WO2017170101A1 - Method for detecting single-base substitution using ion exchange chromatography - Google Patents

Method for detecting single-base substitution using ion exchange chromatography Download PDF

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WO2017170101A1
WO2017170101A1 PCT/JP2017/011644 JP2017011644W WO2017170101A1 WO 2017170101 A1 WO2017170101 A1 WO 2017170101A1 JP 2017011644 W JP2017011644 W JP 2017011644W WO 2017170101 A1 WO2017170101 A1 WO 2017170101A1
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primer
allele
strand
base pair
double
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Japanese (ja)
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海老沼　宏幸
内田　桂
百合子 塚本
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積水メディカル株式会社
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to a specific detection method for mutation such as single base substitution or point mutation contained in a nucleic acid sample.
  • Genetic mutations include genetically inherited germ cell mutations and acquired somatic mutations in individual cells.
  • germline mutations single-nucleotide polymorphisms of specific genes (Single Nucleotide) Polymorphism (SNP) specific genotypes, somatic mutation point mutations (single base substitutions), insertions, deletions, etc. have been reported to be associated with various diseases, and recently their base sequences have been identified Therefore, it is used to select patients who are expected to be effective for a specific drug.
  • UGT1A1 gene polymorphism is used to determine the risk of developing serious side effects of irinotecan, an anticancer drug.
  • UGT1A1 gene polymorphism testing two base sequences (* 6, * 28) are targeted for wild-type without mutation, heterozygote with both wild-type and mutant-type, and homozygous for mutant-type only. It is necessary to determine the joined body.
  • the JAK2 gene mutation used to diagnose polycythemia vera which is one of the genetic mutations in myeloid proliferative disease, is an acquired somatic mutation that gains function. Mutations result in constitutive receptor tyrosine kinase activation. Since this point mutation is not only detected but also quantitative transition is useful in clinical practice, it is required to calculate the allele frequency. That is, as with gene polymorphism detection, it is necessary to quantitatively detect both mutant and wild types in point mutation detection.
  • the MPL (Myeloproliferative leukemia virus) gene mutation set in the WHO classification criteria for primary myelofibrosis has point mutations and deletion / insertion mutations at bases 1543 to 1544 of exon 10 and codon 515. Since there are several mutation patterns at the same position, it is desirable to distinguish and detect these patterns.
  • ion exchange chromatography As a method for separating and detecting nucleic acids with high accuracy in a short time, ion exchange chromatography is used.
  • the advantage of applying this ion exchange chromatography to the detection of nucleic acids is that the nucleic acids can be separated according to their chain lengths.For example, if the length of amplification products by PCR (polymerase chain reaction) is adjusted, multiple It is also possible to separate and detect the amplification products in a single measurement.
  • this principle can theoretically be applied to the detection of multiple gene mutations as described above, in order to detect a difference of only one base such as single base substitution or point mutation, a contrivance is required.
  • Non-patent Document 1 Non-patent Document 1
  • amplification with a pair of primers not related to mutation proceeds, so that components necessary for the amplification are consumed and there is a possibility of affecting the specific reaction.
  • two pairs of primers are used, differences in hybridization and amplification efficiency are likely to occur, and when detecting gene mutations such as JAK2 gene mutation, allele frequency is calculated accurately. It was difficult.
  • this method limits the detection of up to two types of mutations, and there are many types of mutations such as multiple mutations around codon 515 in MPL and point mutations in codons 12 and 13 such as KRAS and NRAS. It cannot be applied to mutation.
  • an object of the present invention is to provide a method for accurately and quantitatively separating and detecting many types of gene mutations, particularly single base substitutions or point mutations.
  • a non-nucleotide component is added to at least one 5 ′ end of an ASP for analyzing gene mutation, particularly single base substitution or point mutation, and its paired primer.
  • the present inventors completed the present invention by amplifying by PCR and separating the amplified products by ion exchange chromatography, so that the amplification products can be separated and detected even if the length is the same. That is, the present invention comprises the following configurations [1] to [8].
  • a method of analyzing a gene amplification product amplified using one or more kinds of allele-specific primers using ion exchange chromatography, wherein 5 ′ of at least one of the allele-specific primer or a primer paired therewith is used.
  • a method for detecting a gene mutation wherein a terminal is added with a non-nucleotide component.
  • the non-nucleotide component is a substance that causes a change in the charge at the 5 ′ end of the primer.
  • a method for detecting the presence of at least one allele at a polymorphic site contained in a double-stranded deoxyribonucleic acid in a sample comprising the following steps: (a) providing a sample containing double-stranded deoxyribonucleic acid containing a polymorphic site; (b) providing a first primer, a second primer, and a third primer;
  • the sequence of the first primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having the first allele at the polymorphic site and any one of the three bases at the 3 ′ end.
  • the sequence of the second primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having the second allele at the polymorphic site and any one or two of the three bases at the 3 ′ end Or one or both of three bases or two bases at the 3 ′ end correspond to the polymorphic site,
  • the sequence of the third primer does not include the polymorphic site and is complementary to the first strand of the double-stranded deoxyribonucleic acid, At least one of the first primer and the second primer has a non-nucleotide component added thereto; (c) performing a polymerase chain reaction,
  • the extension of the strand by the polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the first allele is a double strand having the first allele.
  • Second strand of a double-stranded deoxyribonucleic acid that preferentially occurs compared to the extension of a strand by a polymerase from a second primer hybridized to the second strand of a strand deoxyribonucleic acid and that has a second allele Comparison of polymerase chain elongation from the second primer hybridized to the polymerase with polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid with the second allele Under preferential conditions; (d) subjecting the amplification product of the polymerase chain reaction to ion exchange chromatography;
  • the difference in size between the amplification product of the polymerase chain reaction from the first primer and the third primer and the amplification product of the polymerase chain reaction from the 21st primer and the third primer is 0 base pairs, 1 Base pair, 2 base pair, 3 base pair, 4 base pair, 5 base pair, 6 base pair, 7 base pair, 8 base pair, 9 base pair, or 10
  • step (a) is a step of extracting genomic DNA from a mammalian somatic cell sample such as a human.
  • the polymorphic site is UGT1A1 * 28 polymorphism (rs8175347), UGT1A1 * 6 polymorphism (rs4148323), JAK2 1849G> T (V617F) mutation site (rs77375493), MPL 1589G> T (W515L) mutation site (rs121913615), Alternatively, the method according to [4] or [5] above, wherein MPL 1588: 1599TG> AA (W515K) mutation site (rs121913616).
  • the length of the amplification product is the same, or depending on the sequence, it may differ by 1 to 2 bases, but separation and detection by ion exchange chromatography is generally difficult. is there.
  • the allele-specific primer used in the present invention may be a primer that can specifically bind to the base sequence of the gene polymorphism or gene mutation, and includes bases that include not only single-base substitutions but also insertion and deletion mutations. Any material that is specific to the sequence and applicable to the separation according to the present invention can be used without particular limitation.
  • the non-nucleotide component used in the present invention is preferably a substance that causes a change in the charge at the 5 ′ end of the primer, and a gene amplification product amplified using the allele-specific primer to which the primer is added.
  • a substance that causes a change in the charge at the 5 ′ end of the primer and a gene amplification product amplified using the allele-specific primer to which the primer is added.
  • Preferred non-basic substances include, for example, ionic functional groups themselves or molecules containing at least one ionic functional group.
  • the ionic functional group is not particularly limited, and examples thereof include hydroxy group, aldehyde group, carboxy group, amino group, nitro group, nitroso group, thiol group, sulfonic acid group, fluoro group, chloro group, bromo group, and iodo group. it can.
  • Fluorescent dyes used as primer modifications can also be used as non-nucleotide components, such as Alexa Fluor series, Cy series, ATTO series, DY series, DyLight series, FAM, and TAMRA.
  • addition of functional substances such as digoxin and biotin and amide group modification can be used without limitation.
  • the effects of these modifiers can be further demonstrated by optimizing the length of the gene amplification product amplified using the allele-specific primer. That is, even with the same modifying substance, the difference becomes more noticeable as the length of the gene amplification product is shorter. Therefore, in the present invention, when analyzing a gene amplification product amplified using an allele-specific primer having a non-nucleotide component added to the 5 ′ end using ion exchange chromatography, only the type of non-nucleotide component is used. In addition, it is possible to maximize the effects of the present invention by appropriately combining the lengths of the gene amplification products.
  • cation exchange chromatography or anion exchange chromatography is selected as ion exchange chromatography in consideration of the isoelectric point of the substance to be measured, the pH of the eluent (also referred to as mobile phase), the salt concentration, and the like. can do.
  • anion exchange chromatography it is preferable to use anion exchange chromatography.
  • nucleic acid is a general term for ribonucleic acid (hereinafter also referred to as ribonucleic acid or RNA) and deoxyribonucleic acid (hereinafter also referred to as deoxyribonucleic acid or DNA), and includes bases, sugars, and phosphates.
  • the nucleotides consisting of are linked by phosphodiester bonds.
  • the nucleic acid to be extracted may be either DNA or RNA, and it may be a target whether it is fragmented or not. Examples of the origin of the nucleic acid include, but are not limited to, animals, plants, all organisms including microorganisms, and viruses.
  • nucleic acid inside a cell nucleus the nucleic acid derived from the nucleus which the organelle represented by a mitochondria, a chloroplast, a nucleolus, etc. hold
  • maintain may be sufficient.
  • it may be an artificially synthesized nucleic acid, a plasmid or a viral vector generally used as a vector.
  • a double-stranded deoxyribonucleic acid can be exemplified as a preferred nucleic acid for the method of the present invention, and more preferred nucleic acids include a double-stranded nucleic acid having a nucleotide sequence that includes a single nucleotide polymorphism, a point mutation, and / or a deletion / insertion mutation.
  • a chain deoxyribonucleic acid can be exemplified.
  • the PCR amplification method is not particularly limited, and known methods can be appropriately selected and used according to the sequence, length, amount, etc. of the nucleic acid to be amplified.
  • the chain length of the PCR amplification product can be appropriately selected in consideration of factors such as shortening the PCR amplification time, shortening the analysis time in ion exchange chromatography, and maintaining separation performance.
  • the upper limit of the PCR amplification product chain length is preferably 1000 bp or less, more preferably 700 bp or less, and even more preferably 500 bp or less.
  • the lower limit of the PCR amplification product chain length is preferably 30 bp, more preferably 40 bp.
  • UGT1A1 * 28 polymorphism rs8175347
  • UGT1A1 * 6 polymorphism rs4148323
  • JAK2 1849G> T V617F
  • MPL 1589G> T W515L
  • MPL 1588: 1599TG> AA (W515K) mutation site rs121913616.
  • FIG. 1 shows the result of overwriting the elution peaks of amplification products with three fluorescently labeled primers (SEQ ID NOs: 3, 7, and 8).
  • FIG. 2 shows the separation and detection of amplification products from the * 6 polymorphic site of the UGT1A1 gene using the non-nucleotide component-added ASP.
  • FIG. 3 shows the separation and detection of amplification products from around the codon 515 site of the MPL gene using non-nucleotide component-added ASP.
  • ASP SEQ ID NO: 1
  • SEQ ID NO: 12 reverse primer
  • SEQ ID No. 2 a primer modified with a non-nucleotide component was prepared separately at the 5 'end of ASP (SEQ ID No. 2 was assigned to Thermo Fisher Co., and SEQ ID No.
  • SEQ ID NOS: 4 and 6 are consigned to Integrated DNA Technologies MBL Co., Ltd., other than that, consigned to Sigma-Aldridge Co., Ltd.)
  • Table 1 shows the SEQ ID NO: primer sequence, oligonucleotide length (bp), type of non-nucleotide component, and excitation wavelength and fluorescence wavelength (nm) of the non-nucleotide component.
  • reaction solution containing the following reagents was prepared and amplified by CFX96 (Bio-Rad) by two-step allele-specific PCR.
  • purified DNA collected from a person who was homozygous for the UGT1A1 gene * 6 allele was used.
  • FIG. 1 shows the result of overwriting the elution peak of the amplification product with the three fluorescently labeled primers (SEQ ID NOs: 3, 7, and 8) whose elution time was particularly large.
  • Example 2 Separation and detection of amplification products from * 6 polymorphic site of UGT1A1 gene using non-nucleotide component added ASP
  • the primer for detecting the wild type at the * 6 polymorphic site is an ASP that can specifically amplify the wild type by introducing a non-labeled mismatch base at one site, as in SEQ ID NO: 1 described in Example 1.
  • SEQ ID NO: 13 was prepared separately. In this study, purified DNA collected from a human who was heterozygous for the UGT1A1 gene * 6 gene wild-type, * 6 allele, and homozygous was used.
  • reaction solution containing the following reagents was prepared and amplified by CFX96 (Bio-Rad) by two-step allele-specific PCR. Ion exchange chromatography was measured using the same conditions as in Example 1.
  • sample 1 which is a heterozygote of the allele
  • two elution peaks were observed at the elution position of the unlabeled amplification product (elution time around 8.6 minutes) and the elution position of the FAM-labeled amplification product (elution time 9.2 minutes).
  • sample 2 which is a homozygote of 6 alleles
  • an elution peak was observed only at the elution position of the FAM-labeled amplification product
  • wild-type sample 3 an elution peak was observed only at the elution position of the unlabeled amplification product. It was found that the genotype of the * 6 polymorphic site of UGT1A1 gene can be easily and accurately distinguished.
  • the codon 515 of the MPL gene has three mutation patterns, W515L, W515K, and W515A, each of which has a different base sequence at positions 1543-1544.
  • a forward primer unlabeled ASP (SEQ ID NO: 14 to 16) for detecting each mutant type was prepared, and a reverse primer (SEQ ID NO: 17) to be paired with it was prepared.
  • ASP SEQ ID NOs: 18, 19, and 20
  • plasmid DNAs incorporating the respective gene mutation sequences (SEQ ID NOs: 21 to 23) were prepared for specimens (consigned to Eurofin Genomics Co., Ltd.).
  • SEQ ID NO: 17 (common reverse primer) 5'-GGCGGTACCTGTAGTGTGC-3 '
  • SEQ ID NO: 18 (ASP for biotin-labeled W515K) 5'-Biotin-CTGCTGCTGCTGAGGAA-3 '
  • SEQ ID NO: 19 (ASP for amino group labeling W515K) 5'-NH2-CTGCTGCTGCTGAGGAA-3 '
  • SEQ ID NO: 20 (ASP for Cy3.5 fluorescent dye labeled W515K) 5'-Cy3.5-CTGCTGCTGCTGAGGAA-3 '
  • SEQ ID NO: 21 (W515L gene mutation sequence)
  • SEQ ID NO: 22 (W515K gene mutation sequence)
  • SEQ ID NO: 23 (W515A gene mutation sequence)
  • Reagents, amplification conditions and ion exchange chromatography conditions 25 ⁇ L of a reaction solution containing the following reagents was prepared and amplified by CFX96 (Bio-Rad) by two-step allele-specific PCR.
  • Fig. 3 shows the results of ion exchange chromatography separation and detection of amplification products around the codon 515 site of the MPL gene using the non-nucleotide component-added ASP.
  • the elution position (elution time 3.82 minutes) of the W515A amplification product (45 bp) is the same as the elution position of the W515L and W515K amplification products (each 46 bp).
  • the amplification products of W515L and W515K have almost the same elution position (elution time 4.42 minutes and 4.35 minutes), and the presence or absence of mutation is confirmed, but the pattern cannot be identified. found.
  • the elution positions of the amplification products using ASP (SEQ ID NOs: 18, 19, and 20) added with non-nucleotide components for W515K were 4.16 minutes, 3.91 minutes, and 4.97 minutes, respectively, and the elution positions of W515L and In addition to not overlapping, it was also confirmed that it does not overlap the elution position of the amplified product of W515A.
  • various elutions can be achieved by adding multiple non-nucleotide components that change the elution time by ion exchange chromatography to multiple ASPs, and adding non-nucleotide components to the paired primers. It becomes possible to adjust the time. Further, by using a fluorescent dye as the non-nucleotide component, it is possible to discriminate by the wavelength to be detected even if there is no difference in the elution time by selecting the fluorescent wavelength that does not crosstalk.
  • a method for detecting the amplification product in addition to the method of separating the amplified reagent as it is by ion exchange chromatography, a method of separately preparing a plurality of amplification reagents and separating the mixed solution by ion exchange chromatography is also possible. is there.
  • genotypes and single base substitutions of a plurality of gene polymorphisms that have been difficult with the conventional method can be easily and accurately detected.
  • a method capable of responding to is provided.

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Abstract

The purpose of the present invention is to provide a method for accurately and quantitatively separating and detecting multiple types of genetic mutations, particularly single-base substitutions or point mutations. An allele-specific primer (ASP) for analyzing a genetic mutation, particularly a single-base substitution or a point mutation, wherein a non-nucleotide component is added to the 5' terminus of the ASP and/or a primer complementary thereto, amplification thereof by PCR is performed, and the amplification product is separated by ion exchange chromatography, whereby the amplification product can be separated and detected even when the length thereof is the same.

Description

イオン交換クロマトグラフィーを用いた一塩基置換検出方法Single-base substitution detection method using ion exchange chromatography
　本発明は、核酸試料中に含まれる一塩基置換若しくは点変異などの変異の特異的な検出方法に関する。 The present invention relates to a specific detection method for mutation such as single base substitution or point mutation contained in a nucleic acid sample.
　遺伝子変異には、遺伝的に受け継がれる生殖細胞変異と後天的に一つ一つの細胞内で引き起こされる体細胞変異があるが、生殖細胞変異の中で特定の遺伝子の一塩基多型(Single Nucleotide Polymorphism; SNP)の特定の遺伝子型や体細胞変異の点変異(一塩基置換)、挿入、欠失などが、各種疾病に関連していることが報告されており、近年それらの塩基配列を同定することによって、特定の薬剤に効果が期待される患者の選別に利用されている。例えば、UGT1A1の遺伝子多型は、抗がん剤であるイリノテカンの重篤な副作用の発現のリスクを判断することに利用されている。UGT1A1の遺伝子多型検査においては、2つの塩基配列(*6、*28)を対象に、それぞれ変異を持たない野生型、野生型と変異型の両方を持つヘテロ接合体、変異型のみのホモ接合体を判別する必要がある。骨髄性増殖性疾患の遺伝子変異の1つである真性赤血球増加症の診断に用いられているJAK2遺伝子変異は、機能獲得型の後天的体細胞変異であり、エクソン14の1849 G>Tの点変異により、恒常的な受容体型チロシンキナーゼの活性化をもたらす。この点変異は、検出されるだけでなく、量的推移が診療上有用とされることから、アレル頻度を算出することが要求される。すなわち、遺伝子多型検出と同様に、点変異検出においても変異型及び野生型の両方を定量的に検出する必要がある。さらに、原発性骨髄繊維症でWHO分類の診断基準に設定されているMPL(Myeloproliferative leukemia virus)遺伝子変異は、エクソン10、コドン515の1543～1544番目の塩基に点変異や欠失・挿入変異が入ることから、同じ位置でいくつかの変異パターンがあり、そのパターンを区別して検出することが望ましい。 Genetic mutations include genetically inherited germ cell mutations and acquired somatic mutations in individual cells. Among germline mutations, single-nucleotide polymorphisms of specific genes (Single Nucleotide) Polymorphism (SNP) specific genotypes, somatic mutation point mutations (single base substitutions), insertions, deletions, etc. have been reported to be associated with various diseases, and recently their base sequences have been identified Therefore, it is used to select patients who are expected to be effective for a specific drug. For example, UGT1A1 gene polymorphism is used to determine the risk of developing serious side effects of irinotecan, an anticancer drug. In UGT1A1 gene polymorphism testing, two base sequences (* 6, * 28) are targeted for wild-type without mutation, heterozygote with both wild-type and mutant-type, and homozygous for mutant-type only. It is necessary to determine the joined body. The JAK2 gene mutation used to diagnose polycythemia vera, which is one of the genetic mutations in myeloid proliferative disease, is an acquired somatic mutation that gains function. Mutations result in constitutive receptor tyrosine kinase activation. Since this point mutation is not only detected but also quantitative transition is useful in clinical practice, it is required to calculate the allele frequency. That is, as with gene polymorphism detection, it is necessary to quantitatively detect both mutant and wild types in point mutation detection. Furthermore, the MPL (Myeloproliferative leukemia virus) gene mutation set in the WHO classification criteria for primary myelofibrosis has point mutations and deletion / insertion mutations at bases 1543 to 1544 of exon 10 and codon 515. Since there are several mutation patterns at the same position, it is desirable to distinguish and detect these patterns.
　核酸を短時間に精度良く分離検出できる方法として、イオン交換クロマトグラフィーが利用されている。このイオン交換クロマトグラフィーを核酸の検出に応用する利点としては、核酸をその鎖長に依り分離できることから、例えばPCR(ポリメラーゼ連鎖反応、polymerase chain reaction)による増幅産物の長さを調節すれば、複数の増幅産物を一度の測定で分離及び検出することも可能となることである。この原理は、上記のような複数存在する遺伝子変異の検出への応用も理論的には可能であるが、一塩基置換や点変異のような僅か1塩基の違いを検出するためには、工夫が必要である。一塩基置換検出の場合、単純にSNP部位を挟むようにPCR用のプライマーを設計して、増幅産物を得たとしても、一塩基の違いをイオン交換クロマトグラフィーで分離するのは困難である。これに対して、特許文献1には、アレル特異的プライマー(Allele Specific Primer; ASP)の5’末端に鋳型DNAと不完全相補な配列(タグ配列)を付加させることで、PCRによる増幅産物の長さを人為的に変化させて、イオン交換クロマトグラフィーにてSNPを分離検出する方法が開示されている。しかしながら、付加する塩基配列が長すぎるとプライマーとしてのTm値の変化が大きくなり、特異性が保てなくなる可能性がある。逆に短すぎると、増幅産物長の差が小さくなることでイオン交換クロマトグラフィーの分離が悪くなり、一塩基多型を正確に判定することが出来なくなることが懸念される。 As a method for separating and detecting nucleic acids with high accuracy in a short time, ion exchange chromatography is used. The advantage of applying this ion exchange chromatography to the detection of nucleic acids is that the nucleic acids can be separated according to their chain lengths.For example, if the length of amplification products by PCR (polymerase chain reaction) is adjusted, multiple It is also possible to separate and detect the amplification products in a single measurement. Although this principle can theoretically be applied to the detection of multiple gene mutations as described above, in order to detect a difference of only one base such as single base substitution or point mutation, a contrivance is required. In the case of single-base substitution detection, even if PCR primers are designed so as to sandwich the SNP site and an amplification product is obtained, it is difficult to separate the difference of single bases by ion exchange chromatography. In contrast, in Patent Document 1, by adding a sequence (tag sequence) that is incompletely complementary to the template DNA to the 5 ′ end of an allele-specific primer (All ASP), a PCR amplification product is added. A method for separating and detecting SNPs by ion exchange chromatography by artificially changing the length is disclosed. However, if the base sequence to be added is too long, the change in Tm value as a primer becomes large, and the specificity may not be maintained. On the other hand, if the length is too short, the difference in the length of the amplified product becomes small, so that the separation of the ion exchange chromatography becomes worse, and there is a concern that the single nucleotide polymorphism cannot be accurately determined.
　一方、ASPの設計を、二本鎖のフォワード側及びリバース側に設計し、その対となるプライマーを、変異部位以外の適当な場所に設計することにより、2種類の大きさが異なる増幅産物を得ることで、キャピラリー電気泳動を用いて分離できることが報告されている(非特許文献1)。しかしながら、この方法では変異と関係ないプライマー同士が対となった増幅が進んでしまうことから、増幅に必要な成分が消費され、特異反応に影響する可能性がある。さらに、2組の対となるプライマーを用いていることから、ハイブリダイゼーション及び増幅の効率に差が生じやすく、JAK2遺伝子変異などの遺伝子変異を検出する際に、アレル頻度を正確に算出するのは困難であった。さらに加えて、この方法では、2種類までの変異検出が限界であり、MPLのコドン515周辺の複数変異や、KRASやNRASなどのコドン12やコドン13の点変異のように、多くの種類の変異には適用することができない。 On the other hand, by designing the ASP on the double-stranded forward and reverse sides and designing the paired primers at appropriate locations other than the mutation site, two types of amplified products with different sizes can be obtained. It has been reported that it can be separated using capillary electrophoresis (Non-patent Document 1). However, in this method, amplification with a pair of primers not related to mutation proceeds, so that components necessary for the amplification are consumed and there is a possibility of affecting the specific reaction. Furthermore, because two pairs of primers are used, differences in hybridization and amplification efficiency are likely to occur, and when detecting gene mutations such as JAK2 gene mutation, allele frequency is calculated accurately. It was difficult. In addition, this method limits the detection of up to two types of mutations, and there are many types of mutations such as multiple mutations around codon 515 in MPL and point mutations in codons 12 and 13 such as KRAS and NRAS. It cannot be applied to mutation.
WO2012/133834WO2012 / 133834
　本発明は、上記のような従来の課題を鑑みて、多種類の遺伝子変異、特に一塩基置換若しくは点変異を、正確かつ定量的に分離及び検出する方法を提供することを目的とする。 In view of the conventional problems as described above, an object of the present invention is to provide a method for accurately and quantitatively separating and detecting many types of gene mutations, particularly single base substitutions or point mutations.
　前記課題を解決するための手段として、遺伝子変異、特に一塩基置換若しくは点変異を解析するためのASPにおいて、そのASP及びそれと対をなすプライマーの少なくとも1つの5’末端に、非ヌクレオチド成分を付加してPCRにより増幅し、その増幅産物をイオン交換クロマトグラフィーにより分離することで、増幅産物の長さが同じでも、分離検出できることを見出し、本発明を完成させるに至った。即ち、本発明は以下の〔1〕～〔8〕の構成からなる。
　〔1〕
　1種類以上のアレル特異的プライマーを用いて増幅された遺伝子増幅産物を、イオン交換クロマトグラフィーを用いて分析する方法であって、前記アレル特異的プライマー若しくはそれと対をなすプライマーの少なくとも一方の5’末端が、非ヌクレオチド成分を付加されていることを特徴とする、遺伝子変異を検出する方法。
　〔2〕
　イオン交換クロマトグラフィーが、アニオン交換クロマトグラフィーである上記〔1〕に記載の検出方法。
　〔3〕
　非ヌクレオチド成分が、プライマーの5’末端の電荷に変化を引き起こさせる物質である上記〔1〕又は〔2〕に記載の検出方法。
　〔4〕
　以下の工程を含む、試料中の二本鎖デオキシリボ核酸に含まれる多型部位における少なくとも一つのアレルの存在を検出する方法:
　(a)多型部位を含む二本鎖デオキシリボ核酸を含む試料を提供する工程;
　(b)第1のプライマー、第2のプライマー、及び第3のプライマーを提供する工程、
　ここで、第1のプライマーの配列は、当該多型部位において第1のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖に相補的でありかつ3'末端の3つの塩基の何れか1つ又は2つ又は3つの塩基或いは3'末端の2つの塩基の一方又は両方の塩基が当該多型部位に対応し、
　第2のプライマーの配列は、当該多型部位において第2のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖に相補的でありかつ3'末端の3つの塩基の何れか1つ又は2つ又は3つの塩基或いは3'末端の2つの塩基の一方又は両方の塩基が当該多型部位に対応し、
　第3のプライマーの配列は、当該多型部位を含まずかつ当該二本鎖デオキシリボ核酸の第1の鎖に相補的であり、
　第1のプライマー及び第2のプライマーの少なくとも1つが、非ヌクレオチド成分を付加されている;
　(c)ポリメラーゼ連鎖反応を行う工程、
　ここで、当該ポリメラーゼ連鎖反応は、第1のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第1のプライマーからのポリメラーゼによる鎖の伸長が、第1のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第2のプライマーからのポリメラーゼによる鎖の伸長と比較して優先的に生じ、かつ、第2のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第2のプライマーからのポリメラーゼによる鎖の伸長が、第2のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第1のプライマーからのポリメラーゼによる鎖の伸長と比較して優先的に生じる条件で行われる;
　(d)当該ポリメラーゼ連鎖反応の増幅産物をイオン交換クロマトグラフィーに掛ける工程;
　ここで、第1のプライマー及び第3のプライマーからのポリメラーゼ連鎖反応の増幅産物と第21のプライマー及び第3のプライマーからのポリメラーゼ連鎖反応の増幅産物の大きさの差は、0塩基対、1塩基対、2塩基対、3塩基対、4塩基対、5塩基対、6塩基対、7塩基対、8塩基対、9塩基対、又は10塩基対である;及び
　第1及び第2のアレルの一方又は両方の存在を前記増幅産物の溶出位置又は溶出時間に基づいて検出する工程。
　〔5〕
　前記(a)の工程が、ヒトなどの哺乳類体細胞検体からゲノムDNAを抽出する工程である、上記〔4〕に記載の方法。
　〔6〕
　前記多型部位が、UGT1A1*28多型(rs8175347)、UGT1A1*6多型(rs4148323)、JAK2 1849G>T (V617F)変異部位(rs77375493)、MPL 1589G>T (W515L)変異部位(rs121913615)、又はMPL 1588:1599TG>AA (W515K)変異部位(rs121913616)である、上記〔4〕又は〔5〕に記載の方法。
　〔7〕
　前記非ヌクレオチド成分が、プライマーの5’末端の電荷に変化を引き起こさせる物質である、上記〔4〕～〔6〕の何れかに記載の方法。
　〔8〕
　第3のプライマーが非ヌクレオチド成分を付加されている、上記〔4〕～〔7〕の何れかに記載の方法。 As a means for solving the above-mentioned problems, a non-nucleotide component is added to at least one 5 ′ end of an ASP for analyzing gene mutation, particularly single base substitution or point mutation, and its paired primer. Thus, the present inventors completed the present invention by amplifying by PCR and separating the amplified products by ion exchange chromatography, so that the amplification products can be separated and detected even if the length is the same. That is, the present invention comprises the following configurations [1] to [8].
[1]
A method of analyzing a gene amplification product amplified using one or more kinds of allele-specific primers using ion exchange chromatography, wherein 5 ′ of at least one of the allele-specific primer or a primer paired therewith is used. A method for detecting a gene mutation, wherein a terminal is added with a non-nucleotide component.
[2]
The detection method according to [1], wherein the ion exchange chromatography is anion exchange chromatography.
[3]
The detection method according to [1] or [2] above, wherein the non-nucleotide component is a substance that causes a change in the charge at the 5 ′ end of the primer.
〔Four〕
A method for detecting the presence of at least one allele at a polymorphic site contained in a double-stranded deoxyribonucleic acid in a sample, comprising the following steps:
(a) providing a sample containing double-stranded deoxyribonucleic acid containing a polymorphic site;
(b) providing a first primer, a second primer, and a third primer;
Here, the sequence of the first primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having the first allele at the polymorphic site and any one of the three bases at the 3 ′ end. Or one or both of two or three bases or two bases at the 3 ′ end correspond to the polymorphic site,
The sequence of the second primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having the second allele at the polymorphic site and any one or two of the three bases at the 3 ′ end Or one or both of three bases or two bases at the 3 ′ end correspond to the polymorphic site,
The sequence of the third primer does not include the polymorphic site and is complementary to the first strand of the double-stranded deoxyribonucleic acid,
At least one of the first primer and the second primer has a non-nucleotide component added thereto;
(c) performing a polymerase chain reaction,
Here, in the polymerase chain reaction, the extension of the strand by the polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the first allele is a double strand having the first allele. Second strand of a double-stranded deoxyribonucleic acid that preferentially occurs compared to the extension of a strand by a polymerase from a second primer hybridized to the second strand of a strand deoxyribonucleic acid and that has a second allele Comparison of polymerase chain elongation from the second primer hybridized to the polymerase with polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid with the second allele Under preferential conditions;
(d) subjecting the amplification product of the polymerase chain reaction to ion exchange chromatography;
Here, the difference in size between the amplification product of the polymerase chain reaction from the first primer and the third primer and the amplification product of the polymerase chain reaction from the 21st primer and the third primer is 0 base pairs, 1 Base pair, 2 base pair, 3 base pair, 4 base pair, 5 base pair, 6 base pair, 7 base pair, 8 base pair, 9 base pair, or 10 base pair; and first and second alleles Detecting the presence of one or both of them based on the elution position or elution time of the amplification product.
〔Five〕
The method according to [4] above, wherein the step (a) is a step of extracting genomic DNA from a mammalian somatic cell sample such as a human.
[6]
The polymorphic site is UGT1A1 * 28 polymorphism (rs8175347), UGT1A1 * 6 polymorphism (rs4148323), JAK2 1849G> T (V617F) mutation site (rs77375493), MPL 1589G> T (W515L) mutation site (rs121913615), Alternatively, the method according to [4] or [5] above, wherein MPL 1588: 1599TG> AA (W515K) mutation site (rs121913616).
[7]
The method according to any one of [4] to [6] above, wherein the non-nucleotide component is a substance that causes a change in the charge at the 5 ′ end of the primer.
[8]
The method according to any one of [4] to [7] above, wherein the third primer is added with a non-nucleotide component.
　一塩基置換若しくは点変異を解析するためのASPでは、増幅産物の長さが同じ、若しくは配列によっては、1～2塩基程度異なることはあるが、一般にイオン交換クロマトグラフィーでの分離検出は困難である。 In ASP for analyzing single-base substitutions or point mutations, the length of the amplification product is the same, or depending on the sequence, it may differ by 1 to 2 bases, but separation and detection by ion exchange chromatography is generally difficult. is there.
　これに対して、ASP及びそれと対をなすプライマーの少なくとも1つの5’末端に、非ヌクレオチド成分を付加してPCRにより増幅すると、その増幅産物に1つ若しくは2つの非ヌクレオチド成分が標識されていることになる。この僅かな標識物の物性並びに個数の違いにより、増幅産物のイオン強度が微妙に変化し、イオン交換クロマトグラフィーでの溶出位置が変化し、この特性を用いて増幅産物の分離検出することが可能となると推測される。 In contrast, when amplified by PCR with a non-nucleotide component added to the 5 'end of at least one of ASP and its paired primer, one or two non-nucleotide components are labeled on the amplified product. It will be. Due to the slight difference in physical properties and number of labels, the ionic strength of the amplification product changes slightly, and the elution position in ion-exchange chromatography changes. Using this property, the amplification product can be separated and detected. It is estimated that
　本発明で用いられるアレル特異的プライマーは、その遺伝子多型や遺伝子変異の塩基配列に対して特異的に結合できるプライマーであればよく、一塩基置換だけでなく、挿入や欠失変異を含む塩基配列などに特異的で、かつ本発明による分離に適用できるものであれば特に制限なく利用可能である。 The allele-specific primer used in the present invention may be a primer that can specifically bind to the base sequence of the gene polymorphism or gene mutation, and includes bases that include not only single-base substitutions but also insertion and deletion mutations. Any material that is specific to the sequence and applicable to the separation according to the present invention can be used without particular limitation.
　本発明に用いられる非ヌクレオチド成分としては、プライマーの5’末端の電荷に変化を引き起こさせる物質であることが好適で、それが付加されたアレル特異的プライマーを用いて増幅された遺伝子増幅産物を、イオン交換クロマトグラフィーを用いて分析する場合に、溶出パターンが変化するものであれば特に制限はない。好ましい非塩基物質としては、例えば、イオン性官能基そのもの、若しくはイオン性官能基を少なくとも1つ以上含む分子が挙げられる。イオン性官能基は、特に制限されなく、ヒドロキシ基、アルデヒド基、カルボキシ基、アミノ基、ニトロ基、ニトロソ基、チオール基、スルホン酸基、フルオロ基、クロロ基、ブロモ基、ヨード基などが例示できる。プライマーの修飾として用いられる蛍光色素も非ヌクレオチド成分として利用でき、例えば、Alexa　Fluorシリーズ、Cyシリーズ、ATTOシリーズ、DYシリーズ、DyLightシリーズ、FAM、TAMRAなどが挙げられる。さらに、ジゴキシン、ビオチンなどの機能性物質の付加やアミド基修飾なども制限なく利用可能である。
　これら修飾物質の効果は、アレル特異的プライマーを用いて増幅された遺伝子増幅産物の長さを至適化することで、さらに発揮される。即ち、同じ修飾物質であっても、遺伝子増幅産物の長さが短いほどのその違いが顕著に表れる。従って、本発明では、5’末端に非ヌクレオチド成分が付加されたアレル特異的プライマーを用いて増幅された遺伝子増幅産物を、イオン交換クロマトグラフィーを用いて分析する際に、非ヌクレオチド成分の種類だけでなく、遺伝子増幅産物の長さを適切に組み合わせることによって、本発明の効果を最大限に発揮することが可能となる。 The non-nucleotide component used in the present invention is preferably a substance that causes a change in the charge at the 5 ′ end of the primer, and a gene amplification product amplified using the allele-specific primer to which the primer is added. In the case of analyzing using ion exchange chromatography, there is no particular limitation as long as the elution pattern changes. Preferred non-basic substances include, for example, ionic functional groups themselves or molecules containing at least one ionic functional group. The ionic functional group is not particularly limited, and examples thereof include hydroxy group, aldehyde group, carboxy group, amino group, nitro group, nitroso group, thiol group, sulfonic acid group, fluoro group, chloro group, bromo group, and iodo group. it can. Fluorescent dyes used as primer modifications can also be used as non-nucleotide components, such as Alexa Fluor series, Cy series, ATTO series, DY series, DyLight series, FAM, and TAMRA. Furthermore, addition of functional substances such as digoxin and biotin and amide group modification can be used without limitation.
The effects of these modifiers can be further demonstrated by optimizing the length of the gene amplification product amplified using the allele-specific primer. That is, even with the same modifying substance, the difference becomes more noticeable as the length of the gene amplification product is shorter. Therefore, in the present invention, when analyzing a gene amplification product amplified using an allele-specific primer having a non-nucleotide component added to the 5 ′ end using ion exchange chromatography, only the type of non-nucleotide component is used. In addition, it is possible to maximize the effects of the present invention by appropriately combining the lengths of the gene amplification products.
　本発明の方法において、イオン交換クロマトグラフィーとして、カチオン交換クロマトグラフィーまたはアニオン交換クロマトグラフィーを、測定対象物質の等電点、溶離液(移動相ともいう)のpHや塩濃度等を考慮して選択することができる。核酸など、負の電荷を帯びている測定対象物質の場合には、アニオン交換クロマトグラフィーを用いることが好ましい。 In the method of the present invention, cation exchange chromatography or anion exchange chromatography is selected as ion exchange chromatography in consideration of the isoelectric point of the substance to be measured, the pH of the eluent (also referred to as mobile phase), the salt concentration, and the like. can do. In the case of a measurement target substance having a negative charge such as a nucleic acid, it is preferable to use anion exchange chromatography.
　本明細書において、「核酸」とは、リボ核酸(以下、ribonucleic acid、RNAともいう。)とデオキシリボ核酸(以下、deoxyribonucleic acid、DNAともいう。)の総称であり、塩基、糖、及びリン酸からなるヌクレオチドが、ホスホジエステル結合で連なったものを意味する。本発明において、抽出される核酸は、DNA及びRNAのどちらであってもよく、また断片化しているものであっても、していないものであっても対象となる。該核酸の由来は、動物、植物、微生物を含むあらゆる生物、ウィルスが挙げられるが、これらに限定されない。また、細胞核内の核酸や、ミトコンドリアや葉緑体や核小体等に代表されるオルガネラが保持する核外由来の核酸でもよい。更に、人工的合成された核酸や、一般にベクターとして用いられるプラスミドやウイルスベクターでもよい。本発明の方法に好ましい核酸として二本鎖デオキシリボ核酸が例示でき、より好ましい核酸として、その塩基配列に一塩基多型、点変異、及び/または欠失・挿入変異が入る塩基配列を含む二本鎖デオキシリボ核酸が例示できる。 In the present specification, “nucleic acid” is a general term for ribonucleic acid (hereinafter also referred to as ribonucleic acid or RNA) and deoxyribonucleic acid (hereinafter also referred to as deoxyribonucleic acid or DNA), and includes bases, sugars, and phosphates. The nucleotides consisting of are linked by phosphodiester bonds. In the present invention, the nucleic acid to be extracted may be either DNA or RNA, and it may be a target whether it is fragmented or not. Examples of the origin of the nucleic acid include, but are not limited to, animals, plants, all organisms including microorganisms, and viruses. Moreover, the nucleic acid inside a cell nucleus, the nucleic acid derived from the nucleus which the organelle represented by a mitochondria, a chloroplast, a nucleolus, etc. hold | maintain may be sufficient. Furthermore, it may be an artificially synthesized nucleic acid, a plasmid or a viral vector generally used as a vector. A double-stranded deoxyribonucleic acid can be exemplified as a preferred nucleic acid for the method of the present invention, and more preferred nucleic acids include a double-stranded nucleic acid having a nucleotide sequence that includes a single nucleotide polymorphism, a point mutation, and / or a deletion / insertion mutation. A chain deoxyribonucleic acid can be exemplified.
　PCR増幅の方法としては特に制限はなく、増幅対象の核酸の配列、長さ、量などに応じて、公知の手法を適宜選択して用いることができる。PCR増幅産物の鎖長は、PCRの増幅時間の短縮、ならびにイオン交換クロマトグラフィーでの分析時間の短縮や分離性能の維持等の要素を勘案して適宜選択することができる。例えば、PCR増幅産物の鎖長の上限は、1000bp以下が好ましく、700bp以下がより好ましく、500bp以下がさらに好ましい。一方、PCR増幅産物の鎖長の下限は、30bpが好ましく、40bpがより好ましい。 The PCR amplification method is not particularly limited, and known methods can be appropriately selected and used according to the sequence, length, amount, etc. of the nucleic acid to be amplified. The chain length of the PCR amplification product can be appropriately selected in consideration of factors such as shortening the PCR amplification time, shortening the analysis time in ion exchange chromatography, and maintaining separation performance. For example, the upper limit of the PCR amplification product chain length is preferably 1000 bp or less, more preferably 700 bp or less, and even more preferably 500 bp or less. On the other hand, the lower limit of the PCR amplification product chain length is preferably 30 bp, more preferably 40 bp.
　本発明の方法で検出可能な一塩基多型、点変異、及び/または欠失・挿入変異として、UGT1A1*28多型(rs8175347)、UGT1A1*6多型(rs4148323)、JAK2 1849G>T (V617F)変異部位(rs77375493)、MPL 1589G>T (W515L)変異部位(rs121913615)、MPL 1588:1599TG>AA (W515K)変異部位(rs121913616)が例示できる。 As single nucleotide polymorphism, point mutation, and / or deletion / insertion mutation detectable by the method of the present invention, UGT1A1 * 28 polymorphism (rs8175347), UGT1A1 * 6 polymorphism (rs4148323), JAK2 1849G> T (V617F ) Mutation site (rs77375493), MPL 1589G> T (W515L) mutation site (rs121913615), MPL 1588: 1599TG> AA (W515K) mutation site (rs121913616).
図1は、3つの蛍光標識プライマー(配列番号3、7及び8)による増幅産物の溶出ピークを重ね書きした結果を示す。FIG. 1 shows the result of overwriting the elution peaks of amplification products with three fluorescently labeled primers (SEQ ID NOs: 3, 7, and 8). 図2は、非ヌクレオチド成分付加ASPを用いたUGT1A1遺伝子の*6多型部位からの増幅産物の分離及び検出を示す。FIG. 2 shows the separation and detection of amplification products from the * 6 polymorphic site of the UGT1A1 gene using the non-nucleotide component-added ASP. 図3は、非ヌクレオチド成分付加ASPを用いたMPL遺伝子のコドン515部位周辺からの増幅産物の分離及び検出を示す。FIG. 3 shows the separation and detection of amplification products from around the codon 515 site of the MPL gene using non-nucleotide component-added ASP.
　以下、実施例により本発明を詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to the following examples.
　[実施例1]非ヌクレオチド成分付加ASPを用いたUGT1A1遺伝子の*6多型部位からの増幅産物 [Example 1] Amplification product from * 6 polymorphic site of UGT1A1 gene using non-nucleotide component added ASP
　ヒトUGT1A1遺伝子の*6アレル(211G>A)で、ミスマッチ塩基を一箇所無味入れた当該アレルを特異的に増幅できるASP(配列番号1)及びそのリバースプライマー(配列番号12)を用意し(シグマアルドリッジ株式会社へ委託)、さらにASPの5’末端に、非ヌクレオチド成分を修飾したプライマーを別に用意した(配列番号2は、サーモフィッシャー株式会社へ委託、配列番号8は、ユーロフィンジェノミクス株式会社へ委託、配列番号4及び6は、Integrated DNA Technologies MBL株式会社へ委託、それ以外は、シグマアルドリッジ株式会社へ委託)。配列番号、プライマー配列、オリゴヌクレオチドの長さ(bp)、非ヌクレオチド成分の種類、並びに、非ヌクレオチド成分の励起波長及び蛍光波長(nm)を表1に示す。
Figure JPOXMLDOC01-appb-T000001
 Prepare the ASP (SEQ ID NO: 1) and its reverse primer (SEQ ID NO: 12) that can specifically amplify the allele of the human UGT1A1 gene * 6 allele (211G> A) with a mismatched base in one place. In addition, a primer modified with a non-nucleotide component was prepared separately at the 5 'end of ASP (SEQ ID No. 2 was assigned to Thermo Fisher Co., and SEQ ID No. 8 was assigned to Eurofin Genomics Co., Ltd.) Consignment, SEQ ID NOS: 4 and 6 are consigned to Integrated DNA Technologies MBL Co., Ltd., other than that, consigned to Sigma-Aldridge Co., Ltd.) Table 1 shows the SEQ ID NO: primer sequence, oligonucleotide length (bp), type of non-nucleotide component, and excitation wavelength and fluorescence wavelength (nm) of the non-nucleotide component.
Figure JPOXMLDOC01-appb-T000001
　・試薬、増幅条件及びイオン交換クロマトグラフィー条件 ・ Reagents, amplification conditions and ion exchange chromatography conditions
　以下の試薬を含む25μLの反応溶液を調製し、CFX96(バイオラッド社)にて2ステップのアレル特異的PCRによる増幅を行った。この検討では、UGT1A1遺伝子*6のアレルがホモ接合体の人から採取した精製DNAを用いた。
Figure JPOXMLDOC01-appb-T000002
 A 25 μL reaction solution containing the following reagents was prepared and amplified by CFX96 (Bio-Rad) by two-step allele-specific PCR. In this study, purified DNA collected from a person who was homozygous for the UGT1A1 gene * 6 allele was used.
Figure JPOXMLDOC01-appb-T000002
　その結果を表3に示す。すべての増幅産物の鎖長は117bpとなるが、未標識増幅産物と比較して、各種非ヌクレオチド成分が標識されたプライマーを用いて増幅された産物のイオン交換クロマトグラフィーでの溶出時間(保持時間とも言う。)は、早まったり遅延したりと様々なパターンを示す興味深い知見が得られた。その中から、特に溶出時間の変化が大きかった3つの蛍光標識プライマー(配列番号3、7及び8)による増幅産物の溶出ピークを重ね書きした結果を図1に示す。この結果は、同じ部位にいくつかパターンがある遺伝子多型や遺伝子変異を特定するための特異的なプライマー毎に、標識する非ヌクレオチド成分を変化させることによって、仮にその増幅産物の鎖長が同一になる場合においても、一回のイオン交換クロマトグラフィー分離において、複数変異のマルチプレックス解析が可能であることを支持するものである。
Figure JPOXMLDOC01-appb-T000003
 The results are shown in Table 3. The chain length of all amplification products is 117 bp, but compared to unlabeled amplification products, the elution time (retention time) of products amplified using primers labeled with various non-nucleotide components. (Also known as)) was an interesting finding showing various patterns, such as early and late. Among these, FIG. 1 shows the result of overwriting the elution peak of the amplification product with the three fluorescently labeled primers (SEQ ID NOs: 3, 7, and 8) whose elution time was particularly large. This result shows that the chain length of the amplified product is the same by changing the non-nucleotide component to be labeled for each specific primer for identifying genetic polymorphisms and mutations that have several patterns at the same site. Even in this case, it is supported that a multiplex analysis of a plurality of mutations is possible in one ion exchange chromatography separation.
Figure JPOXMLDOC01-appb-T000003
　[実施例2]非ヌクレオチド成分付加ASPを用いたUGT1A1遺伝子の*6多型部位からの増幅産物の分離及び検出 [Example 2] Separation and detection of amplification products from * 6 polymorphic site of UGT1A1 gene using non-nucleotide component added ASP
　*6アレル検出用のプライマーは、実施例1に記載の配列番号3を用いた。一方、*6多型部位における野生型検出用のプライマーは、実施例1に記載の配列番号1と同様に、非標識でミスマッチ塩基を一箇所に導入して野生型を特異的に増幅できるASP(配列番号13)を別に用意した。この検討では、UGT1A1遺伝子*6の遺伝子多型部位が、野生型、*6アレルのヘテロ接合体及びホモ接合体の人から採取した精製DNAを用いた。 * 6 SEQ ID NO: 3 described in Example 1 was used as a primer for detecting alleles. On the other hand, the primer for detecting the wild type at the * 6 polymorphic site is an ASP that can specifically amplify the wild type by introducing a non-labeled mismatch base at one site, as in SEQ ID NO: 1 described in Example 1. (SEQ ID NO: 13) was prepared separately. In this study, purified DNA collected from a human who was heterozygous for the UGT1A1 gene * 6 gene wild-type, * 6 allele, and homozygous was used.
　配列番号13
　5’-GTTGTACATCAGAGACGAA-3’ SEQ ID NO: 13
5'-GTTGTACATCAGAGACGAA-3 '
　・試薬、増幅条件及びイオン交換クロマトグラフィー条件 ・ Reagents, amplification conditions and ion exchange chromatography conditions
　以下の試薬を含む25μLの反応溶液を調製し、CFX96(バイオラッド社)にて2ステップのアレル特異的PCRによる増幅を行った。イオン交換クロマトグラフィーの測定は、実施例1と同じ条件を用いて実施した。
Figure JPOXMLDOC01-appb-T000004
 A 25 μL reaction solution containing the following reagents was prepared and amplified by CFX96 (Bio-Rad) by two-step allele-specific PCR. Ion exchange chromatography was measured using the same conditions as in Example 1.
Figure JPOXMLDOC01-appb-T000004
　その結果を図2に示す。*6アレルのヘテロ接合体である検体1では、未標識増幅産物の溶出位置(溶出時間8.6分付近)とFAM標識増幅産物の溶出位置(溶出時間9.2分)に2つの溶出ピークを認め、*6アレルのホモ接合体である検体2では、FAM標識増幅産物の溶出位置にのみ溶出ピークを認め、野生型である検体3では、未標識増幅産物の溶出位置のみ溶出ピークを認めたことから、UGT1A1遺伝子の*6多型部位の遺伝子型が容易かつ正確に判別することが可能であることがわかった。 The result is shown in FIG. * 6 In sample 1, which is a heterozygote of the allele, two elution peaks were observed at the elution position of the unlabeled amplification product (elution time around 8.6 minutes) and the elution position of the FAM-labeled amplification product (elution time 9.2 minutes). In sample 2, which is a homozygote of 6 alleles, an elution peak was observed only at the elution position of the FAM-labeled amplification product, and in wild-type sample 3, an elution peak was observed only at the elution position of the unlabeled amplification product. It was found that the genotype of the * 6 polymorphic site of UGT1A1 gene can be easily and accurately distinguished.
　実施例3　非ヌクレオチド成分付加ASPを用いたMPL遺伝子変異(コドン515)の分離検出 Example 3 Separation and detection of MPL gene mutation (codon 515) using non-nucleotide component added ASP
　MPL遺伝子のコドン515には、W515L、W515K、W515Aの3つの変異パターンがあり、それぞれ1543-1544番目の2塩基の配列が異なる。フォワードプライマーとして、それぞれの変異型検出用の未標識ASP(配列番号14～16)を用意し、それと対となるリバースプライマー(配列番号17)を用意した。別に、W515K用の非ヌクレオチド成分を付加したASP(配列番号18、19、20)も用意した。また、それぞれの遺伝子変異配列(配列番号21～23)を組み込んだプラスミドDNAを、検体用として用意(ユーロフィンジェノミクス株式会社へ委託)した。 The codon 515 of the MPL gene has three mutation patterns, W515L, W515K, and W515A, each of which has a different base sequence at positions 1543-1544. As a forward primer, unlabeled ASP (SEQ ID NO: 14 to 16) for detecting each mutant type was prepared, and a reverse primer (SEQ ID NO: 17) to be paired with it was prepared. Separately, ASP (SEQ ID NOs: 18, 19, and 20) to which a non-nucleotide component for W515K was added was also prepared. In addition, plasmid DNAs incorporating the respective gene mutation sequences (SEQ ID NOs: 21 to 23) were prepared for specimens (consigned to Eurofin Genomics Co., Ltd.).
　配列番号14　(W515L用ASP)
　5’-CTGCTGCTGCTGAGGTTTC-3’ Sequence number 14 (ASP for W515L)
5'-CTGCTGCTGCTGAGGTTTC-3 '
　配列番号15　(W515K用ASP)
　5’-CTGCTGCTGCTGAGGAA-3’ Sequence number 15 (ASP for W515K)
5'-CTGCTGCTGCTGAGGAA-3 '
　配列番号16　(W515A用ASP)
　5’-TGCTGCTGCTGAGCGC-3’ Sequence number 16 (ASP for W515A)
5'-TGCTGCTGCTGAGCGC-3 '
　配列番号17　(共通リバースプライマー)
　5’-GGCGGTACCTGTAGTGTGC-3’ SEQ ID NO: 17 (common reverse primer)
5'-GGCGGTACCTGTAGTGTGC-3 '
　配列番号18　(ビオチン標識W515K用ASP)
　5’-Biotin-CTGCTGCTGCTGAGGAA-3’ SEQ ID NO: 18 (ASP for biotin-labeled W515K)
5'-Biotin-CTGCTGCTGCTGAGGAA-3 '
　配列番号19　(アミノ基標識W515K用ASP)
　5’-NH2-CTGCTGCTGCTGAGGAA-3’ SEQ ID NO: 19 (ASP for amino group labeling W515K)
5'-NH2-CTGCTGCTGCTGAGGAA-3 '
　配列番号20　(Cy3.5蛍光色素標識W515K用ASP)
　5’-Cy3.5-CTGCTGCTGCTGAGGAA-3’ SEQ ID NO: 20 (ASP for Cy3.5 fluorescent dye labeled W515K)
5'-Cy3.5-CTGCTGCTGCTGAGGAA-3 '
　配列番号21　(W515L遺伝子変異配列)
Figure JPOXMLDOC01-appb-C000005
 SEQ ID NO: 21 (W515L gene mutation sequence)
Figure JPOXMLDOC01-appb-C000005
　配列番号22　(W515K遺伝子変異配列)
Figure JPOXMLDOC01-appb-C000006
 SEQ ID NO: 22 (W515K gene mutation sequence)
Figure JPOXMLDOC01-appb-C000006
　配列番号23　(W515A遺伝子変異配列)
Figure JPOXMLDOC01-appb-C000007
 SEQ ID NO: 23 (W515A gene mutation sequence)
Figure JPOXMLDOC01-appb-C000007
　・試薬、増幅条件及びイオン交換クロマトグラフィー条件
　以下の試薬を含む25μLの反応溶液を調製し、CFX96(バイオラッド社)にて2ステップのアレル特異的PCRによる増幅を行った。 Reagents, amplification conditions and ion exchange chromatography conditions 25 μL of a reaction solution containing the following reagents was prepared and amplified by CFX96 (Bio-Rad) by two-step allele-specific PCR.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　図3に、非ヌクレオチド成分付加ASPを用いたMPL遺伝子のコドン515部位周辺からの増幅産物のイオン交換クロマトグラフィー分離及び検出結果を示す。先ず、未標識ASPを用いた場合のイオン交換クロマトグラフィー分離結果について、W515Aの増幅産物(45bp)の溶出位置(溶出時間3.82分)は、W515L及びW515Kの増幅産物(それぞれ46bp)の溶出位置と異なることから判別可能であるが、W515LとW515Kの増幅産物は、ほぼ同じ溶出位置(溶出時間4.42分と4.35分)となり、変異の有無は確認されるが、そのパターンの同定は出来ないことが判明した。それに対して、W515K用の非ヌクレオチド成分を付加したASP(配列番号18、19、20)を用いた増幅産物の溶出位置は、それぞれ、4.16分、3.91分、4.97分となり、W515Lの溶出位置と重ならないことに加えて、W515Aの増幅産物の溶出位置にも重ならないことも確認された。 Fig. 3 shows the results of ion exchange chromatography separation and detection of amplification products around the codon 515 site of the MPL gene using the non-nucleotide component-added ASP. First, for the ion exchange chromatography separation results when using unlabeled ASP, the elution position (elution time 3.82 minutes) of the W515A amplification product (45 bp) is the same as the elution position of the W515L and W515K amplification products (each 46 bp). Although it can be distinguished from the difference, the amplification products of W515L and W515K have almost the same elution position (elution time 4.42 minutes and 4.35 minutes), and the presence or absence of mutation is confirmed, but the pattern cannot be identified. found. In contrast, the elution positions of the amplification products using ASP (SEQ ID NOs: 18, 19, and 20) added with non-nucleotide components for W515K were 4.16 minutes, 3.91 minutes, and 4.97 minutes, respectively, and the elution positions of W515L and In addition to not overlapping, it was also confirmed that it does not overlap the elution position of the amplified product of W515A.
　この結果は、ASPを用いた増幅産物の長さが類似して、イオン交換クロマトグラフィーを用いた分離検出で溶出位置による差が認められない場合に、ASPに適当な非ヌクレオチド成分を付加することで、分離検出が可能となることを支持する。 This result shows that when the length of the amplification product using ASP is similar and separation / detection using ion exchange chromatography shows no difference depending on the elution position, an appropriate non-nucleotide component is added to ASP. Therefore, it supports that separation detection is possible.
　上記の知見を踏まえると、複数のASPにイオン交換クロマトグラフィーによる溶出時間が変化する複数の非ヌクレオチド成分を付加し、さらにそれと対をなすプライマーにも非ヌクレオチド成分を付加することによって、様々な溶出時間を調整することが可能となる。さらに、非ヌクレオチド成分に、蛍光色素を用いることによって、蛍光波長がクロストークしないものを選択することで、溶出時間に差が無くとも、検出する波長で判別することも可能となる。 Based on the above findings, various elutions can be achieved by adding multiple non-nucleotide components that change the elution time by ion exchange chromatography to multiple ASPs, and adding non-nucleotide components to the paired primers. It becomes possible to adjust the time. Further, by using a fluorescent dye as the non-nucleotide component, it is possible to discriminate by the wavelength to be detected even if there is no difference in the elution time by selecting the fluorescent wavelength that does not crosstalk.
　増幅産物の検出方法としては、増幅反応した試薬をそのままイオン交換クロマトグラフィーで分離する方法以外に、複数の増幅試薬を別々に準備し、その混合液をイオン交換クロマトグラフィーで分離する方法も可能である。 As a method for detecting the amplification product, in addition to the method of separating the amplified reagent as it is by ion exchange chromatography, a method of separately preparing a plurality of amplification reagents and separating the mixed solution by ion exchange chromatography is also possible. is there.
　従って、本発明によれば、従来法では困難であった複数の遺伝子多型の遺伝子型や一塩基置換を、容易にかつ正確に検出でき、近年需要が高まってきている遺伝子検査のマルチプレックス化に対応可能な方法が提供される。 Therefore, according to the present invention, genotypes and single base substitutions of a plurality of gene polymorphisms that have been difficult with the conventional method can be easily and accurately detected. A method capable of responding to is provided.

Claims (8)

　1種類以上のアレル特異的プライマーを用いて増幅された遺伝子増幅産物を、イオン交換クロマトグラフィーを用いて分析する方法であって、前記アレル特異的プライマー若しくはそれと対をなすプライマーの少なくとも一方の5’末端が、非ヌクレオチド成分を付加されていることを特徴とする、遺伝子変異を検出する方法。 A method of analyzing a gene amplification product amplified using one or more kinds of allele-specific primers using ion exchange chromatography, wherein 5 ′ of at least one of the allele-specific primer or a primer paired therewith is used. A method for detecting a gene mutation, wherein a terminal is added with a non-nucleotide component.
　イオン交換クロマトグラフィーが、アニオン交換クロマトグラフィーである請求項1に記載の検出方法。 2. The detection method according to claim 1, wherein the ion exchange chromatography is anion exchange chromatography.
　非ヌクレオチド成分が、プライマーの5’末端の電荷に変化を引き起こさせる物質である請求項1又は2に記載の検出方法。 3. The detection method according to claim 1 or 2, wherein the non-nucleotide component is a substance that causes a change in the charge at the 5 'end of the primer.
　以下の工程を含む、試料中の二本鎖デオキシリボ核酸に含まれる多型部位における少なくとも一つのアレルの存在を検出する方法:
　(a)多型部位を含む二本鎖デオキシリボ核酸を含む試料を提供する工程;
　(b)第1のプライマー、第2のプライマー、及び第3のプライマーを提供する工程、
　ここで、第1のプライマーの配列は、当該多型部位において第1のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖に相補的でありかつ3'末端の3つの塩基の何れか1つ又は2つ又は3つの塩基或いは3'末端の2つの塩基の一方又は両方の塩基が当該多型部位に対応し、
　第2のプライマーの配列は、当該多型部位において第2のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖に相補的でありかつ3'末端の3つの塩基の何れか1つ又は2つ又は3つの塩基或いは3'末端の2つの塩基の一方又は両方の塩基が当該多型部位に対応し、
　第3のプライマーの配列は、当該多型部位を含まずかつ当該二本鎖デオキシリボ核酸の第1の鎖に相補的であり、
　第1のプライマー及び第2のプライマーの少なくとも1つが、非ヌクレオチド成分を付加されている;
　(c)ポリメラーゼ連鎖反応を行う工程、
　ここで、当該ポリメラーゼ連鎖反応は、第1のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第1のプライマーからのポリメラーゼによる鎖の伸長が、第1のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第2のプライマーからのポリメラーゼによる鎖の伸長と比較して優先的に生じ、かつ、第2のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第2のプライマーからのポリメラーゼによる鎖の伸長が、第2のアレルを持つ二本鎖デオキシリボ核酸の第2の鎖にハイブリダイズした第1のプライマーからのポリメラーゼによる鎖の伸長と比較して優先的に生じる条件で行われる;
　(d)当該ポリメラーゼ連鎖反応の増幅産物をイオン交換クロマトグラフィーに掛ける工程;
　ここで、第1のプライマー及び第3のプライマーからのポリメラーゼ連鎖反応の増幅産物と第2のプライマー及び第3のプライマーからのポリメラーゼ連鎖反応の増幅産物の大きさの差は、0塩基対、1塩基対、2塩基対、3塩基対、4塩基対、5塩基対、6塩基対、7塩基対、8塩基対、9塩基対、又は10塩基対である;及び
　(e)第1及び第2のアレルの一方又は両方の存在を前記増幅産物の溶出位置又は溶出時間に基づいて検出する工程。 A method for detecting the presence of at least one allele at a polymorphic site contained in a double-stranded deoxyribonucleic acid in a sample, comprising the following steps:
(a) providing a sample containing double-stranded deoxyribonucleic acid containing a polymorphic site;
(b) providing a first primer, a second primer, and a third primer;
Here, the sequence of the first primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having the first allele at the polymorphic site and any one of the three bases at the 3 ′ end. Or one or both of two or three bases or two bases at the 3 ′ end correspond to the polymorphic site,
The sequence of the second primer is complementary to the second strand of the double-stranded deoxyribonucleic acid having the second allele at the polymorphic site and any one or two of the three bases at the 3 ′ end Or one or both of three bases or two bases at the 3 ′ end correspond to the polymorphic site,
The sequence of the third primer does not include the polymorphic site and is complementary to the first strand of the double-stranded deoxyribonucleic acid,
At least one of the first primer and the second primer has a non-nucleotide component added thereto;
(c) performing a polymerase chain reaction,
Here, in the polymerase chain reaction, the extension of the strand by the polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid having the first allele is a double strand having the first allele. Second strand of a double-stranded deoxyribonucleic acid that preferentially occurs compared to the extension of a strand by a polymerase from a second primer hybridized to the second strand of a strand deoxyribonucleic acid and that has a second allele Comparison of polymerase chain elongation from the second primer hybridized to the polymerase with polymerase from the first primer hybridized to the second strand of the double-stranded deoxyribonucleic acid with the second allele Under preferential conditions;
(d) subjecting the amplification product of the polymerase chain reaction to ion exchange chromatography;
Here, the difference in size between the amplification product of the polymerase chain reaction from the first primer and the third primer and the amplification product of the polymerase chain reaction from the second primer and the third primer is 0 base pairs, 1 Base pair, 2 base pair, 3 base pair, 4 base pair, 5 base pair, 6 base pair, 7 base pair, 8 base pair, 9 base pair, or 10 base pair; and (e) first and second Detecting the presence of one or both of the two alleles based on the elution position or elution time of the amplification product.
　前記(a)の工程が、ヒトなどの哺乳類体細胞検体からゲノムDNAを抽出する工程である、請求項4に記載の方法。 5. The method according to claim 4, wherein the step (a) is a step of extracting genomic DNA from a mammalian somatic specimen such as a human.
　前記多型部位が、UGT1A1*28多型(rs8175347)、UGT1A1*6多型(rs4148323)、JAK2 1849G>T (V617F)変異部位(rs77375493)、MPL 1589G>T (W515L)変異部位(rs121913615)、又はMPL 1588:1599TG>AA (W515K)変異部位(rs121913616)である、請求項4又は5に記載の方法。 The polymorphic site is UGT1A1 * 28 polymorphism (rs8175347), UGT1A1 * 6 polymorphism (rs4148323), JAK2 1849G> T (V617F) mutation site (rs77375493), MPL 1589G> T (W515L) mutation site (rs121913615), Alternatively, the method according to claim 4 or 5, wherein MPL 1588: 1599TG> AA (W515K) mutation site (rs121913616).
　前記非ヌクレオチド成分が、プライマーの5’末端の電荷に変化を引き起こさせる物質である、請求項4～6の何れかに記載の方法。 The method according to any one of claims 4 to 6, wherein the non-nucleotide component is a substance that causes a change in the charge at the 5 'end of the primer.
　第3のプライマーが非ヌクレオチド成分を付加されている、請求項4～7の何れかに記載の方法。
  The method according to any one of claims 4 to 7, wherein a non-nucleotide component is added to the third primer.
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