of a polymer having a specific structure, and a terminal amino nucleic acid is immobilized by covalent bonding via this functional group. It is good.

[Quantification of complementary base chain amount]
The amount of hybridized complementary base chain is determined by (1) introducing a label and measuring the signal intensity of the label, (2) fixing the repeated base sequence-complementary base chain complex to the solid surface, It can be quantified by measuring the amount of immobilization.

(When using a label)
In the present invention, as a label that can be used, a known substance used for labeling such as a fluorescent dye, a phosphorescent dye, and a radioisotope can be used. Preference is given to fluorescent dyes that are easy to measure and easy to detect signals. Specifically, cyanine (cyanine 2), aminomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indocarbocyanine (cyanine 5), cyanine 5.5, well-known fluorescent dyes, such as cyanine 7, are mentioned. A fluorescent microscope or a fluorescent scanner is used to detect the fluorescent dye. Moreover, you may use the semiconductor fine particle which has luminescent property as a label | marker. Examples of such semiconductor fine particles include cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), silver indium zinc sulfide (AgInZnS), and the like.

  The labeled body may be bonded to a part of the complementary base chain in advance and repeatedly combined with the base sequence, or the labeled body may be introduced after the repeated base sequence-complementary base chain complex is formed. Good. In the former case, the end of the complementary base chain or a label bound to the chain is used. Examples of a method for introducing a label after the formation of a repetitive base sequence-complementary base chain complex include the following methods.

  (1) A label is introduced by an enzymatic method into the terminal part of the complementary base chain of the repetitive base sequence-complementary base chain complex. Specifically, the label is introduced using a base extension reaction, a base ligation reaction, a terminal deoxynucleotidyl transferase reaction that selectively labels the 3 'end of a nucleic acid, and the like.

  (2) A label is introduced into the double-stranded portion using an intercalator that specifically interacts with the double-stranded nucleic acid. An intercalator refers to a compound in which a planar structure portion in a compound can be inserted into a diffusing base pair in a double chain. Specific examples include acridine orange, ethidium bromide, anthracene, pyrene, naphthalene, tetracene, fluorene, and derivatives thereof.

  (3) A reactive functional group is introduced into a complementary base chain that is repeatedly bonded to the base sequence, and the reactive functional group and the labeled body are chemically bonded to introduce the labeled body. Examples of reactive functional groups include amino groups, carboxyl groups, thiol groups, cyano groups, hydroxyl groups, succinimide groups and derivatives thereof, maleimide groups and derivatives thereof.

  (4) A substance that selectively binds to the label is introduced into the repeated base sequence-complementary base chain complex, and the label is bound using the selective binding property. Substances that bind selectively include nucleic acid / nucleic acid, nucleic acid / protein, low molecular weight compound / protein (eg, biotin-avidin, glucocorticoid-glucocorticoid receptor, etc.), sugar chain / sugar chain, sugar chain / Substances that bind selectively between proteins.

(When measuring immobilization amount)
The amount of adsorption to the solid substrate is determined by immobilizing complementary strand nucleic acid on the solid surface, and using the SPR method, QCM method, ellipsometry method, infrared spectroscopy, X-ray photoelectron spectroscopy (XPS) method, etc. Just measure.

  By using the detection method of the present invention, the number of repetitive base sequences can be rapidly and easily detected from the target nucleic acid. The repetitive base sequence is not particularly limited as long as it has a repetitive base sequence. For example, a repetitive base sequence relating to insulin expression regulation can be detected. There are several types of VNTR sequences present in the vicinity of the insulin gene, such as the repeating unit: 5'-ACAggggTgTgggg-3 '. In this case, if different complementary base chains are used corresponding to the repetitive base sequences, the complementary base chains are labeled using different labeling bodies, and the VNTR sequence is detected, the repeats of different types of repetitive base sequences can be performed simultaneously. The number can be detected. According to the present invention, even when the target nucleic acid has a repetitive base sequence that is a different type of repetitive base sequence, the number of repeats of the repetitive base sequence can be detected quickly and easily.

[Detection of SNPs]
By using the present invention, it is possible to detect the number of repeated bases and measure SNPs on a single substrate. That is, according to the present invention, a plurality of amplifications can be sequentially performed using a plurality of primers with a small amount of sample. As a result, it is possible to repeatedly detect a base sequence and measure SNPs on one substrate. Moreover, when a repetitive base sequence and SNPs are present in the vicinity, the number of repetitive base sequences and SNPs can be detected by one amplification operation with one type of target nucleic acid. SNPs can be detected, for example, by detecting a single base mismatch contained in the repetitive nucleotide sequence by using the SPR method (Alexander W. Peterson). , Two others, “Hybridization of Mismatched or Partially Matched DNA at Surfaces”, Journal of American Chemical Society USA, Journal of American Chemical Society, USA Chemical Society), 2002, 124, p.14. 601-14607). Alternatively, the nucleotide sequence is determined by the base extension method and SNPs are measured (Tomi Pastinen) and four others, “Mini-sequencing: a special tool for DNA analysis and diagnosis on oligonucleotide arrays (Minisequencing: A Specific Tool for DNA Analysis and Diagnostics on Volume 7 p., P. 197, Gen. Research, 19 p. 614).

[Detection kit]
The repetitive base sequence detection kit of the present invention comprises a primer for amplifying a target nucleic acid having a repetitive base sequence, a complementary base chain that complementarily binds to the repetitive base sequence, and immobilization complementary to the tag sequence. And a carrier. The primer may have a tag sequence. In addition, the immobilization carrier may be a detection kit that is already immobilized on a substrate. Furthermore, if the detection kit of the present invention contains a label, the amount of the complex can be measured using the label. By using such a kit for detecting a repeated base sequence, the number of repeated base sequences can be easily detected.

  EXAMPLES Examples will be given below to describe the present invention in detail, but the present invention is not limited to the examples.

In the following Examples, synthesis of DNA containing L-type nucleic acid was performed using a DNA / RNA DNA automatic synthesizer (Applied Biosystems). In addition, as L-type nucleic acids, Beta-L-deoxy Adenosine (n-bz) CED phosphoramidite, Beta-L-deoxy Cytidine
(n-bz) CED phosphoramidite, Beta-L-deoxy Guanosine (n-ibu)
CED phosphoramidite, Beta-L-deoxy Thmidine CED phosphoramidite (manufactured by ChemGenes Corporation) as a D-type nucleic acid
Adenosine (n-bz) CED phosphoramidite, deoxy Cytidine (n-bz) CED phosphoramidite, deoxy
Guanosine (n-ibu) CED phosphoramidite and Thymidine CED phosphoramidite (manufactured by ChemGenes Corporation) were used. Synthesis of DNA containing no L-type nucleic acid was performed by Operon Biotechnology Co., Ltd.

(Example 1)
[Detect number of repetitions]
(Creation of a target nucleic acid model having a repetitive base sequence having a tag sequence and a complementary base chain)
A target nucleic acid DNA (SEQ ID NO: 1 to 4) and a nucleic acid DNA (SEQ ID NO: 5) having a complementary base chain shown in Table 1 below were synthesized.

These nucleic acids are D-type nucleic acids. In the table, in the nucleic acid of each SEQ ID NO., The underlined portion indicates a repetitive base sequence and forms a base pair with the complementary base chain of SEQ ID NO: 5. The target nucleic acid DNAs of SEQ ID NOs: 1 to 4 have 1 to 4 repeated base sequences, respectively. That is, the target nucleic acid DNAs of SEQ ID NOs: 1 to 4 have the same sequence as VNTR existing in the vicinity of the insulin gene. Further, in the target nucleic acid DNAs of SEQ ID NOs: 1 to 4, the sequence surrounded by a square indicates a tag sequence. The 5 ′ end of the nucleic acid DNA of SEQ ID NO: 5 is labeled with cyanine 3 (Cy3).

(Repetitive base sequence-complementary base chain complex formation)
10 nmol of the target nucleic acid DNAs of SEQ ID NOs: 1 to 4 were each dissolved in 100 μL of pure water. 2 μL (0.2 nmol) was taken from each of these four types of DNA aqueous solutions and mixed.

98.2 nmol of the DNA of SEQ ID NO: 5 was dissolved in 98.2 μL of pure water. 6 μL (6 nmol) of this aqueous solution and the above mixture were mixed, and 5 × SSC solution (citrate buffer) (0.075 mol / L sodium citrate + 0.75 mol / L sodium chloride, 20 × SSC (manufactured by Sigma) ) Was diluted 4 times) to obtain a diluted solution having a total liquid volume of 80 μL. This diluted solution was heated and cooled for 2 cycles with a thermal cycler (BIO-RAD, trade name: MYCYCLER) under the following conditions. By this operation, a repeated base sequence-complementary base chain complex was formed.
94 ° C (1 minute) → 80 ° C (1 minute) → 70 ° C (1 minute) → 60 ° C (1 minute) → 50 ° C (1 minute) → 40 ° C (1 minute) → 30 ° C (1 minute) → 4 ℃

(Preparation of DNA immobilization carrier)
A polymethyl methacrylate (PMMA) substrate (Cosmo glass extruded plate, thickness: 1 mm, manufactured by Kuraray Co., Ltd.) was immersed in a 10N sodium hydroxide aqueous solution at a temperature of 65 ° C. for 12 hours. The PMMA substrate was washed with pure water, 0.1N hydrochloric acid aqueous solution, and pure water in this order.

D-type DNAs of SEQ ID NOs: 6 to 10 shown in Table 2 below were synthesized and used as immobilization carriers. Each of the D-type DNAs of SEQ ID NOs: 6 to 10 is aminated at the 5 ′ end. Further, the DNA of SEQ ID NO: 10 differs from the DNA of SEQ ID NO: 6 by one base in the underlined portion (single base mismatch).

Each of the DNAs of SEQ ID NOs: 6 to 10 was dissolved in pure water at a concentration of 0.3 nmol / μL to obtain a stock solution. To these DNA aqueous solutions, a phosphate buffer solution (PBS) was added so that the final concentration of immobilized DNA was 0.03 nmol / μL when spotted on the substrate. As PBS, NaCl: 8 g, Na ₂ HPO ₄ · 12H ₂ O: 2.9 g, KCl: 0.2 g, KH ₂ PO ₄ : 0.2 g were dissolved in pure water, and the volume was adjusted to 1 L. For adjustment, hydrochloric acid was added to adjust the pH to 5.5. In addition, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to condense the carboxylic acid on the substrate surface with the terminal amino acid of the immobilized DNA. The final concentration of EDC was 50 mg / mL.

  The mixed solution containing each of the DNAs of SEQ ID NOs: 6 to 9 was spotted on the upper surface of the PMMA substrate using an arrayer (manufactured by Nippon Laser Electronics Co., Ltd .: Gene Stamp-II). The number of spots was 24 in total, 6 points each for DNAs of SEQ ID NOs: 6-9. Next, this substrate was placed in a sealed plastic container and incubated at 37 ° C. and humidity of 100% for about 20 hours. Thereafter, the substrate was washed with pure water to obtain a substrate carrying an immobilization carrier.

(Adjustment of hybridization sample reagent)
Hybridization between the repetitive base sequence-complementary base chain complex and the above-described immobilization carrier was performed as follows. The part enclosed by the square of DNA of sequence number 1-4 is hybridized with sequence number 6-9, respectively, and a bond is formed. Moreover, the SSC solution used below means a solution obtained by diluting 15 × SSC: 20 × SSC (manufactured by Sigma) to 3/4 with pure water.

  An aqueous solution containing 30 mg / mL BSA (bovine serum albumin), 15 × SSC, 0.3 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA (each of the above concentrations is a final concentration). A stock solution was obtained. The repetitive base sequence-complementary base chain complex (hereinafter referred to as “sample nucleic acid”) diluted with pure water and this stock solution were mixed at a ratio of 2: 1 (volume ratio). The sample nucleic acid was diluted with pure water after the sample nucleic acid diluent and the stock solution were mixed and adjusted so that the final concentration of the sample nucleic acid was 3.75 nM.

(Immobilization to immobilization carrier)
20 μL of the hybridization sample solution was placed on a substrate on which a nucleic acid was immobilized as the immobilization carrier, and covered with a cover glass. In order to prevent the hybridization sample solution from drying, the periphery of the cover glass was sealed with a paper bond. The substrate was incubated for 16 hours in a 42 ° C. oven. After incubation, the substrate was removed from the oven, the cover glass was peeled off, and then washed and dried.

(Measurement of complex amount)
The substrate was set on a DNA chip scanner (Axon Instruments, GenePix 4000B), and the signal intensity was measured. Measurement conditions were set such that the laser output was 33% and the sensitivity of the photomultiplier was 500 (maximum value: 1000). Here, the signal intensity means an average value of six spots obtained by dividing the fluorescence intensity in each spot by noise. The results are shown in Table 3.

  Table 3 shows the signal intensities of the repetitive base sequences having 1 to 4 repeats (corresponding to SEQ ID NOs: 4 to 1, respectively). From this table, it can be seen that the signal intensity increases as the number of repetitions increases.

[Detection of single base mismatch]
Next, signal intensity was measured in the same manner as described above except that the nucleic acid of SEQ ID NO: 10 in Table 2 was used as an immobilization carrier. The results are shown in Table 3. From Table 3, it can be seen that when the DNA used as the immobilization carrier is different by one base, the fluorescence intensity is reduced by about 30%. From this, it was confirmed that it is possible to detect a target nucleic acid having a single base mismatch simultaneously with the detection of a repeated base sequence by using the present invention. That is, it was shown that it is possible to detect the number of repeats of SNPs and repetitive base sequences on the same chip substrate.

(Example 2)
[Non-natural nucleic acid immobilization carrier]
(Preparation of L-type nucleic acid solidified carrier)
L-type DNA of SEQ ID NO: 11 shown in Table 4 below was synthesized as an immobilized nucleic acid. The nucleic acid of SEQ ID NO: 6 and the nucleic acid of SEQ ID NO: 11 have the same base sequence. However, the nucleic acid of SEQ ID NO: 6 has a D-type sugar chain part, and the nucleic acid of SEQ ID NO: 11 has a D-type sugar chain part. . The nucleic acid of SEQ ID NO: 6 and the nucleic acid of SEQ ID NO: 11 were immobilized on the same substrate in the same manner as in Example 1.

(Preparation of sample nucleic acid)
A target nucleic acid of SEQ ID NO: 12 shown in Table 5 below was prepared using the L-type nucleic acid and the D-type nucleic acid. In this target nucleic acid, only the portion surrounded by a square corresponding to the tag sequence portion is an L-type nucleic acid, and the other portion is a D-type nucleic acid. This target nucleic acid of SEQ ID NO: 12 corresponds to the target nucleic acid of SEQ ID NO: 1.

The amount of complex was measured in the same manner as in Example 1 except that the nucleic acid of SEQ ID NO: 5 was used as the complementary strand base, and the substrate on which the nucleic acid of SEQ ID NO: 6 and the nucleic acid of SEQ ID NO: 11 were immobilized was used. did. The results are shown in Table 6. In addition, the data of the D-type nucleic acid in Table 6 are those in which the number of repeating base sequences in Example 1 is 4.

  From this table, it was found that the same signal intensity was obtained when either D-type nucleic acid or L-type nucleic acid was used for the immobilization carrier and tag sequence. That is, it was found that both the D-type nucleic acid and the L-type nucleic acid have the same hybridization ability. Therefore, it was found that a non-natural nucleic acid can be used as the immobilization carrier.

(Example 3)
[Detection of VNTR sequences in samples extracted from cells]
(Amplification by PCR of a region containing VNTR sequences in a sample extracted from cells)
As samples, HEK293 (derived from human embryonic kidney cells) and HL60 (derived from human promyelocytic leukemia cells) were used. As primers, DNAs shown in Table 7 below were synthesized and used. In the DNA of SEQ ID NO: 13 and the DNA of 14, 20 bases on the 5 ′ end side have a nucleic acid tag. This nucleic acid tag binds complementarily to the DNA of SEQ ID NOs: 7 and 9, respectively. In the following amplification reaction, the remaining 20 bases of the DNA of SEQ ID NO: 13 (HEK293) and 14 DNA (HL60) were used as a forward primer, and the DNA of SEQ ID NO: 15 was used as a reverse primer.

  Culture with the nucleic acid extraction kit “QIAmp” (Qiagen Co., Ltd.) using the remaining 20 bases of DNA of SEQ ID NO: 13 (HEK293) and DNA of 14 (HL60) as a forward primer and DNA of SEQ ID NO: 15 as a reverse primer. From a human genomic DNA sample extracted from cells (HEK293 and HL60), a VNTR portion present approximately 600 base pairs upstream of the insulin gene was amplified by PCR. For PCR, KOD-Plus-DNA Polymerase (Toyobo) was used, and 35 cycles of amplification reaction were performed under the conditions specified by the manufacturer. Then, it refine | purified by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bioscience Co., Ltd.), and the excess dNTPs was removed.

PCR samples were prepared as follows. That is, 30 μM forward primer 1 μl, 30 μM reverse primer (SEQ ID NO: 15) 1 μl, 10 × PCR buffer for KOD-Plus-10 μl, 2 mM dNTPs
10 μl, 25 mM MgSO _{4 4} μl, KOD-Plus-DNA Polymerase 2 units, and 450 ng of extracted DNA solution were purely made up to 100 μl.

  PCR was performed at 94 ° C. for 2 minutes, 35 cycles of 94 ° C./30 seconds / 55 ° C./30 seconds / 68 ° C./30 seconds, and stored at 4 ° C. after the reaction.

  The length of the amplified portion after the PCR reaction was evaluated by electrophoresis. The electrophoresis conditions were as follows: 1.5% agarose gel, 9.7 g of 2 × TAE (Tris (2-amino-2- (hydroxymethyl) -1,3-propanediol)), acetic acid (glacial acetic acid); Using a developing solution prepared by dissolving 3 ml of EDTA (ethylenediaminetetraacetic acid) · 2Na 0.3 g in 1 L of pure water, electrophoresis was performed at 100 V for 30 minutes. After electrophoresis, the nucleic acid was stained with ethidium bromide. The lengths of the HEK293-derived nucleic acid and HL60-derived nucleic acid portions were 1313 and 1557 base pairs, respectively. From this, it was possible to estimate the number of repetitions as 40 and 50, respectively.

  In the same manner as described above, the amplified DNA was repeatedly formed into a base sequence-complementary base chain complex using the base of SEQ ID NO: 5 as a complementary base chain.

Next, the nucleic acids of SEQ ID NOs: 7 and 9 were immobilized on a PMMA substrate as a solidified carrier in the same manner as in Example 1. The repetitive base sequence-complementary base chain complex obtained above was immobilized on this immobilization carrier. In the same manner as in Example 1, the signal intensity of the repeated base sequence-complementary base chain complex was measured. The results are shown in Table 8.

  From this result, a result indicating a stronger signal was obtained when HL60 was used than when a PCR product amplified from HEK293 was used. Their intensity ratio was 1: 1.35. Since the number of repeats in the target nucleic acid collected from HEK293 and HL60 is 40 and 50, respectively, the ratio of the total length of the repeats is 1: 1.25. Since the total length of the repeated portion increases as the number of repetitions increases, it was shown that the signal intensity increased according to the number of repetitions even when using a target nucleic acid collected from cells.

FIG. 1 is a conceptual diagram for explaining a method for preparing a target nucleic acid using a PCR product. FIG. 2 is a conceptual diagram illustrating the mechanism by which a complementary nucleic acid strand binds to a repetitive base sequence in a target nucleic acid. FIG. 3 is a conceptual diagram illustrating a mechanism for quantifying a complementary nucleic acid chain using a nucleic acid-immobilized carrier.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Primer 2 Target site | part in PCR product 3 Base extension part 4 Base extension nucleic acid 5 Tag sequence 6 Target nucleic acid 7 Repetitive base sequence 8 Complementary base chain which couple | bonds complementarily with a repeat base sequence 9 Immobilization support | carrier 10 Label body

Claims (9)

A first step of amplifying a target nucleic acid having a repetitive base sequence using a primer;
A second step of hybridizing a base chain complementary to the repetitive base sequence of the amplified target nucleic acid to form a repetitive base sequence-complementary base chain complex;
A third step of hybridizing the target nucleic acid to an immobilization carrier and immobilizing the repetitive base sequence-complementary base chain complex;
And a fourth step of quantifying the amount of complementary base chain hybridized from the immobilized repetitive base sequence-complementary base chain complex.   The method for detecting a repetitive base sequence according to claim 1, wherein the primer has a tag sequence. The complementary base strand includes a label,
The method for detecting a repetitive base sequence according to claim 1 or 2, wherein the amount of the complex is measured by measuring the signal intensity of the label.   The method for detecting a repetitive base sequence according to any one of claims 1 to 3, wherein the detection of the repetitive base sequence and the detection of a single nucleotide polymorphism are simultaneously performed.   The method for detecting a repetitive base sequence according to any one of claims 1 to 4, wherein the immobilization carrier is a non-natural nucleic acid. A primer for amplifying a target nucleic acid having a repetitive base sequence;
A complementary base chain that complementarily binds to a repetitive base sequence;
A kit for detecting a repetitive base sequence, comprising the tag sequence and an immobilization carrier complementary thereto.   The repetitive base sequence detection kit according to claim 6, wherein the primer has a tag sequence.   The repetitive base sequence detection kit according to claim 6 or 7, wherein the immobilization carrier is immobilized on a substrate.   The kit for detecting a repeated base sequence according to any one of claims 6 to 8, further comprising a label.