JP5716217B2 - Method for selecting or preferentially recovering genes or gene products containing long chain repeats - Google Patents

Description

  The present invention relates to a method for selecting or preferentially recovering a gene or gene product containing a long chain repeat sequence and use of the recovered gene and the like. More specifically, the present invention relates to abnormal extension of a repeat sequence such as triplet repeat disease. The present invention relates to a method for selectively or preferentially collecting a mutant allele that is a causative gene of a disease caused by the cause, and its use.

  A typical example of a disease caused by an abnormal extension of a repetitive sequence is triplet repeat disease (also called tribasic repeat disease). Triplet repeat disease is a disease group that develops when a repeated sequence of three bases such as CAG, CTG, and CGG in a disease-causing gene becomes longer than usual. The extension of three bases in the untranslated region of the gene causes an abnormality at the RNA transcript level, and the extension of three bases in the translated region mainly causes an abnormality at the protein level. The severity of the disease depends on the degree of extension (number of repeats) of the three bases. Since these diseases are inherited to the next generation and tend to progress abnormally, the symptoms are more serious in children and grandchildren than in parents, and the onset age is earlier.

  When the CAG tribase of the CAG repeat sequence (CAG repeat) is translated into glutamine, it is also called polyglutamine disease. In that case, the translated polyglutamine chain is contained in the gene product. Polyglutamine disease is thought to develop due to the cause of an abnormally elongated polyglutamine chain of this gene product.

  Because triplet repeat disease is a disease caused by inheritance of a disease-causing gene, the onset of the disease is abnormal by PCR amplification of the disease-causing gene and confirmation by electrophoresis, or by Southern blotting using genomic DNA It is possible by genetic diagnosis such as confirming an extended disease-causing gene. However, since the onset mechanism of triplet repeat disease is not known in detail, a fundamental treatment method has not yet been established.

  As a promising therapeutic method in the future, there is a mutant allele-specific RNAi knockdown by RNA interference (hereinafter abbreviated as “RNAi”). In this method, only the target mutant allele is specifically suppressed by targeting a base sequence specific to the mutant allele causing the disease. Specific RNAi knockdown of the mutant allele causing the disease can be an important technique for realizing a safe treatment with few side effects. In fact, attempts have been made to expand the RNAi induction technology to triplet repeat disease (Patent Documents 1 and 2). In Patent Document 1, a specific sequence of mRNA in the upstream vicinity region of the CAG repeat in the huntingtin gene (hereinafter abbreviated as “HTT gene”), which is a causative gene of Huntington's disease, is targeted. It is described that the expression of the huntingtin gene is suppressed using a single-stranded RNA.

  Since the disease-causing (allelic) gene of triplet repeat disease has an abnormality only in the extension degree of the triplet repeat, the mutation in the disease-causing allele, that is, the extension degree of the repeated sequence varies from patient to patient. Patients also inherit (normal) alleles with normal repeat elongation by inheritance. When a triplet repeat, which is a mutation site, is directly targeted for RNAi, even the normal allele is knocked down, leading to complete loss of function of the gene. Therefore, it is necessary to suppress the expression of only the mutant allele that causes pathogenesis. When RNAi is applied to a disease that occurs due to abnormal extension of a repeated sequence such as triplet repeat disease, a new base index for identifying a long-chain mutant allele that differs for each living body (patient) Necessary.

  As an index, single nucleotide polymorphism (hereinafter abbreviated as “SNP”) has been studied (Non-patent Document 1). In the technique of Non-Patent Document 1, in order to find the relationship between the length of the CAG repeat sequence and the heterogeneous SNP, a technique called SLiC method including intermolecular ligation operation of Long-range PCR amplification products is used. According to this technique, SNPs specific to mutant alleles can be found within 2 weeks of analysis.

WO2004 / 101787 WO2004 / 013280

Nature Methods Vol. 5 No. 11 November 2008 pp 951-953

  Isolating a long-chain mutant allele containing a long extended repeat sequence using conventional techniques based on cloning and determining the SNP of that mutant allele is very time consuming and labor intensive It is a difficult task that requires. Moreover, sufficient attention must be paid to the reproducibility and accuracy.

  As described above, in a disease that occurs due to abnormal extension of a repetitive sequence typified by triplet repeat disease, identifying and characterizing the long-chain mutant allele that caused the abnormal extension, It is extremely important in the treatment strategy. Therefore, an object of the present invention is to select a gene or gene product such as a mutant allele having a long repetitive sequence and causing a disease rather than a gene or gene product such as a normal allele having a short repetitive sequence or It is to provide a method of preferentially collecting.

  The present invention also relates to the use of a mutant allele in which a repetitive sequence obtained by the above selection or preferential recovery method causes abnormal extension.

The present inventors have conducted extensive research on the recovery and identification of disease-causing alleles. Surprisingly, mutants having a long repetitive sequence have been obtained by a novel method using a repetitive sequence-containing synthetic labeled RNA probe. Alleles were successfully recovered preferentially over normal alleles with short repeats. Based on the above findings, the present invention is a method for selecting or preferentially recovering a mutant allele or gene product having an abnormal extension of a repetitive sequence, which causes a three-base repeat disease, and comprises the following steps:
(A) genomic DNA which the disease is acquired from a living body suspected, the cDNA synthesized from total RNA or total RNA, synthetic labeling containing tribasic repeat sequence in the sense or antisense strand nucleotide sequence of a disease gene Hybridizing the RNA probe, and (b) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label,
A method for selecting or preferentially recovering a mutant allele or gene product having an abnormal extension of a repetitive sequence that causes a three-base repeat disease is included.

Hereinafter, the method for selecting or preferentially recovering a gene or gene product of the present invention, particularly the method for selecting or preferentially recovering a mutant allele may be simply referred to as the method for selective recovery of the present invention. In addition, in the present specification, the mutant allele means a gene that extends more than the repetitive sequence of the normal allele (that is, has a large number of repeats) and induces a disease due to the extension. Used for.

The present invention is also a method for identifying, for each living organism , a mutant allele having an abnormal extension of a repetitive sequence, which causes a trinucleotide repeat disease, and comprises the following steps:
(A) genomic DNA which the disease is acquired from a living body suspected, the cDNA synthesized from total RNA or total RNA, synthetic labeling containing tribasic repeat sequence in the sense or antisense strand nucleotide sequence of a disease gene Hybridizing RNA probes,
(B) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label; and (d) DNA-synthesis recovered in step (b). For the labeled RNA probe conjugate, determine the SNP site typing of the mutant allele and the haplotype based on it
A method for identifying the mutant allele is provided.

The present invention also provides a method for preparing a tailor-made medicine for a tribasic repeat disease , comprising the following steps:
(A) genomic DNA which the disease is acquired from a living body suspected, the cDNA synthesized from total RNA or total RNA, synthetic labeling containing tribasic repeat sequence in the sense or antisense strand nucleotide sequence of a disease gene Hybridizing RNA probes,
(B) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label;
(D) SNP typing of the allele and the haplotype based on it for the conjugate of the DNA recovered in step (b) and the synthetic labeled RNA probe, and (e) the allele identified in step (d) There is provided a method for preparing the tailor-made medicament, comprising preparing a double-stranded RNA molecule that causes RNAi to a SNP site of a gene that is heterozygous to the other allele. In the present specification, the “double-stranded RNA molecule” means an RNA molecule obtained by hybridizing the single-stranded RNAs in whole or in part.

  According to the selective recovery method of the present invention, it is possible to select or preferentially recover a mutant allele having a long extended repetitive sequence, etc., by an extremely simple operation and procedure as compared with conventional techniques. According to the present invention, mutant alleles can be recovered in an analysis period of about one day. Further, by amplifying the collected nucleic acid by PCR and analyzing it by electrophoresis, it becomes very easy to detect a band having a long repetitive sequence (that is, a mutant type). Obtaining mutant alleles that differ from patient to patient very quickly and accurately is very effective in identifying disease-causing genes for each patient. In addition, it is also useful for therapeutic research on diseases in which a gene containing a long-chain repeat sequence or the like is an etiological gene, research during disease treatment (confirmation of therapeutic effect, etc.), or prognosis research.

  The selective recovery method of the present invention enables selective recovery using the same synthetic labeled RNA probe as long as the causative repeat sequence (CAG or the like) is the same even if the disease-causing gene is different. This is a major feature and advantage of the present invention.

  According to the method for identifying a mutant allele of the present invention, it is quick and easy to identify a mutant allele that is different for each patient. According to the present invention, it is possible to identify a patient's SNP haplotype within an analysis period of 1 day, including selection of a mutant allele or preferential recovery. The identified information is useful for the preparation of the tailor-made medicine shown below.

  According to the method for preparing a tailor-made medicine of the present invention, optimal tailor-made medicine for each patient is made possible by using the mutant allele obtained by the selection or preferential recovery method and identification method. This can be done by examining a predetermined SNP site of the mutant allele for each living body (patient) and, if it is heterozygous, creating siRNA (small interfering RNA) for RNAi.

It is a figure which shows the experiment procedure according to the selective collection | recovery method of this invention, a required reagent composition, etc. Untreated cDNA (−) prepared from two types of immortalized cells ((1) C0142; (2) C0221) derived from Huntington's disease (HD) patients, and cDNA obtained by the selective recovery method of the present invention (+ Is an electrophoretic diagram after performing PCR amplification on the CAG repeat sequence of the huntingtin (HTT) gene. It is the figure which analyzed the effect of the selective collection method using the biotinylated RNA probe of this invention by the allele discrimination test. Allele discrimination tests were performed on four SNP sites present in the HTT gene transcription sequence. The sample (●) obtained by the selective recovery method was plotted at a position away from the calibration curve, compared to the calibration curve prepared using the untreated sample (○) that was not subjected to the selective recovery method. . It is a figure which shows the haplotype (SNP structure of an allele) estimated from the typing of four SNP site | parts of FIG. 3A. It is a figure which shows the result of having performed IC50 test about siRNA (Table 6A, Table 7) which showed the strongest inhibitory effect with respect to the allele of the SNP site | part of rs3633099 of an HTT gene, and high SNP discrimination | determination ability. It is a figure which shows the result of having performed IC50 test about siRNA (Table 6B, Table 7) which showed the strongest inhibitory effect with respect to the allele of the SNP site | part of rs362331 of an HTT gene, and high SNP discrimination ability. It is a figure which shows the result of having conducted IC50 test about siRNA (Table 6C) which showed the strongest inhibitory effect with respect to the allele of the SNP site | part of rs362273 of HTT gene, and high SNP discrimination | determination ability. It is a figure which shows the result of having conducted IC50 test about siRNA (Table 6D) which showed the strongest inhibitory effect with respect to the allele of the SNP site | part of rs362272 of HTT gene, and high SNP discrimination | determination ability. After introducing siRNAs (individually or in a mixture of four types shown in Table 8) selected based on the mutant HTT gene haplotype of HD patient-derived immortalized cells determined according to the method of the present invention into RNA patient-derived immortalized cells This is a result of conducting an allele discrimination test on SNP site rs363030. After introducing siRNAs (individually or in a mixture of four types shown in Table 8) selected based on the mutant HTT gene haplotype of HD patient-derived immortalized cells determined according to the method of the present invention into RNA patient-derived immortalized cells This is the result of conducting an allele discrimination test on SNP site rs362331. After introducing siRNAs (individually or in a mixture of four types shown in Table 8) selected based on the mutant HTT gene haplotype of HD patient-derived immortalized cells determined according to the method of the present invention into RNA patient-derived immortalized cells This is a result of conducting an allele discrimination test on SNP site rs362273. After introducing siRNAs (individually or in a mixture of four types shown in Table 8) selected based on the mutant HTT gene haplotype of HD patient-derived immortalized cells determined according to the method of the present invention into RNA patient-derived immortalized cells This is a result of conducting an allele discrimination test on the SNP site rs362272. Using the untreated cDNA (−) prepared from SH-SY5Y cells, which are human-derived cell lines, and the cDNA (+) obtained by the selective recovery method of the present invention, CAG of the DRPLA causative gene (ATN1) It is an electrophoretic diagram after performing PCR amplification with respect to a repetitive sequence. Cause of SCA3 using untreated cDNA (-) prepared from HEK293 cells, HeLa cells and SH-SY5Y cells, which are human-derived cell lines, and cDNA (+) obtained by the selective recovery method of the present invention. It is an electrophoretic diagram after performing PCR amplification with respect to the CAG repeat sequence of a gene (ATXN3). CAG repeat sequence of SCA6 causative gene (CACNA1A) using untreated cDNA (−) prepared from HD patient-derived immortalized cells (C0221) and cDNA (+) obtained by the selective recovery method of the present invention It is the electrophoretic diagram after performing PCR amplification with respect to. It is a figure which shows the outline of the disease gene specific knockdown by RNAi to the disease allele discriminated according to the method of this invention. It is the schematic which knocks down a disease gene specifically by introduce | transducing RNAi to the multiple SNP site | part of the disease allele for every patient according to the method of this invention.

  Hereinafter, the selective recovery method, the mutant allele identification method, and the tailor-made pharmaceutical preparation method of the present invention will be described. The selective recovery method of the present invention selectively or preferentially recovers a gene or gene product containing a long chain repeat sequence such as a mutant allele that causes a disease caused by the expression of a long repeat sequence. The method comprises (a) genomic DNA, total RNA, or cDNA synthesized from total RNA obtained from a living body, particularly a living body suspected of the disease, in the sense strand or antisense strand base sequence of the gene, particularly a disease gene. A step of hybridizing a synthetic labeled RNA probe containing a repetitive sequence in the sense strand or antisense strand base sequence of (1), and (b) a DNA or RNA obtained in step (a) and a synthetic labeled RNA probe; The step of recovering and purifying the conjugate of is based on the label.

Step (a): Hybridization In step (a), hybridization between a mutant allele having a long repetitive sequence and a synthetic labeled RNA probe is performed. Here, examples of diseases that are caused by abnormal extension of repetitive sequences targeted by this method include Huntington's disease (HD), spinocerebellar degeneration type 1 (SCA1), spinocerebellar degeneration type 2 (SCA2) Spinocerebellar degeneration type 3 (also called Machado-Joseph disease) (SCA3), spinocerebellar degeneration type 6 (SCA6), spinocerebellar degeneration type 7 (SCA7), spinocerebellar degeneration type 8 (SCA8), spinal cord Cerebellar degeneration type 12 (SCA12), dentate nucleus red nucleus pallidal louis atrophy (DRPLA), spinal and spinal muscular atrophy (SBMA) [above, CAG tribasic elongation], Friedreich ataxia (FRDA) ) [GAA tribase extension], myotonic dystrophy (DM) [CTG tribase extension], fragile X syndrome [CGG tribase extension] and the like. Of these, triplet repeats are present in gene untranslated regions in myotonic dystrophy and fragile X syndrome, and are present in introns in Friedreich schizophrenia and in translated regions in other diseases.

  Polyglutamine diseases in which triplet repeats of abnormally elongated CAG tribases (hereinafter sometimes referred to as triplet bases) are all translated into glutamine are Huntington's disease, bulbar spinal muscular atrophy, hereditary spinocerebellar degeneration (SCA1). 2, 3, 6, 7, 8, 12, etc.) and dentate nucleus red nucleus pallidal Louis atrophy.

  Living organisms using the selective recovery method of the present invention are living organisms including humans. Preferred are humans and experimental animals. The disease-causing gene from the living body may be collected as long as it contains genomic DNA or RNA, and blood, oral cells, part of body fluid, hair with root sheath, skin, bone, gingiva In addition, tissue pieces such as organs or formalin-fixed tissues are used. Preferred are blood, skin, biopsy tissue, cultured cells derived from the living body, and the like.

  In the selective recovery method of the present invention, genomic DNA or total RNA obtained from a living body may be used as it is, and further, cDNA obtained from the obtained total RNA may be used. When genomic DNA is used, depending on the target disease-causing gene, the genome may be fragmented with a restriction enzyme or the like as necessary.

  When cDNA is used, cDNA is obtained from a total RNA of a living body by performing a reverse transcription reaction. For obtaining total RNA, a conventionally known method or a commercially available kit can be used without particular limitation. Specifically, according to the acid guanidinium-phenol-chloroform (AGPC) method or the guanidine-cesium chloride ultracentrifugation method, a protein denaturant is added to a biological sample such as a cell solution or tissue to extract total RNA. In the AGPC method, for example, a commercially available TRIzol reagent (registered trademark, Invitrogen) or the like can be used.

  CDNA is reverse-transcribed from the extracted total RNA. To start the synthesis reaction by reverse transcriptase, a primer that is the starting point of the reaction is annealed on RNA to synthesize cDNA in the 3 'to 5' direction. Examples of the primer include an oligo dT primer targeting a polyA structure characteristic of mRNA, a primer specific to a known RNA sequence, and a random primer. As the reverse transcriptase, a reverse transcriptase derived from Avian Myeloblastosis Virus (AMV), a reverse transcriptase derived from Moloney murine leukemia virus (MoMuLV), SuperScript reverse transcriptase (Invitrogen), etc. are used. After cDNA synthesis, alkali treatment with alkali hydroxide or RNase H is added to degrade template RNA.

  Next, a synthetic labeled RNA probe containing a repetitive sequence in a disease gene is hybridized to genomic DNA, total RNA, or cDNA obtained above. The repeated sequence in the disease gene is a CAG repeat if the disease is Huntington's disease, and a GAA repeat if the disease is Friedreich ataxia. When repeat disease is suspected in a patient (living body), a single type of RNA probe is used and the selective recovery method of the present invention is carried out only once. For example, it is possible to perform PCR analysis and SNP typing for various repeat disease causative genes (HD, SCA3, etc.) having CAG). That is, if the repeat sequences are the same, it is possible to determine the repeat length and determine the haplotype of the mutant allele regardless of the type of repeat disease (HD, SCA3, etc.).

  The length of the synthetic RNA probe is usually 10 to 40 bases, preferably 15 to 25 bases, and particularly preferably 20 bases. If the probe length is shorter than 10 bases, there may be a problem that the specificity to the repeat sequence is lowered. When the total RNA and the synthetic RNA probe are hybridized, the sequence of the synthetic RNA probe is a complementary sequence of the target repeat sequence.

  The label of the probe is not particularly limited as long as it is necessary for recovery and purification after hybridization and can be used for recovery. Examples of the label include a label using biotin, avidin or digoxigenin, a fluorescent label such as FITC that can be recovered later by an antigen-antibody reaction, and a label using a nucleic acid analog such as bromodeoxyuridine (BrdU). Further, a probe and latex particles, magnetic particles, or the like that are directly or indirectly bound can be used. The binding position of the marker for labeling may be either the 5'-side or 3'-side end of the RNA probe, but the 5'-side end is preferred.

  The hybridization operation is performed in a solution. The hybridization buffer is not particularly limited as long as it is a buffer solution containing a salt concentration at which nucleic acids can hybridize with each other. For example, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 3.1 M sodium chloride and 1 M sodium citrate are used. A surfactant such as SDS, formamide, or the like can be added to the hybridization buffer as a solution for adjusting the binding between nucleic acids.

  For the hybridization work, a mixed solution of DNA or the like and a synthetic labeled RNA probe is usually heated to 80 to 100 ° C., preferably 95 ° C., and kept at that temperature for usually 1 to 10 minutes, preferably 5 minutes. To do. Thereafter, annealing is usually performed at 4 to 50 ° C., preferably 20 to 30 ° C., usually for 2 minutes or longer, preferably 30 to 60 minutes. The reaction volume is usually 10 to 200 μL, preferably 20 to 100 μL.

  Surprisingly, the above hybridization revealed that a mutant allele having a long repetitive sequence can be selectively or preferentially bound to an RNA probe over a normal allele having a short repetitive sequence. The evidence is as shown in Example 1.

Step (b): Recovery and Purification of Conjugate of cDNA and Synthetic Labeled RNA Probe Hereinafter, examples using cDNA synthesized from total RNA will be described in detail. The conjugate of the cDNA and the synthetic labeled RNA probe is recovered based on the label. For example, when the label is biotin, it may be recovered and purified by biotin-avidin reaction. Specifically, an avidin-resin carrier (product name: SoftLink Soft Release Avidin Resin, manufactured by Promega) is allowed to act on a conjugate of cDNA and a synthetic labeled RNA probe.

  In addition, a blocking buffer is added to efficiently recover and purify the conjugate of the cDNA and the labeled probe. For example, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM sodium chloride, 1% bovine serum albumin and 1 mg / mL yeast RNA are used as the blocking buffer. The composition of the blocking buffer is not particularly limited as long as it is used for nucleic acid hybridization and used for each labeling method. Nucleic acid reagents such as yeast RNA and Cot1 DNA, serum or albumin derived therefrom, protein reagents such as skim milk or casein derived therefrom, and the like can be used.

  In order to allow the avidin-resin carrier to act efficiently, a blocking buffer is added and usually stirred at a constant temperature of 25 to 50 ° C., preferably 30 to 37 ° C., usually 30 to 120 minutes, preferably 60 minutes or more. Make it work.

  After stirring, the mutant allele DNA or RNA is collected based on each labeling method. Specifically, when an avidin-resin carrier is allowed to act on a conjugate of cDNA and a synthetic labeled RNA probe, it is collected as a precipitate by centrifugation. If the label is a magnetic particle, it can be recovered by a magnet, and if it is a fluorescent substance such as FITC or a chemical substance such as BrdU, it can be recovered by a known specific antibody and a known recovery method (such as protein sepharose).

  The avidin-resin carrier is dissociated from the recovered cDNA-bound biotinylated RNA probe-avidin-resin carrier. Usually, cDNA can be dissociated by adding an alkaline solution or heating, but it is preferably dissociated by a biotin solution. Specifically, when SoftLink Soft Release Avidin Resin (Promega) is used, the avidin-resin complex is dissociated with 10 μL of 5 mM biotin solution. Thereafter, the resin carrier is removed by a known recovery method such as centrifugation described above to obtain a cDNA-synthetic labeled RNA probe conjugate.

Step (c): PCR amplification Repeatedly as appropriate for cases such as when it is desired to confirm early whether the cDNA bound to the biotinylated RNA probe recovered in step (b) is a normal or mutant allele. The harvest may be PCR amplified using specific primers that allow amplification of the region containing the sequence. For example, if the disease is Huntington's disease, as shown in Table 2 of Example 1, a forward primer and a reverse primer that sandwich the CAG repeat sequence of the huntingtin gene are selected. As PCR conditions, conventionally known conditions are appropriately modified according to the disease gene and its primers.

  The PCR amplification product is subjected to gel electrophoresis to confirm the expression of a mutant allele having a long repetitive sequence. As shown in FIG. 2 of Example 1 and FIGS. 6 to 8 of Example 2, only the mutant allele having a long repetitive sequence was detected in the gel electropherogram, and the normal allele having a short repetitive sequence was recovered. It is confirmed that it is not. In addition, the extension degree (number of repeats) of the repetitive sequence is grasped from the position of the detection band.

A method for identifying an allele having a long repetitive sequence, wherein a conjugate of the cDNA recovered in step (b) and the synthetic labeled RNA probe causes disease caused by abnormal extension of the repetitive sequence for each living body Can be used for That is, the present invention is a method for identifying, for each living body, a mutant allele having a long chain repeat sequence that causes a disease caused by abnormal extension of a repeat sequence, and includes the following steps:
(A) A synthetic labeled RNA probe containing a repetitive sequence in the sense strand or antisense strand base sequence of a disease gene in genomic DNA, total RNA, or cDNA synthesized from total RNA obtained from a living body suspected of the disease Hybridization
(B) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label; and (d) DNA-synthesis recovered in step (b). For the labeled RNA probe conjugate, determine the SNP site typing of the mutant allele and the haplotype based on it
A method for identifying the mutant allele is provided.

  Steps (a) and (b) of the identification method are the same as steps (a) and (b) in the selection or preferential recovery method.

Step (d): SNP Typing of Long Variant Allele In step (d), the haplotype is identified by examining the SNP in the allele for the DNA-synthetic labeled RNA probe conjugate of step (b). To do. In addition, instead of the DNA-synthetic labeled RNA probe conjugate recovered in step (b), the PCR amplification product in step (c) can also be used for SNP typing.

  For SNP typing, a known method such as TaqMan method, Invader method, single base extension method, SSCP method, RFLP method, direct sequence method, or the like can be used. Commercially available kits can also be used, and examples thereof include TaqMan allelecition assay (Applied Biosystems).

  The SNP site of each disease-causing gene is known from a haplotype database (HapMap database, http://www.hapmap.org/index.html.ja). A plurality of highly heterozygous SNP sites are selected from the database. Preferably, two or more SNP sites are selected.

The haplotype for each living body of the SNP site of the mutant allele obtained by the above identification method can be used in a method for preparing a tailor-made medicine for a disease caused by abnormal extension of a repetitive sequence. That is, the present invention is a method for preparing a tailor-made medicine for a disease caused by abnormal extension of a repetitive sequence, which comprises the following steps:
(A) hybridization of a synthetic labeled RNA probe containing a repetitive sequence in the sense strand or antisense strand base sequence of a disease gene to genomic DNA total RNA obtained from a living body or cDNA synthesized from total RNA;
(B) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label;
(D) SNP typing of the allele and the haplotype based on it for the conjugate of the DNA recovered in step (b) and the synthetic labeled RNA probe, and (e) the allele identified in step (d) There is provided a method for preparing the tailor-made medicament, comprising preparing a double-stranded RNA molecule that causes RNAi to a SNP site of a gene that is heterozygous to the other allele.

Steps (a) to (d) of the preparation method are the same as those of the selection or priority recovery method and identification method.
including.

Step (e): Preparation of double stranded RNA molecule that causes RNAi to SNP site In step (e), SNPs of mutant alleles with long repeat sequences identified by steps (d) and (c) A double-stranded RNA molecule that causes RNAi against a mutant allele is prepared by targeting the SNP site that is heterozygous for a normal allele that is a site and has a short repeat sequence.

  RNAi is a phenomenon in which mRNA having a base sequence complementary to double-stranded RNA is degraded. The RNAi method is a method of specifically suppressing the expression of an arbitrary gene by artificially introducing or expressing a double-stranded RNA into a cell using the phenomenon of RNAi. This double-stranded RNA that causes RNAi is named small interfering RNA (hereinafter abbreviated as “siRNA”). If the base sequence of the target mutant allele can be known, the target mutant allele can be specifically knocked down by synthesizing and introducing siRNA that induces RNAi into cells. The principle of gene expression suppression by RNAi will be described with reference to FIG. When siRNA having a length of about 21 nucleotides is chemically synthesized and introduced into a cell, the siRNA forms a RISC (RNA-induced silencing complex) together with a plurality of proteins. RISC recognizes mRNA of a target gene having a sequence complementary to the incorporated siRNA, and cleaves the siRNA-mRNA binding site on the mRNA side by nuclease activity in RISC. Thereby, the expression of the target gene is blocked.

  If the haplotype of the mutant allele SNP is identified, it is also possible to design siRNA that identifies the difference in the single nucleotide sequence of the SNP using the high sequence specificity of RNAi. In summary, siRNA molecules are designed such that their antisense strand is complementary to the sequence surrounding the SNP of the target allele. The length of the siRNA molecule is usually 18 to 23 bases, preferably 19 to 21 bases in the double-stranded part. The antisense strand of siRNA may protrude from the double-stranded portion at the 3 'end, and preferably protrudes by 2 bases. The sense strand of siRNA may protrude from the double-stranded portion at the 3 'end, and preferably protrudes from 0 to 2 bases.

  In order to improve the allele discrimination ability of siRNA and enhance the knockdown ability, mismatch bases can be introduced into the siRNA. In the mismatch siRNA, one or more, preferably 1 to 4 nucleotides mismatching with the antisense strand are introduced on the 3 ′ end side of the sense strand in the double-stranded portion, and The number of nucleotides complementary to the antisense strand allows for hybridization of both strands in the cell. The creation of mismatched siRNA is described in detail in WO 2005/015154 by the inventors. The contents of the specification of WO 2005/015154 are inserted herein for reference.

  In addition to the double-stranded RNA molecule itself, the siRNA molecule may be an expression vector incorporating a DNA sequence capable of transcribing each RNA strand of siRNA. Expression vectors include known plasmids and viral vectors. In addition, modified nucleic acids such as Locked Nucleotide Acid (LNA) and Peptide Nucleotide Acid (PNA), which have a nucleotide sugar-phosphate skeleton modified, can be used in the same manner as RNA.

  When siRNA molecules that cause RNAi against a specific SNP of each disease are stocked in advance, the siRNA stocked when the haplotype for each patient is determined by the method for identifying a long mutant allele of the present invention. It is possible to quickly supply a tailor-made medicine consisting of

  As shown in FIG. 10, it is preferable to induce RNAi knockdown to a plurality of heterozygous SNP sites, preferably 2 to 4 SNP sites, in order to make the knockdown effect strong and stable. . Therefore, in the method for preparing a tailor-made medicine of the present invention, it is preferable to prepare a cocktail in which a plurality of siRNA molecules are mixed. In that case, when the haplotype for each patient is determined by the method for identifying long-chain mutant alleles of the present invention, a plurality of siRNA molecules stocked in advance are appropriately mixed according to the haplotype of the patient to make a cocktail. Thus, an effective tailor-made medicine can be quickly provided for each patient.

  Whether or not the designed siRNA molecule induces a mutant allele-specific knockdown can be confirmed using a conventional method.

  The cell is preferably a mammalian cell capable of causing an RNAi phenomenon by siRNA and expressing a mutant allele. Specific examples include those derived from humans, mice, rats, hamsters, rabbits, dogs, monkeys and the like. Examples of established cultured cells include patient-derived immortalized cells, HeLa cells, HEK293 cells, SH-SY5Y cells, NIH / 3T3 cells, and COS-7 cells.

  Co-introduction of siRNA molecules and genes or parts thereof into cells is known. For example, those inserted into a plasmid or the like are introduced into cells by the lipofection method, electroporation method or the like.

  A reporter assay that constructs a reporter gene containing a base sequence that is a target of siRNA, and introduces it into a cell to verify the presence or absence and the effect of mutant allele-specific RNAi knockdown by siRNA There is. This reporter assay is also known. As the reporter gene, for example, a firefly luciferase gene, a Renilla luciferase gene, or the like is used.

  In use as a tailor-made medicine such as a double-stranded RNA molecule, a known introduction method can be appropriately employed for introduction into a living body, tissue or cell based on the introduction site. For example, a double-stranded RNA molecule or an expression vector thereof is injected into the brain, extremities, blood vessels, intraperitoneal cavity, etc., or percutaneously absorbed from the nasal mucosa. Furthermore, it is also possible to introduce using virus infection.

Hereinafter, the present invention will be described in more detail by showing examples of the present invention. However, the present invention is not limited to the following examples.
[Example 1]
1. Selective Recovery Method The selective recovery method of the present invention will be described with reference to the flowchart of FIG. The test disease was Huntington's disease (HD). HD is an inherited chronic progressive disease of autosomal dominant inheritance that develops after middle age. The clinical symptoms include involuntary movements, intelligence impairment, dementia, psychiatric symptoms, behavioral abnormalities, gait disturbance and the like. The causative gene for HD is the huntingtin (HTT) gene present in the short arm of chromosome 4 (4p16.3). The CAG repeat sequence of the base present in Exon1 of the HTT gene is about 20 times on average in the normal type, but it extends to an average of 40 times or more, resulting in abnormal conformation of the mutant protein, which accumulates in the nucleus It is said that it causes apoptosis of neurons and leads to onset.

  First, total RNA was extracted from immortalized cells derived from HD patients ((1) C0142 and (2) C0221, obtained from National Psychiatric and Neurological Center Hospital, Department of Neurology) using TRIzol reagent (registered trademark, Invitrogen). . CDNA was synthesized from total RNA using oligo dT primer (15 bases, Promega) and reverse transcriptase (trade name SuperScript III, Invitrogen), and then template RNA was digested using RNAseH (Invitrogen). It was.

  The synthesized cDNA (100 ng / 10 μL) and a synthetic biotinylated RNA probe (100 pmol) in which biotin is added to the 5 ′ side of the 20 base sequences shown in Table 1 are mixed in a hybridization buffer (10 mM Tris-HCl) to a final volume of 40 μL. , PH 7.5, 1 mM EDTA, 3.1 M sodium chloride, 1 M sodium citrate). The mixed solution was heat treated at 95 ° C. for 5 minutes, and then annealed at room temperature for 30 minutes.

  After annealing, 10 μL of avidin-resin carrier (trade name: SoftLink Soft Release Avidin Resin, Promega) was added, and further blocking buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM sodium chloride, 1% (w / vol) Bovine serum albumin, 1 mg / mL yeast RNA) was added to a final volume of 400 μL. The avidin-resin carrier used was pretreated with the above blocking buffer (1 mL) at 37 ° C. for 30 minutes.

  The mixed solution having a final volume of 400 μL was stirred at 180 ° C. for 30 minutes (180 rpm) to carry out a binding reaction between biotin and avidin. Thereafter, 1 mL of a washing solution (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM sodium chloride) was added for washing, and centrifugation (1500 g, 1 minute) was performed. After removing the supernatant, 1 mL of a washing solution was added in the same manner, and the operations of centrifugation and supernatant removal were repeated twice to obtain a precipitate (cDNA-binding biotinylated RNA probe-avidin-resin carrier).

  Finally, 10 μL of 5 mM biotin solution is added to the precipitate and left at room temperature for 10 minutes to dissociate the cDNA-bound biotinylated RNA probe from the avidin-resin carrier side of the cDNA-bound biotinylated RNA probe-avidin-resin carrier. I let you. Thereafter, centrifugation (1500 g, 1 minute) was performed, and the supernatant was collected as a sample (cDNA-binding biotinylated RNA probe). This recovered material was subjected to subsequent PCR analysis and allele discrimination test.

2. PCR analysis In order to confirm the effect of the selective recovery method of the present invention, it was examined by PCR analysis whether the cDNA recovered by the above-described method is a normal type or a mutant type allele.

  PCR amplification was performed using PrimeSTAR HS DNA polymerase with GC buffer (TaKaRa) and specific primers (Table 2) that amplify the CAG repeat of the huntingtin gene. In the PCR reaction, initial denaturation (95 ° C., 5 minutes), denaturation (98 ° C., 10 seconds), annealing (60 ° C., 5 seconds), extension reaction (72 ° C., 30 seconds) were repeated 26 times. .

  PCR amplification products were analyzed by electrophoresis (5% polyacrylamide gel, 50 mA, 2 hours). As shown in FIG. 2, the PCR product obtained by amplifying untreated cDNA (-) derived from an HD patient-derived immortalized cell shows two bands of different CAG repeat lengths, that is, a normal type and a mutant type. The HTT gene was detected. On the other hand, from the PCR product amplified from the cDNA (+) obtained by the selective recovery method of the present invention, a long (ie, mutant) band of the CAG repeat sequence was detected, and the short CAG repeat sequence ( That is, the normal type) band was very little or hardly recognized.

3. Allele Discrimination Test The cDNA (−) prepared from the HD patient-derived immortalized cells (C0142, C0221) and the cDNA (+) obtained by the selective recovery method were then used for the allelic discrimination test (TaqMan allelecition assay, Applied Bio System)) to determine whether the abundance ratio of normal and mutant huntingtin alleles with different CAG repeat lengths changed. Within the HTT gene transcription sequence, there are only 23 SNP sites with obvious heterozygosity. In this allele discrimination test, four SNP sites, rs36399, rs362331, rs362273, and rs362272, which have a high degree of heterozygosity, were targeted.

  A method of creating the discrimination test result in FIG. 3A will be briefly described. A graph is created in which the X-axis and Y-axis are the presence levels of alleles in a heterojunction relationship with each other. A standard curve is prepared using untreated cDNA (10, 1, 0.01, and 0 ng) that has not been subjected to the selective recovery method of the present invention as a comparative control. In the case of heterojunction, the calibration curve shows a straight line having a concentration-dependent slope (about 45 degrees slope) from the origin, whereas in the case of homojunction, the calibration curve is parallel to the X axis or Y axis. A straight line is shown. A calibration curve is also necessary to evaluate the effect of the selective recovery method regardless of variations in sample concentration or TaqMan Probe reaction bias.

  When the abundance ratio of normal and mutant HTT alleles is changed by the selective recovery method, the result of the SNP typing is displayed as a discrimination result on the upper left or lower right with respect to the calibration curve. The farther the typing result is from the calibration curve, the greater the change in the abundance ratio. This determination makes it possible to determine not only the haplotype of the HTT gene obtained by the selective recovery method (SNP configuration of alleles) but also whether or not there is a bias in the abundance ratio.

  FIG. 3A shows the results of an allele discrimination test for four SNP (rs363030, rs362231, rs362273, and rs362272) sites present in the HTT gene transcription sequence.

  In FIG. 3A, the sample (●) obtained by the selective recovery method of the present invention is located at a distance from the calibration curve prepared using the untreated cDNA sample (◯) that has not been subjected to the selective recovery method. Plotted. Thus, it was confirmed that the abundance ratio between the normal type and the mutant HTT gene was biased by performing the selective recovery method of the present invention.

  Moreover, the allele discrimination | determination test with respect to four SNP site | parts was done like the above using HD patient-derived immortalized cell genomic DNA. The results are shown in Table 3.

  The results of the allele discrimination test performed using genomic DNA coincided with the results of the paired gene discrimination test using cDNA. The typing result of rs362331 site in C0221 cells means that the SNP is a “C / C” homozygote. The other 3 sites of C0221 cells and all 4 sites of C0142 cells mean “A / G” or “C / T” heterozygotes.

  Table 4 shows the frequency of haplotypes for the four SNPs in all races and Japanese of the HTT gene obtained from the haplotype database (HapMap database).

1; C0142
2; C0221

  The haplotype (SNP configuration of alleles) derived from FIG. 3A is shown in FIG. 3B. The results in FIG. 3B are suggested to be consistent with the genomic DNA typing results shown in Table 3 and the haplotype frequency statistics in Japanese and all races shown in Table 4.

4). Construction of reporter gene expression plasmid containing HTT gene SNP For selection and evaluation of siRNA capable of suppressing allele-specific expression, pGL3-TK plasmid (Ohnishi et al., 2005) and Renilla luciferase expressing firefly luciferase gene A phRL-TK plasmid (Promega) that expresses the gene was used.

  As an allele sequence, a synthetic oligo DNA consisting of 41 bases containing a highly heterozygous SNP site (rs363030, rs362331, rs362273 or rs362272) in the HTT gene transcription sequence was selected. In order to be able to cope with either base of SNP, a synthetic oligo DNA for both bases of SNP was designed. Table 5 shows SNP peripheral sequences (synthetic oligo DNA sequences) in the huntingtin gene transcription sequence. This was inserted into the untranslated region (3′UTR) of each reporter gene to construct a reporter gene expression plasmid containing a huntingtin gene SNP.

In the above table, restriction enzyme sites and SNPs are indicated by lowercase letters and underlines, respectively.
The degree of heterozygosity for each SNP was referenced from the NCBI SNP database.
ss; sense strand (nucleotide strand having a sequence homologous to the coding strand of the gene)
as; antisense strand (nucleotide strand having a sequence complementary to the coding strand of the gene)

  Allele-specific siRNAs designed for the SNP sites in the HTT gene transcription sequence and their sequences are shown in Tables 6A to 6D.

The SNP site is underlined.
ss; sense strand as; antisense strand

5. Cell culture Human-derived cell lines, HeLa cells and HEK293 cells, were cultured in DMEM culture medium (Invitrogen) containing 10% fetal bovine serum (FBS, Invitrogen) and antibiotics (100 units / ml penicillin, 100 μg / ml streptomycin, Wako). In the presence of the company). Human neuroblastoma-derived cell line SH-SY5Y cells were cultured in the presence of the above-mentioned serum and antibiotic-containing DMEM / F-12 medium (Invitrogen).

6). Gene transfer and reporter assay The day before gene transfer, HeLa cells and HEK293 cells were dispersed by trypsin digestion, seeded in a 24-well culture plate at a cell density of 1 × 10 ⁵ cells / cm ² , and cultured in an antibiotic-free DMEM culture solution Went.

  24 hours later, siRNA targeting each SNP (final concentration 20 nM), HTT gene SNP-containing reporter allele expression plasmid [pGL3-TK-backbone plasmid (300 ng / well), phRL-TK-backbone plasmid (100 ng / well) And a β-galactosidase gene expression plasmid [pSV-beta-Galactosidase control plasmid (50 ng / well, Promega): standardized plasmid for standardization of luciferase activity] that was not subjected to RNAi knockdown.

  As a comparative control, siRNA that does not induce RNAi (siControl; final concentration 20 nM, Qiagen) was also used for the same gene transfer.

  24 hours after the introduction, cell extracts were prepared, and the activity of the expressed reporter alleles (two luciferases) and the β-galactosidase gene as an external standard were measured respectively in Dual-Luciferase reporter assay system (Promega), and It measured using Beta-Glo assay system (Promega). A value (standardized value) obtained by dividing the measured value of luciferase activity by the measured value of β-galactosidase activity was evaluated as the expression level of the reporter.

  Furthermore, in order to eliminate the bias due to the difference in the activity of the two luciferases with respect to the reporter, an experiment was also conducted in which the reporter gene was replaced for each SNP site.

7). Improvement of allele discrimination ability by mismatch siRNA Compared to SNP sites (rs363030 C-allyl, rs362331 C-allyl) where allele-specific RNAi knockdown could not be induced by the above reporter assay, the allele discrimination ability was low. In addition, using siRNAs having strong inhibitory effects on both alleles, it was examined whether new mismatches could be introduced into their siRNA sequences to improve the allele discrimination ability. Evaluate these effects using the same method as above. The sequence of the improved siRNA (mismatched siRNA) is shown in Table 7.

SNP sites and mismatched nucleotides are underlined and lowercase, respectively.
ss; sense strand as; antisense strand

8). IC50 test of selected siRNA An IC50 (50% inhibitory concentration) test was performed on siRNA that had strong allele discrimination ability and suppression effect. Table 8 shows allele-specific siRNAs having strong inhibitory effects and high SNP discrimination ability and their sequences.

SNP sites and mismatched nucleotides are indicated by underlining and lowercase letters, respectively.
ss; sense strand as; antisense strand

  HeLa cells were cultured in a 96-well culture plate, and after 24 hours, three types of plasmids and siRNA were introduced under the same conditions as described above. At this time, siRNA is adjusted to each final concentration (40, 20, 10, 5, 1.25, 0.32, 0.08, 0.02, 0.005, 0.001, 0 nM) Introduced with.

  Thereafter, the expression of the reporter gene and the external standard gene was measured by the same method as described above. The graphing and numerical calculation in the IC50 test were obtained by fitting to a sigmoid curve with a Hill coefficient of 1 (FIGS. 4A to 4D). 4A to 4D, ● represents the activity of firefly luciferase, and ■ represents the activity of Renilla luciferase, which are shown as values normalized by the activity of the external standard galactosidase. Similarly to the above, an experiment was also conducted in which the reporter gene was replaced with respect to each SNP site in order to eliminate the bias due to the difference between the two kinds of luciferase activities.

  siRNAs exhibiting the strongest suppressive effect and high SNP discriminating ability against the alleles at the SNP sites of rs363030 (FIG. 4A), rs362331 (FIG. 4B), rs362273 (FIG. 4C), and rs362272 (FIG. 4D) (Table) As a result of conducting an IC50 test on 8), the tested siRNA showed allele-specific and strong RNAi-suppressing effects on both alleles of the targeted SNP site.

9. Specific Expression Suppression of Endogenous Mutant Huntingtin Gene Using HD Patient-Derived Immortal Cells Based on SNP haplotype information of mutant HTT gene determined by selective recovery method, allele discrimination test and PCR analysis of the present invention And select functional siRNAs (siRs099_T10, siRs331_C9 (C15), siRs273_G9, and siRs272_A9) for the four SNP sites (rs363030, rs362231, rs362273, and rs362272), and select the long mutant allele-specific RNA down went. In addition, an introduction test of an siRNA cocktail (siCocktail) in which equal amounts of all four types were mixed was also conducted. As a comparative control, an introduction test of siRNA that does not induce RNAi (siControl) was also conducted.

About 1 × 10 ⁶ HD patient-derived immortalized cells (C0142), siRNA having a final concentration of 5 μM was introduced into an electroporation method (gene introduction system Nucleofector, Amaxa) to induce RNAi. Cells were harvested 48 hours after introduction.

Total RNA was extracted, cDNA synthesis, and allelic discrimination tests were performed on each SNP site to evaluate the allele-specific RNAi effect of the selected siRNA. Similar to the above-described allele discrimination test, a calibration curve is prepared using an untreated sample in which no siRNA has been introduced, and the sample to which siRNA has been introduced is plotted at a distance from the standard curve. Evaluated. The results are shown in FIGS. 5A to 5D, the meanings of the marks are as follows.
○: Untreated sample without siRNA (for creating calibration curve)
●: Sample with one type of siRNA introduced ◎: Sample with a siRNA cocktail (siCocktail) mixed with an equal amount of four types of siRNA ■ Sample with siRNA (siControl) that does not induce RNAi

  As shown in FIGS. 5A to 5D, the sample (●) into which one type of siRNA was introduced and the sample (◎) into which an siRNA cocktail (siCocktail) in which equal amounts of four types of siRNA were mixed were contrasted with FIG. 3A. Shifted to the opposite side of the calibration curve, that is, the expression of the mutant HTT allele was significantly suppressed compared to the normal allele. In particular, siRs099_T10 (●) and siCocktail (◎) in which equal amounts of four types of siRNA were mixed showed the strongest allele-specific expression suppression effect.

[Example 2] Analysis of CAG triplet repeat disease-causing genes other than HTT gene The method of the present invention is effective not only for HD but also in diseases caused by abnormal extension of CAG repeats. In order to confirm, the selective recovery method of the present invention and PCR analysis were performed in the same manner as in Example 1 for CAG triplet repeat diseases other than HD shown in Table 9.

1. Selective collection method HD patient-derived immortalized cells and three types of human-derived cell lines (HeLa cells, HEK293 cells, SH-SY5Y cells) were used. Hybridization was performed with cDNA obtained from each cell using the same synthetic biotinylated RNA probe as in Example 1.

2. PCR analysis From the cDNA-binding biotinylated RNA probe-avidin-resin carrier obtained by the above hybridization, after collecting the cDNA-binding biotinylated RNA probe, primers specific to each disease gene that amplifies the CAG repeat sequence (Table 10). ).
PCR amplification was performed under the same conditions as HD for amplification of the causative genes of SCA3 and DRPLA, but the amplification of the causal gene of SCA6 (CACNA1A) was changed to 36 times.

  The electrophoresis was performed under the same conditions as in Example 1. The results of electrophoresis are shown in FIGS. In FIG. 6, as a result of analyzing the expression of the ATN1 gene in HD patient-derived immortalized cells and three types of human-derived cell lines, the expression of two ATN1 transcripts having different CAG repeat sequence lengths (PCR) in SH-SY5Y cells (PCR) Product). When the same experiment as described above was performed using the cDNA of the SH-SY5Y cell, it was found that most of ATN1 amplified from the obtained cDNA (+) was a PCR product having a long CAG repeat sequence.

  In FIG. 7, the expression of ATXN3 in untreated (−) cells ((1) HEK293 cells, (2) HeLa cells, (3) SH-SY5Y cells) was examined. As a result, the CAG repeat sequence was long in HeLa cells. ATXN3 was expressed. Thus, an equal amount of untreated cDNA from HeLa cells ((2); long repeats) and HEK293 cells ((1); short repeats) or SH-SY5Y cells ((3); short repeats) When mixed ((1) + (2), (2) + (3)) and subjected to selective recovery method and PCR analysis, most of the PCR products amplified from cDNA (+) are long type of repetitive sequences. It was found to be ATXN3 (derived from HeLa cells).

  In FIG. 8, the HD patient-derived immortalized cell C0221 expressed two types of CACNA1A having different lengths, but most of the PCR products obtained by amplifying the obtained cDNA (+) were CACNA1A having a long CAG repeat sequence. It has been shown.

Claims (7)

A method for selecting or preferentially recovering a mutant allele or gene product having an abnormal extension of a repetitive sequence that causes tribasic repeat disease, comprising the following steps:
(A) genomic DNA which the disease is acquired from a living body suspected, the cDNA synthesized from total RNA or total RNA, synthetic labeling containing tribasic repeat sequence in the sense or antisense strand nucleotide sequence of a disease gene Hybridizing the RNA probe, and (b) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label,
A method for selecting or preferentially recovering a mutant allele or gene product having an abnormal extension of a repetitive sequence, which causes a three-base repeat disease . In step (a), as the synthetic labeled RNA probe, a synthetic biotinylated RNA probe in which biotin is added as a label to the 5 ′ side of a repetitive sequence in a gene or gene product is used, and in step (b), DNA or A mutant allele having an abnormal extension of a repetitive sequence, which causes a tribase repeat disease according to claim 1 , wherein a conjugate of RNA and a synthetic biotinylated RNA probe is recovered using a biotin-avidin reaction. Or gene product selection or preferential recovery methods. In addition, the following steps:
(C) PCR amplification is performed on the conjugate of the DNA recovered in step (b) and the synthetic labeled RNA probe using a primer specific for the disease gene that amplifies the repetitive sequence.
A method for selecting or preferentially recovering a mutant allele or gene product having an abnormal extension of a repetitive sequence, which causes a tribasic repeat disease according to claim 1 or 2 . The disease is Huntington's disease, hereditary spinocerebellar degeneration, dentate nucleus red nucleus pallidal Louis atrophy, bulbar spinal muscular atrophy, Friedreich ataxia, myotonic dystrophy or fragile X syndrome A method for selecting or preferentially recovering a mutant allele or gene product having an abnormal extension of a repetitive sequence, which causes the three-base repeat disease according to any one of claims 1 to 3 . A method for identifying, for each organism , a mutant allele having an abnormal extension of a repetitive sequence, which causes a trinucleotide repeat disease, comprising the following steps:
(A) genomic DNA which the disease is acquired from a living body suspected, the cDNA synthesized from total RNA or total RNA, synthetic labeling containing tribasic repeat sequence in the sense or antisense strand nucleotide sequence of a disease gene Hybridizing RNA probes,
(B) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label; and (d) DNA-synthesis recovered in step (b). For the labeled RNA probe conjugate, determine the SNP site typing of the mutant allele and the haplotype based on it
A method for identifying the mutant allele, comprising: A method for preparing a tailor-made medicine for a tribasic repeat disease , comprising the following steps:
(A) a cDNA where the disease is synthesized from genomic DNA total RNA or total RNA obtained from a biological suspected, synthesized labeled RNA containing tribasic repeat sequence of the sense strand or antisense strand nucleotide sequence of a disease gene Hybridize the probe,
(B) recovering and purifying the conjugate of the DNA or RNA obtained in step (a) and the synthetic labeled RNA probe based on the label;
(D) SNP typing of the allele and the haplotype based on it for the conjugate of the DNA recovered in step (b) and the synthetic labeled RNA probe, and (e) the allele identified in step (d) A method for preparing a tailor-made medicament, comprising preparing a double-stranded RNA molecule that causes RNAi to a SNP site of a gene that is heterozygous to the other allele. The method for preparing a tailor-made medicine according to claim 6 , wherein in the step (e), two or more kinds of double-stranded RNA molecules that cause RNAi to the SNP site are mixed.