WO2012133834A1 - 一塩基多型検出用の試料核酸、一塩基多型検出試料調製用のpcrプライマー及びイオン交換クロマトグラフィー分析に用いる一塩基多型検出用試料の調製方法 - Google Patents
一塩基多型検出用の試料核酸、一塩基多型検出試料調製用のpcrプライマー及びイオン交換クロマトグラフィー分析に用いる一塩基多型検出用試料の調製方法 Download PDFInfo
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- single nucleotide
- nucleotide polymorphism
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/10—Detection mode being characterised by the assay principle
- C12Q2565/137—Chromatographic separation
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Definitions
- the present invention relates to a sample nucleic acid for detecting a single nucleotide polymorphism used in a rapid and simple method for detecting a single nucleotide polymorphism.
- the present invention also relates to a PCR primer for preparing a single nucleotide polymorphism detection sample and a method for preparing a sample for single nucleotide polymorphism detection used for ion exchange chromatography analysis.
- SNPs single nucleotide polymorphisms
- an RFLP method Restriction Fragment Length Polymorphism
- a restriction enzyme that recognizes a gene mutation site in a PCR (Polymerase Chain Reaction) amplification product
- a primer is set at the common sequence site, and polymorphism is present within that PCR amplification product.
- the amplified PCR product is cleaved with a restriction enzyme, and the presence or absence of a polymorphism is determined by the length of the fragment.
- a restriction enzyme since a restriction enzyme is used, there are problems such as an increase in analysis cost and a long analysis time.
- the difference in chain length is detected by electrophoresis, there are problems such as complicated operations and a long analysis time.
- Non-Patent Document 1 discloses a method for separating nucleic acid-related compounds by high performance liquid chromatography. However, even the method disclosed in Non-Patent Document 1 has a problem that it is difficult to sufficiently separate nucleic acids having different chain length differences such as single nucleotide polymorphisms.
- the present invention is a sample nucleic acid for detecting a single nucleotide polymorphism having the following characteristics (a) to (c).
- (c) compares the presence or absence of a single nucleotide polymorphism
- the present invention will be described in detail below, in which the difference in chain length from the target sample nucleic acid is 10 bp or less.
- the present inventors have found that a single nucleotide polymorphism can be detected quickly and easily by using a sample nucleic acid having specific characteristics, and have completed the present invention.
- the AS-PCR (Allele Specific-PCR) method is a method for detecting a gene polymorphism (particularly a single nucleotide polymorphism) using a sequence-specific amplification reaction. Specifically, PCR is performed so that the nucleotide sequence of the single nucleotide polymorphism to be detected is the 3 'end of the primer. When the base sequence of the target nucleic acid and the primer are completely complementary, an extension reaction is caused by DNA polymerase. On the other hand, when the base sequence of the target nucleic acid and the primer are incompletely complementary, the extension reaction of DNA polymerase is inhibited.
- this is a method for determining a single nucleotide polymorphism based on the result of an amplification reaction using two kinds of primers having a single nucleotide polymorphism wild-type or mutant nucleotide sequence at the 3 'end.
- AS-PCR method the method disclosed in Non-Patent Document 2 can be used.
- the primers used have the following characteristics.
- Two types of forward primers are used for the mutant type and the wild type.
- the mutant type has a single nucleotide polymorphic base sequence at the 3 ′ end, and the wild type has a 3 ′ end (single base multiple). (Corresponding to the type site) has a wild-type base sequence.
- One of the two types of Forward primers has a sequence (hereinafter sometimes referred to as “tag sequence”) that is incompletely complementary to the base sequence of the target nucleic acid at the 5 ′ end.
- the chain length of the tag sequence is 10 bp or less.
- the above two types of Forward primers are designed so that the chain length of the amplified product is 200 bp or less.
- a common primer is used for the mutant type and the wild type.
- the reverse primer has a tag sequence at the 3 ′ end after the second cycle of PCR amplification. The amplification product it has can be replicated.
- the characteristics of the first cycle of PCR amplification are as follows. The two types of primers for the mutant type and the wild type are annealed to the mutant type template DNA and the wild type template DNA, respectively, and then the extension reaction proceeds. Duplicated. At this time, the replicated mutant DNA has a total chain length longer than the replicated wild-type DNA by the length of the tag sequence.
- the characteristics after the second cycle of PCR amplification are as follows.
- the Reverse primer anneals to the replication product of the mutant DNA (having a tag sequence at the 5 ′ end) and the replication product of the wild type DNA, and then the extension reaction proceeds to replicate the mutant DNA and the wild type DNA, respectively. Is done. At this time, the mutant DNA replicated with the Reverse primer also has a total chain length longer than the replicated wild-type DNA by the chain length of the tag sequence.
- Two types of PCR amplification products having different total chain lengths by the tag sequence thus obtained are separated and analyzed by ion exchange chromatography described later.
- a tag sequence may be added to both the mutation type primer and the wild type primer as long as two kinds of PCR amplification products can be separated and analyzed by ion exchange chromatography.
- the terms forward primer and reverse primer are used in the ordinary sense in the technical field of the present invention.
- the forward primer is used for the mutant type, the wild type is used as a common primer, and the reverse primer is a mutant type.
- a form in which a tag sequence is added to the 5 ′ end for use is also included in the scope of the present invention.
- Patent Document 1 when a tag sequence similar to that of the present invention is added to a primer, the purpose is to avoid non-specific reactions (Patent Document 1) and to detect using a tag recognition probe (Patent Document 2).
- Patent Document 2 There are no examples of samples, particularly single nucleotide polymorphism detection samples.
- the present invention provides a sample nucleic acid for single nucleotide polymorphism detection, a PCR primer for preparing a single nucleotide polymorphism detection sample, and a method for preparing a sample for single nucleotide polymorphism detection used for ion exchange chromatography analysis. It is done.
- ion exchange chromatography is used. It is preferable that the eluent used for ion exchange chromatography contains a guanidine salt derived from guanidine represented by the following formula (1).
- guanidine salts include guanidine hydrochloride, guanidine sulfate, guanidine nitrate, guanidine carbonate, guanidine phosphate, guanidine thiocyanate, guanidine sulfamate, aminoguanidine hydrochloride, aminoguanidine bicarbonate, and the like. It is done. Of these, guanidine hydrochloride and guanidine sulfate are preferably used.
- the concentration of the guanidine salt in the eluent at the time of analysis may be appropriately adjusted according to the substance to be detected, but is preferably 2000 mmol / L or less. Specifically, a method of performing gradient elution with a guanidine salt concentration in the range of 0 to 2000 mmol / L can be mentioned. Therefore, the concentration of guanidine salt at the start of analysis need not be 0 mmol / L, and the salt concentration of guanidine salt at the end of analysis need not be 2000 mmol / L.
- the gradient elution method may be a low pressure gradient method or a high pressure gradient method, but a method of eluting while performing precise concentration adjustment by the high pressure gradient method is preferred.
- the guanidine salt may be added to the eluent alone or in combination with other salts.
- the salt that can be used in combination with the guanidine salt include, for example, a salt composed of a halide such as sodium chloride, potassium chloride, sodium bromide, potassium bromide and an alkali metal, calcium chloride, calcium bromide, magnesium chloride.
- salts of halides such as magnesium bromide and alkaline earth metals, and inorganic acid salts such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, etc.
- organic acid salts such as sodium acetate, potassium acetate, sodium succinate, potassium succinate and the like may be used.
- buffers and organic solvents can be used. Specifically, for example, Tris hydrochloric acid buffer, TE buffer composed of Tris and EDTA, Tris, acetic acid and EDTA are used. And a TBA buffer solution consisting of Tris, boric acid and EDTA.
- the pH of the eluent is not particularly limited as long as the nucleic acid chain can be separated by anion exchange.
- those having a cationic group introduced on at least the surface of the base particle are preferable, and those having a strong cationic group and a weak anionic group on at least the surface of the base particle. Is more preferable.
- the “strong cationic group” means a cationic group that dissociates in a wide range of pH from 1 to 14. That is, the strong cationic group can be kept dissociated (cationized) without being affected by the pH of the aqueous solution.
- a quaternary ammonium group is mentioned as a strong cationic group.
- Specific examples include trialkylammonium groups such as a trimethylammonium group, a triethylammonium group, and a dimethylethylammonium group.
- examples of counter ions of strong cationic groups include halide ions such as chloride ions, bromide ions, and iodide ions.
- the amount of the strong cationic group is not particularly limited, but the preferable lower limit per dry weight of the filler is 1 ⁇ eq / g, and the preferable upper limit is 500 ⁇ eq / g.
- the amount of the strong cationic group is less than 1 ⁇ eq / g, the holding power of the filler is weakened, and the separation performance may be deteriorated.
- the amount of the strong cationic group exceeds 500 ⁇ eq / g, the retention of the filler becomes too strong, the detection target substance cannot be easily eluted, and problems such as a long analysis time may occur.
- the “weak anionic group” means an anionic group having a pKa of 3 or more. That is, the weak anionic group is affected by the pH of the aqueous solution, and the dissociation state changes. When the pH is higher than 3, the protons of the carboxy group are dissociated and the proportion of negative charges increases. On the other hand, when the pH is lower than 3, the proportion of the non-dissociated state in which the carboxy group protons are bonded increases.
- the weak anionic group include a carboxy group and a phosphate group. Of these, a carboxy group is preferable.
- Examples of a method for introducing a carboxy group into at least the surface of the base particle include, for example, a method of copolymerizing a monomer having a carboxy group, a method of hydrolyzing an ester portion in the monomer, and a carboxy group by treatment with ozone water.
- a method of forming a carboxy group by ozone gas, a method of forming a carboxy group by plasma treatment, a method of reacting a silane coupling agent having a carboxy group, an epoxy group by copolymerizing a monomer having an epoxy group A known method such as a method of forming a carboxy group by ring opening of can be used.
- the base particle has a hydrophobic structure portion, particularly a carbon-carbon double bond, it is preferable to use a method of forming a carboxy group by treatment with ozone water.
- Ozone is highly reactive with a double bond, and ozone that has reacted with the double bond forms an ozonide that is an intermediate, and then a carboxy group or the like is formed.
- Ozone water means that ozone gas is dissolved in water.
- the particle surface can be easily oxidized simply by dispersing the particles in ozone water.
- hydrophilic groups such as a carboxy group, a hydroxyl group, an aldehyde group, and a keto group are formed.
- Ozone has a strong oxidizing effect, but by treating with ozone water, the particle surface can be oxidized more uniformly than by treating with ozone gas, and carboxy groups are formed more uniformly. preferable.
- concentration of the dissolved ozone in ozone water is not specifically limited, A preferable minimum is 20 ppm.
- concentration of dissolved ozone is less than 20 ppm, it takes a long time to form a carboxy group, or the formation of a carboxy group becomes insufficient, and the nonspecific adsorption of the detection target substance is sufficiently suppressed. I cannot do it.
- a more preferable lower limit of the concentration of dissolved ozone is 50 ppm.
- ozone water is a method in which raw water and ozone gas are brought into contact with each other through an ozone gas permeable film that allows only gas to pass and blocks liquid from passing through. Can be prepared.
- the carboxy group introduced on the surface of the substrate particle is almost dissociated, and it is considered that a weak cation exchange interaction occurs with a few cations in the nucleobase.
- hydrophilic groups such as hydroxyl groups, aldehyde groups and keto groups are formed in addition to carboxy groups, and the presence of these hydrophilic groups acts between the surface of the filler and the nucleic acid. It is thought that the hydrophobic interaction is weakened.
- the amount of the weak anionic group introduced into at least the surface of the base particle is not particularly limited as long as it is equal to or less than the strong cationic group amount.
- the base particles for example, synthetic polymer fine particles obtained using a polymerizable monomer, inorganic fine particles such as silica-based particles can be used, and hydrophobic crosslinked polymer particles made of organic synthetic polymers. And a layer made of a hydrophilic polymer having an ion exchange group copolymerized on the surface of the hydrophobic crosslinked polymer particles.
- Hydrophobic crosslinked polymer is a hydrophobic crosslinked polymer obtained by homopolymerizing one kind of hydrophobic crosslinkable monomer, and obtained by copolymerizing two or more kinds of hydrophobic crosslinkable monomers. Any of a crosslinked polymer and a hydrophobic crosslinked polymer obtained by copolymerizing at least one hydrophobic crosslinking monomer and at least one hydrophobic non-crosslinking monomer may be used.
- the hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one monomer molecule.
- ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate , Propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate and other di (meth) acrylates tetramethylol methane tri (meth) acrylate, trimethylol propane tri (meth) acrylate, tetramethylol methane tetra
- Examples include tri (meth) acrylic acid esters such as (meth) acrylate or tetra (meth) acrylic acid esters, and aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene.
- (meth) acryl means “acryl or methacryl”
- (meth) acrylate” means “acrylate or
- the hydrophobic non-crosslinkable monomer is not particularly limited as long as it is a non-crosslinkable polymerizable organic monomer having hydrophobic properties.
- methyl (meth) acrylate, ethyl (meth) acrylate, propyl examples thereof include (meth) acrylates such as (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate and t-butyl (meth) acrylate, and styrene monomers such as styrene and methylstyrene.
- the hydrophobic cross-linked polymer consists of a copolymer of a hydrophobic cross-linkable monomer and a hydrophobic non-cross-linkable monomer
- the inclusion of segments derived from the hydrophobic cross-linkable monomer in the hydrophobic cross-linked polymer is 10% by weight, and the more preferable lower limit is 20% by weight.
- the hydrophilic polymer having an ion exchange group is composed of a hydrophilic monomer having an ion exchange group, and includes a segment derived from a hydrophilic monomer having one or more ion exchange groups.
- Good That is, as a method for producing a hydrophilic polymer having an ion exchange group, a method in which a hydrophilic monomer having an ion exchange group is polymerized alone, a hydrophilic monomer having an ion exchange group and an ion exchange group are used. And a method of copolymerizing with a hydrophilic monomer not to be used.
- the hydrophilic monomer having an ion exchange group preferably has a strong cationic group, and more preferably has a quaternary ammonium group.
- Examples include acrylamidoethyltrimethylammonium chloride, acrylamidoethyltriethylammonium chloride, and acrylamidoethyldimethylethylammonium chloride.
- the average particle diameter of the filler is not particularly limited, but a preferable lower limit is 0.1 ⁇ m and a preferable upper limit is 20 ⁇ m.
- a preferable lower limit is 0.1 ⁇ m and a preferable upper limit is 20 ⁇ m.
- the average particle size of the packing material is less than 0.1 ⁇ m, the internal pressure of the column becomes high, which may cause poor separation. If the average particle size of the packing material exceeds 20 ⁇ m, the dead volume in the column becomes too large and may cause poor separation.
- an average particle diameter shows a volume average particle diameter, and can be measured using a particle size distribution measuring apparatus (made by AccuSizer780 / Particle Sizing Systems).
- the size of the product amplified by the AS-PCR method is preferably 200 bp or less. If the size of the product amplified by the AS-PCR method exceeds 200 bp, the PCR amplification time or the analysis time in ion exchange chromatography may become long, or sufficient separation performance may not be obtained.
- the size of the product amplified by the AS-PCR method is more preferably 100 bp or less.
- the difference in product size (chain length difference) between the wild type and the mutant type amplified by the AS-PCR method is preferably 10 bp or less. Even if the AS primer is designed so that the difference in size between the amplified wild-type product and the mutant product exceeds 10 bp, a desired amplification product may not be obtained in a non-specific amplification reaction or the like.
- the sample nucleic acid for single nucleotide polymorphism detection used for the rapid and simple detection method of single nucleotide polymorphism can be provided.
- Example 1 it is the chromatogram obtained by isolate
- FIG. 1 it is the chromatogram obtained by isolate
- FIG. 1 it is a chromatogram obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region using an anion exchange column 1.
- ozone water is 400 hollow-tube ozone gas permeable membranes having an inner diameter of 0.5 mm, a thickness of 0.04 mm, and a length of 350 cm made of perfluoroalkoxy resin in a jacket having a cylindrical shape with an inner diameter of 15 cm and a length of 20 cm. It was prepared using an ozone water production system (manufactured by Sekisui Chemical Co., Ltd.) containing the accommodated ozone dissolution module. The obtained filler for ion exchange chromatography was measured using a particle size distribution analyzer (Particulate Sizing Systems, “Accurizer 780”), and the average particle size was 10 ⁇ m. The following column (anion exchange column 1) was prepared using the obtained packing material for ion exchange chromatography. Column size: inner diameter 4.6 mm x 20 mm Ion exchange group: quaternary ammonium group
- Example 1 In Example 1, separation detection of wild type 76 bp and mutant type 79 bp of the UGT1A1 * 6 region was performed.
- reaction C1000 Bio-Rad Laboratories
- the temperature cycle is as follows.
- the template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
- Reference Example 1 separation detection of wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region was performed.
- reaction C1000 Bio-Rad Laboratories
- the temperature cycle is as follows.
- the template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
- Comparative Example 1 In Comparative Example 1, an attempt was made to separate and detect wild type 76 bp and mutant type 96 bp of the UGT1A1 * 6 region.
- reaction C1000 Bio-Rad Laboratories
- the temperature cycle is as follows.
- the template was thermally denatured at 94 ° C. for 30 seconds, 40 cycles of 94 ° C. for 15 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 30 seconds were performed, and finally the temperature was kept at 68 ° C. for 5 minutes. Samples were stored at 4 ° C. until use.
- Reference Example 2 separation detection of wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region was performed.
- HPLC analysis was performed using anion exchange column 2 in the same manner as in Example 1 except that sodium chloride was used instead of guanidine hydrochloride as the salt added to eluent B.
- Example 1 chromatograms obtained by separating and detecting wild type 76 bp and mutant type 79 bp of UGT1A1 * 6 region are shown in FIG. 1 (when anion exchange column 1 is used) and FIG. When used). From the results shown in FIGS. 1 and 2, both columns were able to successfully separate and detect wild-type 76 bp and mutant-type 79 bp of UGT1A1 * 6 region amplified by AS-PCR. In particular, when the anion exchange column 1 was used, almost complete separation and detection could be achieved in a short time.
- FIG. 3 chromatograms obtained by separating and detecting wild type 271 bp and mutant type 274 bp in the UGT1A1 * 6 region are shown in FIG. 3 (when anion exchange column 1 is used) and FIG. When used). From the results of FIGS. 3 and 4, in contrast to Example 1, it was not possible to separate the wild type 271 bp and the mutant type 274 bp of the UGT1A1 * 6 region amplified by AS-PCR. This is considered to be because the difference in chain length between the wild type and the mutant type was small with respect to the size of the AS-PCR amplification product.
- FIG. 5 shows a chromatogram obtained by separating and detecting wild type 76 bp and mutant type 79 bp in the UGT1A1 * 6 region using the anion exchange column 2 in Reference Example 2.
- sodium chloride was added to eluent B instead of guanidine hydrochloride, wild type 76 bp and mutant type 79 bp could not be separated.
- the sample nucleic acid for single nucleotide polymorphism detection used for the rapid and simple detection method of single nucleotide polymorphism can be provided.
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Abstract
Description
(a)全鎖長200bp以下である
(b)5’末端、3’末端のいずれかに鋳型DNAと不完全相補な配列(タグ配列)を有する
(c)一塩基多型の有無を比較すべき試料核酸との鎖長差が10bp以下である
以下に本発明を詳述する。
(1)Forwardプライマーは、変異型用、野生型用の2種類を用い、変異型用は3’末端に一塩基多型の塩基配列を有し、野生型用は3’末端(一塩基多型部位に相当)に野生型の塩基配列を有する。
(2)上記2種類のForwardプライマーのうち一方のプライマーは、5’末端に標的核酸の塩基配列とは不完全相補である配列(以下「タグ配列」ということがある。)を有する。
(3)上記タグ配列の鎖長は、10bp以下である。
(4)上記2種類のForwardプライマーは、増幅産物の鎖長が200bp以下になるよう設計されている。
(5)Reverseプライマーは、変異型用、野生型用で共通のプライマーを使用する。
(6)上記Reverseプライマーは、変異型用Forwardプライマーにより、5’末端にタグ配列を有するPCR増幅産物が複製されていた場合には、PCR増幅の2サイクル目以降、3’末端にタグ配列を有する増幅産物を複製することができる。
(A)PCR増幅の1サイクル目の特徴は以下である。変異型用、野生型用の2種類のプライマーは、変異型の鋳型DNA、野生型の鋳型DNAに対してそれぞれアニーリングした後、それぞれ伸長反応が進行し、変異型DNA、野生型DNA、それぞれが複製される。この時、複製された変異型DNAは、複製された野生型DNAに対して、タグ配列の鎖長分だけ全鎖長が長い。
(B)PCR増幅の2サイクル目以降の特徴は以下である。Reverseプライマーは、変異型DNAの複製産物(5’末端にタグ配列を有する)及び野生型DNAの複製産物、それぞれにアニーリングした後、伸長反応が進行し、変異型DNA、野生型DNAそれぞれが複製される。この時、Reverseプライマーで複製された変異型DNAも、複製された野生型DNAに対して、タグ配列の鎖長分だけ全鎖長が長い。
なお、Forwardプライマー、Reverseプライマーの用語は、本発明の技術分野における通常の意味で使用しており、Forwardプライマーを変異型用、野生型用で共通のプライマーを使用し、Reverseプライマーのうち変異型用の5’末端にタグ配列を付加するといった形態も本発明の範囲に含まれることはいうまでもない。
イオン交換クロマトグラフィーに用いる溶離液は、下記式(1)で示されるグアニジンから誘導されるグアニジン塩を含有することが好ましい。
具体的には、グアニジン塩の濃度を0~2000mmol/Lの範囲でグラジエント溶出させる方法を挙げることができる。従って、分析開始時のグアニジン塩の濃度は0mmol/Lである必要はなく、また、分析終了時のグアニジン塩の塩濃度も2000mmol/Lである必要はない。
グラジエント溶出の方法は、低圧グラジエント法であっても高圧グラジエント法であってもよいが、高圧グラジエント法による精密な濃度調整を行いながら溶出させる方法が好ましい。
また、強カチオン性基のカウンターイオンとしては、例えば、塩化物イオン、臭化物イオン、ヨウ化物イオン等のハロゲン化物イオンが挙げられる。
上記弱アニオン性基としては、例えば、カルボキシ基、リン酸基等が挙げられる。なかでも、カルボキシ基であることが好ましい。
オゾンは二重結合との反応性が高く、二重結合と反応したオゾンは、中間体であるオゾナイドを形成し、その後、カルボキシ基等が形成される。
オゾン水を用いることにより、オゾン水中に粒子を分散させるだけで粒子表面を簡便に酸化させることができる。その結果、基材粒子における疎水性の構造部分が酸化され、カルボキシ基、水酸基、アルデヒド基、ケト基等の親水性基が形成されると考えられる。
オゾンには強力な酸化作用があるが、オゾン水を用いて処理することにより、オゾンガスを用いて処理するよりも粒子表面を均一に酸化させることができ、より均一にカルボキシ基が形成されるので好ましい。
また、オゾン水によって処理することで、カルボキシ基の他、水酸基、アルデヒド基、ケト基等の親水性基が形成され、これらの親水性基の存在によって充填剤の表面と核酸との間に働く疎水性相互作用が弱まると考えられる。
従って、少なくとも表面に強カチオン性基と弱アニオン性基とを有する充填剤を用いた場合、主たる相互作用である充填剤表面と核酸との間に働くアニオン交換相互作用に加え、上述したように、弱いカチオン交換相互作用が働いたり、疎水性相互作用が弱まったりすることによって分離性能が向上するものと考えられる。
なお、本明細書において、「(メタ)アクリル」とは、「アクリル又はメタクリル」を意味し、「(メタ)アクリレート」とは、「アクリレート又はメタクリレート」を意味する。
なお、本明細書において平均粒子径は体積平均粒子径を示し、粒度分布測定装置(AccuSizer780/Particle Sizing Systems社製)を用いて測定することができる。
(アニオン交換カラム1)
攪拌機付き反応器中にて、3重量%ポリビニルアルコール(日本合成化学社製)水溶液2000mLに、テトラエチレングリコールジメタクリレート(新中村化学工業社製)300g、トリエチレングリコールジメタクリレート(新中村化学工業社製)100g、及び、過酸化ベンゾイル(キシダ化学社製)1.0gの混合物を添加した。攪拌しながら加熱し、窒素雰囲気下にて80℃で1時間重合した。次に、強カチオン性のイオン交換基(4級アンモニウム基)を有する単量体として、メタクリル酸エチルトリメチルアンモニウムクロリド(和光純薬工業社製)100gをイオン交換水に溶解し、得られた溶液を上記反応器中に更に添加した。次いで、攪拌しながら窒素雰囲気下にて80℃で2時間重合し、重合体組成物を得た。得られた重合体組成物を水及びアセトンで洗浄することにより、基材粒子の表面に4級アンモニウム基を有する親水性の被覆重合体粒子を得た。
得られた被覆重合体粒子10gを溶存オゾン濃度100ppmのオゾン水300mLに浸漬し、30分間攪拌した。攪拌終了後、遠心分離機(日立製作所社製、「Himac CR20G」)を用いて遠心分離し、上澄みを除去した。この操作を2回繰り返し、被覆重合体粒子にオゾン水処理を施し、4級アンモニウム基とカルボキシ基が共存するイオン交換クロマトグラフィー用充填剤を得た。
なお、オゾン水は、内径15cm×長さ20cmの円柱形を有する外套内に、パーフルオロアルコキシ樹脂からなる内径0.5mm×厚さ0.04mm×長さ350cmの中空管状のオゾンガス透過膜400本収容されたオゾン溶解モジュールを含むオゾン水製造システム(積水化学工業社製)を用いて調製した。
得られたイオン交換クロマトグラフィー用充填剤について、粒度分布計(Particle Sizing Systems社製、「Accusizer780」)を用いて測定したところ、平均粒子径は10μmであった。
得られたイオン交換クロマトグラフィー用充填剤を用いて以下のカラム(アニオン交換カラム1)を準備した。
カラムサイズ:内径4.6mm×20mm
イオン交換基:4級アンモニウム基
市販されているカラムとして、以下のカラムを準備した。
品名:TSK-gel DNA-STAT(東ソー社製)
カラムサイズ:内径4.6mm×長さ100mm
イオン交換基:4級アンモニウム基
実施例1では、UGT1A1*6領域の野生型76bpと変異型79bpとの分離検出を行った。
以下に示すAS-PCR条件によって野生型と変異型の増幅産物を得た。
(1)試薬
AccuPrime Taq DNA Polymerase High Fidelity(Invitorgen社製、Lot.760816)
10×AccuPrime PCR Buffer I
AccuPrime Taq DNA Polymerase High Fidelity(5U/μL)
UGT1A1*6 primer(Operon Biotechnologies社製)
Forward(野生型)(10pmol/μL):5’-(cgcctcgttgtacatcagagcgg)-3’(配列番号1)
Forward(変異型)(10pmol/μL):5’-(ctgacgcctcgttgtacatcagagcga)-3”(配列番号2)
Reverse(10pmol/μL):5’-(cacatcctccctttggaatggca)-3”(配列番号3)
Nuclease-free Water(not DEPC-treated)(Ambion社製、Lot.0803015)
UGT1A1遺伝子 野生型配列挿入プラスミド(1×106コピー/μL)
UGT1A1遺伝子 変異型配列挿入プラスミド(1×106コピー/μL)
5μLの10×AccuPrime PCR Buffer I、1μLのForward primer、1μLのReverse primerに総量が49μLとなるようNuclease-free Waterで調整した溶液に、1μLのUGT1A1遺伝子配列挿入プラスミドを添加し反応溶液とした。
C1000(バイオ・ラッド ラボラトリーズ社製)を用いて、PCR反応を行った。温度サイクルは以下に示す通りである。
94℃、30秒でテンプレートを熱変性させ、94℃を15秒、62℃を15秒、68℃を30秒の増幅サイクルを40サイクル行い、最後に68℃、5分で保温した。サンプルは使用するまで4℃で保存した。
準備したアニオン交換カラムを用いて、以下の条件でAS-PCR増幅産物を分離検出した。
システム:LC-20Aシリーズ(島津製作所社製)
溶離液:溶離液A 25mmol/Lトリス塩酸緩衝液(pH7.5)
溶離液B 25mmol/Lトリス塩酸緩衝液(pH7.5)+1mol/Lグアニジン塩酸塩
分析時間:アニオン交換カラム1を用いたときの分析時間は10分
アニオン交換カラム2を用いたときの分析時間は20分
溶出法:以下に示すグラジエント条件により、溶離液Bの混合比率を直線的に増加させた。
アニオン交換カラム1を用いたときの条件
0分(溶離液B40%)→10分(溶離液B50%)
アニオン交換カラム2を用いたときの条件
0分(溶離液B70%)→20分(溶離液B90%)
検体:UGT1A1*6領域の野生型76bp
UGT1A1*6領域の変異型79bp
流速:0.5mL/min(アニオン交換カラム1を用いたとき)
1.0mL/min(アニオン交換カラム2を用いたとき)
検出波長:260nm
試料注入量:10μL
参考例1では、UGT1A1*6領域の野生型271bpと変異型274bpとの分離検出を行った。
以下に示すAS-PCR条件によって野生型と変異型の増幅産物を得た。
AccuPrime Taq DNA Polymerase High Fidelity(Invitorgen社製、Lot.760816)
10×AccuPrime PCR Buffer I
AccuPrime Taq DNA Polymerase High Fidelity(5U/μL)
UGT1A1*6 primer(Operon Biotechnologies社製)
Forward(野生型)(10pmol/μL):5’-(cgcctcgttgtacatcagagcgg)-3’(配列番号1)
Forward(変異型)(10pmol/μL):5’-(ctgacgcctcgttgtacatcagagcga)-3”(配列番号2)
Reverse(10pmol/μL):5’-(gaaagggtccgtcagcatgac)-3”(配列番号4)
Nuclease-free Water(not DEPC-treated)(Ambion社製、Lot.0803015)
UGT1A1遺伝子 野生型配列挿入プラスミド(1×106コピー/μL)
UGT1A1遺伝子 変異型配列挿入プラスミド(1×106コピー/μL)
5μLの10×AccuPrime PCR Buffer I、1μLのForward primer、1μLのReverse primerに総量が49μLとなるようNuclease-free Waterで調整した溶液に、1μLのUGT1A1遺伝子配列挿入プラスミドを添加し反応溶液とした。
C1000(バイオ・ラッド ラボラトリーズ社製)を用いて、PCR反応を行った。温度サイクルは以下に示す通りである。
94℃、30秒でテンプレートを熱変性させ、94℃を15秒、62℃を15秒、68℃を30秒の増幅サイクルを40サイクル行い、最後に68℃、5分で保温した。サンプルは使用するまで4℃で保存した。
準備したアニオン交換カラムを用いて、以下の条件でAS-PCR増幅産物を分離検出した。
システム:LC-20Aシリーズ(島津製作所社製)
溶離液:溶離液A 25mmol/Lトリス塩酸緩衝液(pH7.5)
溶離液B 25mmol/Lトリス塩酸緩衝液(pH7.5)+1mol/Lグアニジン塩酸塩
分析時間:アニオン交換カラム1を用いたときの分析時間は10分
アニオン交換カラム2を用いたときの分析時間は20分
溶出法:以下に示すグラジエント条件により、溶離液Bの混合比率を直線的に増加させた。
アニオン交換カラム1を用いたときの条件
0分(溶離液B60%)→10分(溶離液B80%)
アニオン交換カラム2を用いたときの条件
0分(溶離液B80%)→20分(溶離液B100%)
検体:UGT1A1*6領域の野生型271bp
UGT1A1*6領域の変異型274bp
流速:0.5mL/min(アニオン交換カラム1を用いたとき)
1.0mL/min(アニオン交換カラム2を用いたとき)
検出波長:260nm
試料注入量:10μL
比較例1では、UGT1A1*6領域の野生型76bpと変異型96bpとの分離検出を試みた。
AccuPrime Taq DNA Polymerase High Fidelity(Invitorgen社製、Lot.760816)
10× AccuPrime PCR Buffer I
AccuPrime Taq DNA Polymerase High Fidelity(5U/μL)
UGT1A1*6 primer(Operon Biotechnologies社製)
Forward(野生型)(10pmol/μL):5’-(cgcctcgttgtacatcagagcgg)-3’(配列番号1)
Forward(変異型)(10pmol/μL):5’-(atagttgtcctagcacctgacgcctcgttgtacatcagagcga)-3”(配列番号5)
Reverse(10pmol/μL):5’-(cacatcctccctttggaatggca)-3”(配列番号3)
Nuclease-free Water(not DEPC-treated)(Ambion社製、Lot.0803015)
UGT1A1遺伝子 野生型配列挿入プラスミド(1×106コピー/μL)
UGT1A1遺伝子 変異型配列挿入プラスミド(1×106コピー/μL)
5μLの10×AccuPrime PCR Buffer I、1μLのForward primer、1μLのReverse primerに総量が49μLとなるようNuclease-free Waterで調整した溶液に、1μLのUGT1A1遺伝子配列挿入プラスミドを添加し反応溶液とした。
C1000(バイオ・ラッド ラボラトリーズ社製)を用いて、PCR反応を行った。温度サイクルは以下に示す通りである。
94℃、30秒でテンプレートを熱変性させ、94℃を15秒、62℃を15秒、68℃を30秒の増幅サイクルを40サイクル行い、最後に68℃、5分で保温した。サンプルは使用するまで4℃で保存した。
参考例2では、UGT1A1*6領域の野生型76bpと変異型79bpとの分離検出を行った。
Claims (5)
- 下記(a)~(c)の特性を有する一塩基多型検出用の試料核酸。
(a)全鎖長200bp以下である
(b)5’末端、3’末端のいずれかに鋳型DNAと不完全相補な配列(タグ配列)を有する
(c)一塩基多型の有無を比較すべき試料核酸との鎖長差が10bp以下である - さらに、(d)タグ配列の鎖長が10bp以下である、請求項1記載の試料核酸。
- イオン交換クロマトグラフィー分析に用いられるものである、請求項1又2記載の試料核酸。
- 下記(a)~(c)の特性を有する一塩基多型検出試料調製用のPCRプライマー。
(a)AS-PCR法に用いた場合に全鎖長200bp以下の増幅産物を複製する
(b)5’末端に鋳型DNAと不完全相補な配列(タグ配列)を有する
(c)タグ配列の鎖長が10bp以下である - 請求項4に記載のPCRプライマーを用いることを特徴とする、イオン交換クロマトグラフィー分析に用いる一塩基多型検出用試料の調製方法。
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EP12765405.1A EP2692863B1 (en) | 2011-03-31 | 2012-03-30 | Pcr primers for preparing samples for single nucleotide polymorphism detection comprising allele-specific pcr and ion exchange chromatographic detection |
CN2012800147838A CN103443274A (zh) | 2011-03-31 | 2012-03-30 | 单核苷酸多态性检测用的试样核酸、单核苷酸多态性检测试样制备用的pcr引物以及用于离子交换色谱法分析的单核苷酸多态性检测用试样的制备方法 |
JP2013507820A JPWO2012133834A1 (ja) | 2011-03-31 | 2012-03-30 | 一塩基多型検出用の試料核酸、一塩基多型検出試料調製用のpcrプライマー及びイオン交換クロマトグラフィー分析に用いる一塩基多型検出用試料の調製方法 |
KR1020137027899A KR20140026426A (ko) | 2011-03-31 | 2012-03-30 | 단일 염기 다형 검출용의 시료 핵산, 단일 염기 다형 검출 시료 조제용의 pcr 프라이머 및 이온 교환 크로마토그래피 분석에 사용하는 단일 염기 다형 검출용 시료의 조제 방법 |
US14/008,770 US20140349284A1 (en) | 2011-03-31 | 2012-03-30 | Sample nucleic acid for single nucleotide polymorphism detection purposes, pcr primer for preparing sample for single nucleotide polymorphism detection purposes, and method for preparing sample for single nucleotide polymorphism detection purposes which can be used in ion exchange chromatographic analysis |
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WO2015129916A1 (ja) * | 2014-02-28 | 2015-09-03 | 国立研究開発法人国立がん研究センター | 腎細胞癌の予後判定方法 |
WO2017170101A1 (ja) * | 2016-03-31 | 2017-10-05 | 積水メディカル株式会社 | 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 |
WO2019064483A1 (ja) | 2017-09-29 | 2019-04-04 | 積水メディカル株式会社 | 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 |
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EP2664917B1 (en) | 2011-01-12 | 2017-09-06 | Sekisui Medical Co., Ltd. | Method for detecting single nucleotide polymorphisms |
CN104946626A (zh) | 2011-01-12 | 2015-09-30 | 积水医疗株式会社 | 离子交换色谱法用洗脱液以及核酸链的分析方法 |
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KR20140026426A (ko) | 2014-03-05 |
US20140349284A1 (en) | 2014-11-27 |
EP2692863B1 (en) | 2018-06-20 |
JPWO2012133834A1 (ja) | 2014-07-28 |
EP2692863A4 (en) | 2015-02-25 |
EP2692863A1 (en) | 2014-02-05 |
CN103443274A (zh) | 2013-12-11 |
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