JP2008193984A - Method for counting sequence number of repeating base sequence, and sequence number counting kit for repeating base sequence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and quantitatively counting the repetition number of a repeating base sequence from a number of target nucleic acids at the same time, and to provide a counting kit therefor. <P>SOLUTION: The method for counting the repetition number of a repeating base sequence includes a first step of introducing a label to each of a base sequence, in the repeating base sequence and a base sequence from among the repeating base sequence; and a second step of determining the quantities of the introduced labels from the labeled base sequence in the repeating base sequence and the labeled base sequence out of the repeating base sequence and determining the ratio of the labeled quantities of the labels, introduced into the base sequence in the repeating base sequence and the labels, introduced into the base sequence out of the repeating base sequence. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for measuring the number of sequences of repeated base sequences and a kit for measuring the number of sequences of repeated base sequences.

  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. A gene polymorphism is a single nucleotide polymorphism (hereinafter referred to as “SNP”) in which one base is replaced with another base, a microsatellite polymorphism having 2 to 4 repeat units, and a number of repeat units. A VNTR (variable nucleotide tandem repeat) polymorphism of several tens of bases to a base is known.

  Genetic polymorphism is spread within a population with a certain frequency. For this reason, it does not adversely affect survival (reproduction), but has attracted attention as a factor that affects the constitution without any changes in traits. 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 Huntington's disease, the number of repeats of the microsatellite polymorphism (repeating 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. In addition, there are VNTR polymorphisms (repeating units: several types such as 5'-ACAggggTgTgggg-3 ') having a repeating unit of 14 bases in the vicinity of the insulin gene that controls blood glucose level. 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).

As a method for detecting such a repetitive base sequence, a target nucleic acid containing a repetitive base sequence existing in the genome is amplified from the genome, repeatedly detected using electrophoresis, and the number of repeats is measured from the sequence length. The method is known. Or the method of detecting the repetitive base sequence in a genome using the labeled probe is known (for example, refer patent document 1, 2). In Patent Document 1, a telomere size is measured by measuring a labeling signal of a hybridized DNA probe. Further, in Patent Document 2, 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 JP 2001-95586 A JP 2005-237334 A

  In these conventional methods for detecting repetitive base sequences (VNTR and microsatellite), a repetitive region is amplified and then detected by Southern hybridization or the like. For this reason, since detection time takes 1-2 days, there exists a problem that a quick detection cannot be performed.

  In the methods described in Patent Documents 1 and 2, the labeled molecule is measured in a solution. For this reason, it is necessary to measure one sample in one reaction system, and it is not easy to measure many samples simultaneously. In addition, multiple items cannot be detected using one specimen.

  In recent years, the DNA chip method has been frequently used to detect multiple specimens and multiple items in genetic diagnosis and identification of pathogenic bacteria. In this method, a DNA chip having a large number of probe nucleic acids immobilized on the same carrier is used. Each immobilized probe nucleic acid selectively hybridizes with a multi-item target nucleic acid. Thereby, a plurality of target nucleic acids can be detected. However, the DNA chip has low quantitativeness. For this reason, there exists a problem that it is not suitable for the detection by which the high quantitative property is calculated | required, such as calculating | requiring the number of repetitions of a repetitive base sequence.

  Furthermore, it is not easy to examine a large number of genetic polymorphisms simultaneously with conventional detection methods. In particular, SNPs and repetitive base sequences 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 thereof is to provide a method and a detection kit for rapidly and quantitatively detecting the number of repetitive base sequence repeats from a plurality 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 problems, the present inventors have hybridized a base sequence chain complementary to a repetitive sequence unit, and then labeled the nucleic acid in the repetitive sequence unit and repeated it as an internal standard. It was found that nucleic acids outside the base sequence were labeled, the amount of labeling was measured, and the number of repeats of the repetitive base sequence was detected rapidly and quantitatively, and the present invention was completed. That is, the present invention is as follows.

  The method for measuring the number of repetitions of a repetitive base sequence according to the present invention includes a first step of introducing a label into each of a base sequence within a repetitive base sequence and a base sequence outside the repetitive base sequence, and the labeled repetitive base The amount of label introduced into each of the base sequence within the sequence and the base sequence outside the labeled repeat base sequence is measured, and the label and repeat introduced into the base sequence within the repeat base sequence from the measured label amount And a second step of determining the ratio of the amount of label introduced into the base sequence outside the base sequence.

  Before the first step, including a step of hybridizing a base sequence complementary to a repetitive sequence unit of a target nucleic acid having a repetitive base sequence to form a repetitive sequence unit-complementary base chain complex; preferable. A step of amplifying a target nucleic acid having a repetitive sequence using a primer may be included before this step. A label may be introduced into the primer. In this case, the first step may extend a labeled base to the end of the hybridized complementary base chain. Moreover, you may hybridize to the repeating sequence unit of a target nucleic acid using the complementary base chain to which the labeled base is added. Alternatively, it may be hybridized to a repetitive sequence unit of the target nucleic acid using a complementary base chain to which a labeled base is added. Furthermore, it may be hybridized to a portion other than the repetitive sequence of the target nucleic acid using a complementary base chain to which a labeled base is added. In this case, after the complementary base chain to which the labeled base is added is dissociated from the complex, the amount of the labeled base added to the complementary base chain is measured, thereby repeatedly in the base sequence. The labeled amount of the label introduced into each of the base sequence and the base sequence outside the labeled repeated base sequence may be measured. Alternatively, the labeled amount may be measured together with the labeled nucleic acid without dissociating the complementary base chain from the complex. The complementary base strand or target nucleic acid may have a tag sequence. The tag sequence may contain a non-natural nucleic acid. By using the method of the present invention, it is possible to simultaneously measure the number of repetitive nucleotide sequences and to detect single nucleotide polymorphisms.

  The kit for measuring the number of repetitions of a repetitive base sequence according to the present invention comprises a complementary base chain that complementarily binds to a repetitive sequence unit of a target nucleic acid having a repetitive base sequence, and an immobilization carrier for immobilizing the target nucleic acid or the complementary base chain Is included. The complementary base chain may have a tag sequence. Furthermore, a labeled nucleic acid for extending a labeled nucleic acid to the complementary base chain may be included, or the complementary base chain may be a complementary base chain to which a labeled nucleic acid is added.

  In the present invention, different labels are introduced at the repetitive site and other sites. Measure the labeling amount of each label and determine the ratio of the two. From this quantitative ratio, the number of repeated base sequences is detected. As a result, it is possible to provide a method and a detection kit for rapidly and quantitatively detecting the number of repeated base sequences. In the present invention, the number of repetitive base sequences is detected from the ratio of the amounts of different labels introduced into the repetitive site and other sites. As a result, multiple items such as samples with different numbers of repetitions and different types of repetitive sequences, which have conventionally been separately performed, can be simultaneously measured on the DNA chip. Also, SNPs can be detected simultaneously.

[principle]
The method and the detection kit for detecting the number of repeats of a repetitive base sequence of the present invention utilize the following regularity that a repetitive base sequence generally has. FIG. 1 is a diagram schematically showing a nucleic acid having a repetitive base sequence. In the example of this figure, a nucleic acid having a repetitive base sequence has n repetitive sequence units 1. As shown in FIG. 1A, a nucleic acid at the 3 ′ end of each repeating sequence unit (T in this example) and a nucleic acid outside the 5 ′ end of the repeating sequence (A in this example) Generally differ in the type of nucleic acid. A complementary base chain is hybridized to each repeating sequence unit 1. In the example shown in FIG. 1 (b), a complementary base chain 2 that is complementary to at least a part of the 5 ′ end side of the repetitive sequence unit is used. Next, a labeled nucleic acid is introduced. As shown in FIG. 1 (c), the labeled nucleic acid A is introduced into the complementary base chain bound to the 1st to (n-1) th of the repeating sequence unit, and the complementary base bound to the nth of the repeating sequence unit. A labeled nucleic acid T is introduced into the chain. Alternatively, a labeled nucleic acid can be similarly introduced even if a labeled nucleic acid having A or T bound to the end of a complementary base chain is used. Complementary base strands into which the labeled nucleic acid A or T has been introduced are detected with different labels, and the ratio of the amounts of both labels is determined. In the example of this figure, the signal intensity of the complementary base chain into which A is introduced is divided by the signal intensity of the complementary base chain into which T, which is an internal standard, is introduced. Therefore, from the target nucleic acid having n repeating units, Is (n-1) signal intensity. Therefore, even if the target nucleic acids have different numbers of repeat sequences, the number of repeats can be measured simultaneously.

  In the above example, the example in which the internal standard is introduced into the nth repetitive sequence unit has been described. The internal standard is not limited to the repetitive sequence unit, and may be a site other than the repetitive sequence portion in the labeled nucleic acid, for example, as shown in FIG. In the example of this figure, a labeled nucleic acid is introduced into a sequence 2 'complementary to a site other than the repeated sequence portion in the labeled nucleic acid. Alternatively, as shown in FIG. 2B, the label 14 may be introduced into a site other than the repetitive sequence portion in the labeled nucleic acid. The example of FIG. 2B is suitable for the case where the target nucleic acid is immobilized and the number of repeated sequences is measured. In the example shown in FIG. 2, the signal intensity is n or (n-1) from the target nucleic acid having n repeating units. In addition, the base chain complementary to the repetitive sequence unit may be the whole, not a part of the repetitive sequence unit.

  According to the present invention, for example, there are several types of VNTR sequences present in the vicinity of the insulin gene, such as a repetitive sequence unit: 5'-ACAggggTgTgggg-3 '. Even in this case, if different complementary base chains are used corresponding to the repetitive base sequences and different labels are introduced, the number of repetitive base sequences can be measured simultaneously.

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

[Target nucleic acid]
The target nucleic acid is not particularly limited as long as it has a repetitive base sequence, and a genomic gene extracted from a cell or the like or a target region amplified using an enzymatic reaction may be used. In consideration of the amount of gene, it is preferable to use an amplified target region. The method for amplifying the target region is not particularly limited, and a known amplification method can be used. A preferred amplification method is a polymerase chain reaction (hereinafter referred to as “PCR reaction”) using a thermostable DNA polymerase. When a PCR reaction is used, amplification efficiency is excellent and it is easy to introduce a label. As shown in FIG. 3, the target nucleic acid 4 having the repetitive base sequence 3 is amplified from the annealed site 6 in the genome using the primer 5 (base extension portion). The primer 5 to be used is not particularly limited as long as it can amplify a repetitive base sequence to be detected. Moreover, you may use two types of primers, a forward primer and a reverse primer. Furthermore, the primer may be capable of amplifying a target nucleic acid having two or more types of repetitive sequences.

[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. 4, the complementary base chains 7 and 8 bind to the repetitive sequence unit 9 in the repetitive base sequence in the target nucleic acid 4 to form a repetitive sequence unit-complementary base chain complex. The complementary base chains 7 and 8 only need to hybridize with all or part of the repetitive sequence unit 9. Further, the complementary base chain 7 is not limited to one type, and two or more types may be used. For example, the complementary base strand 8 serving as an internal standard may be hybridized with a portion other than the repetitive base sequence of the target nucleic acid 4. In this case, the hybridization efficiency of the complementary base chain 7 that hybridizes to the repetitive sequence unit 9 and the complementary base chain 8 that becomes an internal standard to hybridize to a part other than the repetitive base sequence of the target nucleic acid are equal. Required. The length and sequence of the complementary base strands 7 and 8 are not particularly limited, but may be any sequence that has a characteristic of hybridizing with a target nucleic acid at 60 ° C. or lower and sufficiently dissociating at 95 ° C. This is because the duplex stability is improved. The complementary base chains 7 and 8 may be aggregates such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA). A preferred complementary base strand is deoxyribonucleic acid (DNA).

[Introduction of labeled base]
The labeled base 10 is introduced by (1) hybridizing an unlabeled complementary base chain and then extending the labeled base by an enzyme reaction or the like to introduce a label at the end of the base chain. (2) Labeled complementary Hybridization using a base chain, (3) Generating a complementary base chain using a primer having a labeled base and introducing a label (in the case of introducing an internal standard), etc. . Specifically, a base extension reaction, a base ligation reaction, a terminal deoxycleotidyl transferase reaction that selectively labels the 3 ′ end of a nucleic acid, and the like are used. More preferably, it is an extension reaction using an enzyme polymerase. In carrying out this reaction, dideoxynucleotide may be used as a labeled base. Polymerase has high base selectivity and can introduce a label accurately. As a result, the number of repeated base sequences can be accurately detected. When dideoxynucleotide is used as the labeling base, only one base is extended. As a result, detection with higher quantitativeness becomes possible. The label may be added to the labeled base using a base to which the label is added or a complementary base chain, or after extending the base or after hybridizing the complementary base chain.

[Quantification of label]
The labeled base is not particularly limited as long as it can detect bases or base sequences within and outside the repeated sequence with different labels. As a label that can be used, a known substance used for labeling such as a dye label, a radioisotope label, a fluorescent dye, a phosphorescent dye, and a luminescent dye can be used. Preferred are fluorescent dyes that are excellent in safety and detection sensitivity and that are 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. Any of these fluorescent dyes can be used. Preference is given to cyanine fluorescent dyes that can be easily recognized due to differences in structure. 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), silicon (Si), and the like. When using different labels, it is preferable to use one that can be measured with the same apparatus. Further, in order to bind a base or base sequence to which a label is added to a complementary base chain, a structure capable of binding to the complementary base chain is required.

[Measurement of label strength]
The label strength may be measured using a complementary base chain dissociated from the repeating sequence unit-complementary base chain complex, or may be measured for the entire repeating sequence-complementary base chain complex. The labeling strength of the dissociated complementary base chain may be measured in a solution, or may be measured after immobilization on an immobilization carrier. When measuring the labeling intensity of the entire repeating sequence unit-complementary base chain complex, measurement is performed after immobilization on an immobilization carrier. When detecting signals of a plurality of labels at a high density, it is preferable to measure the signals by immobilizing them on an immobilization carrier such as a DNA chip. As shown in FIG. 5, the immobilization on the immobilization carrier is performed by hybridizing the tag sequence of the complementary base chain to which the tag sequence is added and the immobilization carrier 13 immobilized on the substrate 12. . The tag sequence 11 may form a complex using a complementary base chain in which the tag sequence is added to the complementary base chains 7 and 8 in advance, and the labeled base sequence-complementary base chain complex to the complementary base A tag sequence may be added after the strand is dissociated. In addition, when a repetitive sequence-complementary base chain complex is immobilized, if a tag sequence is added to the target nucleic acid, it can be similarly immobilized on the substrate.

[Immobilized carrier]
The immobilization carrier only needs to be immobilized on a base material such as a substrate or beads. Immobilizing a complementary base chain on a substrate is preferable because it allows multiple samples to be processed.

  The immobilization carrier may be a nucleic acid aggregate. This is because the nucleic acid can be complementarily bound to the complementary base chain or a part of the target nucleic acid (including the tag sequence). The immobilization carrier and a part of the complementary base chain or the target nucleic acid (including the tag sequence) may be a natural nucleic acid or a non-natural nucleic acid. A natural nucleic acid means a nucleic acid that exists in nature, and refers to a nucleic acid in which the sugars contained in the nucleic acid are all D-forms. Non-natural nucleic acid means a nucleic acid that does not exist in nature, and refers to a nucleic acid in which at least one of the sugars contained in the nucleic acid is L-form, or a part of the sugar is modified. In particular, a nucleic acid in which at least one of the sugars contained in the nucleic acid is L-form is referred to as an L-type nucleic acid. 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. Some non-natural nucleic acids do not hybridize with natural nucleic acids. Therefore, when a non-natural nucleic acid is used, it is possible to design a tag sequence that does not cause cross-hybridization.

  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.

[Measurement of label strength]
The number of repetitive base sequences is quantified by measuring the signal intensity of the target nucleic acid hybridized on the carrier or the labeled complementary base chain. In order to achieve quantification that does not depend on the detector specifications, the concentration should be set so that it is an indicator of the hybridization efficiency of two or more complementary base strands and the standardization of the polymerase extension reaction when a label is introduced. The signal intensity may be measured by adding a labeled polynucleotide having a known concentration or a target nucleic acid having a known concentration and a complementary base strand.

  By using the detection method of the present invention, the number of repetitive base sequences can be rapidly and quantitatively 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 repetitive base sequences can be rapidly and quantitatively detected.

[Detection of SNPs]
If this invention is used, the measurement of the number of repetitions of a repeating base and the measurement of SNPs can be performed on one fixed support | carrier. That is, according to the present invention, a plurality of labels can be simultaneously performed using a plurality of target nucleic acids with a small amount of sample. As a result, it is possible to measure the number of repeated base sequences and to detect the SNPs type on one immobilization carrier. In addition, when a repetitive base sequence and SNPs are present in the vicinity, one type of target nucleic acid is amplified from one type of primer set, and the repetitive base sequence and SNPs are made identical using a plurality of types of complementary base chains. From the target nucleic acid. SNPs can be detected, for example, by detecting a mismatch of one base contained in the repetitive base sequence by using the SPR method (Reference 1: Alexander W. Peterson (Alexander). W. Peterson, et al., “Hybridization of Mismatched or Partially Matched DNA at Surfaces,” Journal of American Chemical Society, Journal of American America, USA. Chemical Society (2002), 124th, Chemical Society (American Chemical Society) Volume, p. 14601-14607). Alternatively, the base sequence is determined by the base extension method, and SNPs are measured (Reference 2: Tomi Pastinen) and four others, “Mini sequencing: for DNA analysis and diagnosis on oligonucleotide arrays ”Special Tool: A Specific Tool for DNA Analysis and Diagnostics, Vol. 7”, ”Bor, B. p.606-614).

[Detection kit]
The repetitive base sequence repeat number measurement kit of the present invention includes a complementary base chain that complementarily binds to a repetitive sequence unit of a target nucleic acid having a repetitive base sequence, and an immobilization carrier that fixes the complementary base chain. Yes. The complementary base chain may have a tag sequence. A labeled nucleic acid for extending a labeled nucleic acid to the complementary base chain may be included, or the complementary base chain may be a complementary base chain to which a labeled nucleic acid is added. The immobilization carrier may be a measurement kit that is already immobilized on a substrate. By using such a kit for measuring the number of repeats of a repeated base sequence, the number of repeats of the repeated base sequence 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.

(Example 1)
[Detection of repeat number of VNTR contained in human IL1RN (IL-1 receptor antagonist)] (when dissociating complementary base chain)
(Synthesis of polynucleotide)
In order to amplify the VNTR region (SEQ ID NO: 1) contained in IL1RN, a forward primer and a reverse primer that can cover the repeated region of SEQ ID NO: 1 were synthesized. This primer can be amplified in any region having VNTR of 2 to 6 repeats. In addition, a complementary base chain (SEQ ID NO: 2) for the repeating sequence unit was synthesized. The synthesis was outsourced to Operon Biotechnology Co., Ltd. using D-type nucleic acid.
The complementary base chain of SEQ ID NO: 2 was designed so that it could be used not only for detecting the number of VNTR repeats but also for detecting an internal standard.

(Amplification of region containing VNTR sequence by PCR)
Genomic DNA was extracted with a nucleic acid extraction kit “QIAmp” (manufactured by Qiagen) using HEK293 (derived from human embryonic kidney cells) cells and HL60 (derived from human promyelocytic leukemia cells) cells. The following reagent 1 was added to the genomic DNA, and the VNTR sequence contained in IL1RN was amplified under the following condition 1.
<Reagent 1>
A 50 μl solution containing the following reagents was prepared for each sample.
Forward primer 15 pmol
Reverse primer 15 pmol
Buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
KOD-plus-DNA polymerase (TOYOBO) 1 U
Extracted DNA solution 50 ng
<Amplification condition 1>
95 ℃, 5 minutes
95 ℃, 30 seconds, 55 ℃, 30 seconds, 68 ℃, 30 seconds (35 cycles)

(Evaluation by agarose gel electrophoresis)
The length of the amplified portion after the PCR reaction was evaluated by electrophoresis. Electrophoresis was performed by running a 2% agarose gel at 100 V for 30 minutes using a developing solution in which 20 × TAE Buffer (manufactured by Invitrogen) was diluted 20 times. After electrophoresis, the nucleic acid was stained with an aqueous ethidium bromide solution, and the length of the amplified target nucleic acid was determined. As a result, the target nucleic acid fragment derived from HEK293 cells was 851 bp / 1023 bp, and the target nucleic acid fragment derived from HL60 cells was 1023 bp. From this, it was estimated that the number of each iteration was 2 and 4 hetero and 4 homo.

(Incorporation of labeled base using labeled base)
The PCR reaction product was purified using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bioscience Co., Ltd.). The following reagent 2 was added to the purified product, and a labeled base was incorporated by the enzymatic method under the following condition 2. Polynucleotides 3 and 4 are polynucleotides for confirming whether Cy3-labeled ddATP and Cy5-labeled ddUTP are normally incorporated. The polynucleotides 5 and 6 for standardization bind to the polynucleotides 3 and 4, and the bases of the polynucleotides 5 and 6 for standardization are labeled with the bases 3 and 4 as templates, and the reaction proceeds normally. It is a polynucleotide for confirming that it exists. The base sequences of polynucleotides 3 to 6 are base sequences corresponding to SEQ ID NOs: 3 to 6 in Table 2, respectively.

After incorporating the labeled base, the base sequence-complementary base chain complex was repeatedly dissociated using a thermal cycler and kept at 95 ° C. By this operation, a complementary base chain with a tag sequence to which a labeled nucleic acid was added was obtained.
<Reagent 2>
A 20 μl solution containing the following reagents was prepared for each sample.
PCR purified product (target nucleic acid) 100 ng
Complementary base chain with tag sequence 20.3 pmol
10 mM Cy3-labeled ddATP 1 μl
10 mM Cy5-labeled ddUTP 1 μl
1 mM 10 mM ddGTP
1 mM 10 mM ddCTP
Buffer 2 μl
Thermo sequence polymerase 8 U
Polynucleotide 3 12.5 ng
Polynucleotide 4 12.5 ng
Polynucleotide 5 for standardization 0.3 pmol
Polynucleotide 6 for standardization 0.3 pmol
<Reaction condition 2>
95 ℃, 2 minutes
95 ℃ for 20 seconds, 60 ℃ for 15 seconds (30 cycles)
95 ℃, 5 minutes
0 ℃, 3 minutes

(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 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.

  Four types of anti-tag sequence D-type nucleic acid complementary strands designed so as to avoid cross-hybridization between the anti-tag sequences and aminated at the 5 'end were synthesized and used as an immobilization carrier (anti-tag sequence).

  Each of the four types of synthesized complementary DNAs of the anti-tag sequence was dissolved in pure water at a concentration of 0.3 nmol / μL, and then diluted to 0.03 nmol / μL using a phosphate buffer (pH 5.5). Further, in order to condense the carboxylic acid on the substrate surface with the terminal amino group of the immobilized DNA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is added so that the final concentration is 50 mg / mL. added.

(Preparation of hybridization solution)
Hybridization of the complementary base chain with a tag sequence to which a labeled nucleic acid was added and the immobilized anti-tag sequence was performed as follows. 15 × SSC used below means 20 × SSC (0.3 M sodium citrate dihydrate, 3 M NaCl solution: manufactured by Sigma) diluted 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) was prepared. . This is mixed with a complementary nucleotide chain solution with a tag sequence to which the labeled nucleic acid is added at a ratio of 3: 1, and a Cy3 labeled polynucleotide (polynucleotide 7) or a Cy5 labeled polynucleotide, which is the same tag sequence as the tag sequence (Polynucleotide 8) was added in an amount of 0.01 pmol, and the total amount was 40 μL.

(Immobilization to immobilization carrier)
40 μL of the hybridization solution was placed on the substrate carrying the immobilization carrier, covered with a cover glass, incubated at 42 ° C. for 4 hours, and then the cover glass was peeled off, washed and dried.

(Measurement of labeling amount)
The substrate was set on a DNA chip scanner (Perkin Elmer, Scan Array Express), and the signal intensity was measured. The measurement conditions were such that the laser output was 90% and the sensitivity of the photomultiplier was adjusted so that the signal intensities of Cy3 and Cy5 detected by the polynucleotides 7 and 8 were comparable. Here, the signal intensity refers to an average value of fluorescence intensity in each spot. The results are shown in Table 3.

  From Table 3, the signal intensity ratio (Cy3 / Cy5) derived from HEK293 cells is 2, and it is determined that the number of repeats of the repeated nucleotide sequence is 3 times or 2 times and 4 times heterozygous by comparison with the theoretical value. it can. Similarly, the signal intensity ratio (Cy3 / Cy5) derived from HL60 cells is 3, and it can be determined that the number of repetitions is 4 from comparison with the theoretical value. From this table, it was found that when the labeled nucleic acid was introduced into the complementary base strand after the formation of the repeated sequence unit-complementary strand complex, the number of repeats of the repeated base sequence could be accurately quantified.

(Example 2)
[Detection of the number of repeats of VNTR contained in human IL1RN using a labeled primer] (When labeled nucleic acid is immobilized and analyzed)
(Synthesis of polynucleotide)
In order to amplify the VNTR region (SEQ ID NO: 1) contained in IL1RN, a forward primer capable of covering the repeat region of SEQ ID NO: 1, a reverse primer and a complementary base sequence polynucleotide (SEQ ID NO: 2) for the repeat sequence unit were synthesized. .

  A tag sequence using a non-natural nucleic acid (L-type nucleic acid) was added to the forward primer. A Cy3 label was introduced at the 5 'end of the forward primer. The reverse primer was synthesized with D-type nucleic acid. This primer can be amplified in any region having VNTR of 2 to 6 repeats.

  The complementary base introduced a Cy5 label at the 5 'end.

  Synthesis of DNA containing L-type nucleic acid was performed using a DNA / RNA DNA automatic synthesizer (Applied Biosystems). Further, as L-type nucleic acids, Beta-L-deoxy Adenosine (n-bz) CED phosphoramidite, Beta-L-deoxy Cytidine (n-bz) CED phosphoramidite, Beta-L-deoxy guanidine (B-L-deoxy guanidine) -L-deoxy Thmidine CED phosphoramidite (manufactured by ChemGenes Corporation) as a D-type nucleic acid, deoxy Adenosine (n-bz) CED phosphoramidite, deoxy Cytidine (n-bz) sphoramidite, using deoxy Thmidine CED phosphoramidite (manufactured by ChemGenes Corporation).

  The reverse primer and the forward primer introduced with Cy3 label at the 5 'end and the complementary base chain introduced with Cy5 label at the 5' end were commissioned to Operon Biotechnology Co., Ltd.

(Amplification of region containing VNTR sequence by PCR)
The following reagent 3 was added to human genomic DNA extracted in the same manner as in Example 1, and the VNTR sequence contained in IL1RN was amplified under the above condition 1.
<Reagent 3>
A 50 μl solution containing the following reagents was prepared for each sample.
Cy3-labeled forward primer with 20 pmol
Reverse primer 0.2 pmol
Buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
KOD-plus-DNA polymerase (TOYOBO) 1 U
Extracted DNA solution 50 ng

  Further, electrophoresis was performed in the same manner as in Example 1. After the electrophoresis, a target nucleic acid having 2 repeats was extracted.

(Repetitive base sequence-complementary chain complex formation)
The PCR reaction product was purified using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bioscience Co., Ltd.).

  To 100 ng of the purified target nucleic acid, 1 μL of the Cy5-labeled complementary base chain dissolved in 100 μM was added, and 5 × SSC diluted with pure water was added so that the total amount became 20 μL. This solution was incubated at 95 ° C. for 5 minutes and then allowed to stand on ice for 5 minutes.

(Immobilization to immobilization carrier)
A sequence complementary to the tag sequence was synthesized using L-type nucleic acid. Further, the obtained sequence complementary to each tag sequence has an amino group added to the 5 ′ end, and was immobilized on the substrate using the method described in Example 1.

  10 μL of the repetitive base sequence-complementary base chain complex solution, 30 μL of the hybridization reagent described in Example 1, and Cy3 labeled polynucleotide (polynucleotide 7) or Cy5 labeled polynucleotide (polynucleotide 8) were each 0.01 pmol. Added and mixed.

  The hybridization solution was added to a substrate on which a sequence complementary to the L-type tag sequence was immobilized, and immobilization was performed by the method described in Example 1.

(Measurement of labeling amount)
The substrate was set on a DNA chip scanner (Perkin Elmer, Scan Array Express), and the signal intensity was measured. The measurement conditions were such that the laser output was 90% and the sensitivity of the photomultiplier was adjusted so that the signal intensities of Cy3 and Cy5 detected by the polynucleotides 7 and 8 were comparable. Here, the signal intensity refers to an average value of fluorescence intensity in each spot. The results are shown in Table 4.

  From Table 4, it was shown that the increase in the fluorescence intensity ratio with the number of repetitions of the repeated base sequence was also measured in the measurement of the number of repetitions using the target nucleic acid-complementary base chain complex using the labeled primer. . Further, it was found that even a tag sequence using an L-type nucleic acid can have the same sensitivity as that of the D-type.

(Example 3)
[Simultaneous detection of VNTR repeat number and SNPs]
(Synthesis of polynucleotide)
Primers capable of amplifying four SNPs regions were synthesized as polynucleotides for detecting SNPs. In addition, a single-base extension method (Reference Document 2), which is a known technique, was used to detect each SNP, and a polynucleotide suitable for this technique was synthesized. The synthesis was commissioned to Operon Biotechnology Co., Ltd.

  Complementary base strands for measuring the number of sequences of each primer and repetitive base sequence are synthesized with D-type nucleic acid, and D-type nucleic acid tag sequence designed so that there is no cross-hybridization between tag sequences in the complementary base strand. Added.

(Amplification of region containing SNPs by PCR)
HEK293 (human embryonic kidney cell-derived) genomic DNA was extracted by the method described in Example 1. The following reagent 4 was added, and conditions 1 described in Example 1 were used to amplify the VNTR sequence contained in IL1RN and the region containing 4 SNPs, respectively.
<Reagent 4>
A 50 μl solution containing the following reagents was prepared for each sample to obtain a total of five reaction solutions.
Forward primer 15 pmol
Reverse primer 15 pmol
Buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
KOD-plus-DNA polymerase (TOYOBO) 1 U
Extracted DNA solution 50 ng

(Incorporation of complementary bases using labeled bases)
The PCR reaction product was purified using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bioscience Co., Ltd.).

The following reagent 5 was added to the purified product thus obtained, and a labeled base was incorporated by an enzymatic method under the condition 2 described in Example 1.
<Reagent 5>
A 20 μl solution containing the following reagents was prepared for each sample to obtain a total of five reaction solutions.
PCR purified product (target nucleic acid) 100 ng
Tagged complementary base chain 0.3 pmol
10 mM Cy3-labeled ddATP 1 μl
10 mM Cy5-labeled ddUTP 1 μl
1 mM 10 mM ddGTP
1 mM 10 mM ddCTP
Buffer 2 μl
Thermo sequence polymerase 8 U
Polynucleotide 3 12.5 ng
Polynucleotide 4 12.5 ng
Polynucleotide 5 for standardization 0.3 pmol
Polynucleotide 6 for standardization 0.3 pmol

(Immobilization to immobilization carrier)
A strand complementary to the tag sequence was synthesized with a D-type nucleic acid having an amino group added to the 5 ′ end. The synthesis was commissioned to Operon Biotechnology Co., Ltd. Next, using the method described in Example 1, a substrate having strands complementary to these tag sequences immobilized thereon was prepared.

  The above reaction solution was mixed with 2 μL of each sample to prepare 10 μL of a labeled polynucleotide solution, which was mixed with 30 μL of the hybridization reagent described in Example 1 and 0.01 pmol of each of polynucleotides 7 and 8. A solution was made.

  The hybridization solution was added to the DNA immobilization substrate, and immobilization was performed by the method described in Example 1.

(Measurement of labeling amount)
The substrate was set on a DNA chip scanner (Perkin Elmer, Scan Array Express), and the signal intensity was measured. The measurement conditions were such that the laser output was 90% and the sensitivity of the photomultiplier was adjusted so that the signal intensities of Cy3 and Cy5 detected by the polynucleotides 7 and 8 were comparable. Here, the signal intensity refers to an average value of fluorescence intensity in each spot. The results are shown in Table 5.

The SNP type binds complementarily to a complementary labeled base. From the results of Table 5, it is determined that the SNP types of SNP1 and SNP2 are A / T hetero, and the SNP types of SNP3 and SNP4 are A homo. This result was consistent with the result of a sequence method which is a known method. Further, in the determination of the number of VNTR repeats, the fluorescence intensity ratio was 2, similarly to the above result, and the number of repeats could be discriminated as 3 or 2 and 4 hetero. From this, it was shown that SNPs and VNTR sequences can be detected simultaneously by using an immobilized carrier on which a tag sequence is immobilized.

FIG. 1 is a schematic diagram for explaining the principle of the present invention. FIG. 2 is a schematic diagram for explaining a method for introducing an internal standard into a site other than the repetitive sequence of a labeled nucleic acid. FIG. 3 is a conceptual diagram for explaining a method for preparing a target nucleic acid using a PCR product. FIG. 4 is a conceptual diagram illustrating a mechanism in which a complementary base chain binds to a repetitive sequence unit in a target nucleic acid. FIG. 5 is a conceptual diagram illustrating a mechanism for detecting and quantifying labeled nucleic acid using an immobilized carrier.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Repeating sequence unit 2 Complementary base chain 3 Repeating base sequence 4 Target nucleic acid 5 Primer 6 Annealing site 7, 8 Complementary base chain 9 Repeating base sequence 10 Labeled base 11 Tag sequence 12 Substrate 13 Immobilization carrier 14 Label

Claims (15)

A first step of introducing a label into each of the base sequence within the repeat base sequence and the base sequence outside the repeat base sequence;
The labeled amount of the label introduced into each of the base sequence within the labeled repetitive base sequence and the base sequence outside the labeled repetitive base sequence is measured, and the measured label amount is converted into the base sequence within the repetitive base sequence. A method for measuring the number of repetitions of a repetitive base sequence, comprising: a second step of obtaining an amount ratio of the amount of label introduced to a base sequence outside the repetitive base sequence and the introduced label. Before the first step,
The repeat of a repetitive base sequence according to claim 1, comprising a step of hybridizing a base chain complementary to a repetitive sequence unit of a target nucleic acid having a repetitive base sequence to form a repetitive sequence unit-complementary base chain complex. Number measurement method. Before the first step,
Amplifying a target nucleic acid having a repetitive sequence using a primer, and hybridizing a base chain complementary to the repetitive sequence unit of the target nucleic acid having the repetitive base sequence to form a repetitive sequence unit-complementary base chain complex The measuring method of the repetition number of the repetitive base sequence of Claim 1 including the process to perform.   The method for measuring the number of repetitions of a repetitive base sequence according to claim 3, wherein a label is introduced into the primer.   The method for measuring the number of repetitions of a repetitive base sequence according to any one of claims 2 to 4, wherein in the first step, a labeled base is extended to the end of the hybridized complementary base chain.   The method for measuring the number of repetitions of a repetitive base sequence according to any one of claims 2 to 4, wherein a complementary base chain to which a labeled base is added is used to hybridize to the repetitive sequence unit of the target nucleic acid.   The method for measuring the number of repetitions of a repetitive base sequence according to claim 6, further comprising hybridizing to a portion other than the repetitive sequence of the target nucleic acid using a complementary base chain to which a labeled base is added. After dissociating the complementary base chain to which the labeled base has been added from the complex,
By measuring the amount of the labeled base added to the complementary base chain, the labeled amount of the label introduced into each of the base sequence within the repeated base sequence and the base sequence outside the labeled repeated base sequence is measured. The method for measuring the number of repetitions of a repetitive base sequence according to claim 5 or 6.   The repeat base sequence labeled with the base sequence in the repeat base sequence by measuring the amount of the labeled base added to the complementary base chain of the target nucleic acid formed with the repeat sequence unit-complementary base chain complex. The method for measuring the number of repetitions of a repetitive base sequence according to any one of claims 4 to 7, wherein the amount of label introduced into each of the other base sequences is measured.   The method for measuring the number of repetitions of a repetitive base sequence according to any one of claims 2 to 9, wherein the complementary base chain or the target nucleic acid has a tag sequence.   The method according to claim 10, wherein the tag sequence includes a non-natural nucleic acid.   The method for measuring the number of repetitions of a repetitive base sequence according to any one of claims 1 to 11, wherein the measurement of the number of repetitions of the repetitive base sequence and the detection of a single nucleotide polymorphism are performed simultaneously. A complementary base chain that complementarily binds to a repetitive sequence unit of a target nucleic acid having a repetitive base sequence;
A kit for measuring the number of repetitive base sequences, comprising an immobilization carrier for immobilizing the target nucleic acid or complementary base chain.   The kit for measuring the number of repetitions of a repetitive base sequence according to claim 13, wherein the complementary base chain has a tag sequence. Furthermore, it contains the labeled nucleic acid for extending | stretching a labeled nucleic acid to the said complementary base chain, or the said complementary base chain is a complementary base chain to which the labeled nucleic acid is added. A kit for measuring the number of repeated nucleotide sequences.