JP2002272463A - Method for judging form of monobasic polymorphism - Google Patents

Method for judging form of monobasic polymorphism

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JP2002272463A
JP2002272463A JP2001083704A JP2001083704A JP2002272463A JP 2002272463 A JP2002272463 A JP 2002272463A JP 2001083704 A JP2001083704 A JP 2001083704A JP 2001083704 A JP2001083704 A JP 2001083704A JP 2002272463 A JP2002272463 A JP 2002272463A
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nucleotide
polynucleotide
single
method
labeled
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Kunio Hori
邦夫 堀
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Olympus Optical Co Ltd
オリンパス光学工業株式会社
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Abstract

PROBLEM TO BE SOLVED: To provide a method for simply judging the form of a monobasic substitution in a short time. SOLUTION: This method for judging the form of monobasic polymorphism comprises a process for preparing a polynucleotide which is a polynucleotide obtained by hybridizing a target polynucleotide having a monobasic polymorphism part at, n-position with a primer and in which a nucleotide located at 3' end of the primer is hybridized with a nucleotide at (n+1) position of the target polynucleotide, a process for carrying out an elongation reaction using a labeled modified nucleotide which is a modified nucleotide complementary to a nucleotide species, which exists at the polymorphism part and is to be detected, and modified to stop a further elongation reaction, a process for assaying a position change in a minute space of the labeling with time and a process for analyzing the assayed result of the previous process using a fluorescent correlation analysis method and determining whether the labeled nucleotide is taken in the polynucleotide or not to judge the form of a monobasic polymorphism.

Description

【発明の詳細な説明】 DETAILED DESCRIPTION OF THE INVENTION

【0001】 [0001]

【発明の属する技術分野】本発明は、一塩基多型の型を判定する方法に関する。 The present invention relates to a method for determining the type of single nucleotide polymorphisms.

【0002】 [0002]

【従来の技術】疾患感受性遺伝子や薬物反応に関する遺伝子などを効率よく同定していくためには、信頼性の高いマーカー(Genetic Landmark)が必要であり、そのために制限酵素断片長多型(RFLP) Etc. To continue to efficiently identify the Related Art genes for disease susceptibility genes and drug response, a reliable marker (Genetic Landmark) is required, restriction fragment length polymorphism for the (RFLP)
やVNTR(VariableNumber of T And VNTR (VariableNumber of T
andem Repeat)、マイクロサテライトマーカー等のマーカーが使われてきた。 andem Repeat), markers such as microsatellite markers have been used. しかし、各マーカーともその分布が偏っていたり、数が少ないため遺伝子領域を数Mb程度に絞るのが限界であった。 However, in each marker or it its distribution biased, that squeeze the gene region for a small number about several Mb was limited. この点1塩基変異に基づく多型である一塩基多型(Single N Single nucleotide polymorphism is a polymorphism based on the point 1 nucleotide mutation (Single N
ucleotide Polymorphism;以下、SNPと称する)は、候補遺伝子の領域を30kb ucleotide Polymorphism; hereinafter referred to as SNP) is, 30 kb region of the candidate gene
程度まで絞ることが可能であるため、様々な手法が開発されている。 Since it is possible to narrow extent, various methods have been developed.

【0003】このような1塩基多型を検出する方法として、プライマー伸長によるものがある(米国特許第6, As a method of detecting such single nucleotide polymorphism is by primer extension (U.S. Patent No. 6,
013,431号)。 No. 013,431). 本方法は、標的遺伝子を一本鎖にした後、固相表面に、例えばビオチン−アビジン反応を介して固相し、1塩基変異のある部分のすぐ隣の3'側からある領域にわたって相補的となるようなプライマーをハイブリダイズさせる。 This method is characterized by the single strand of the target gene, to a solid surface, such as biotin - avidin reaction and solid phase via a complementary over a region that is 3 'to the immediately adjacent portion of the single base mutation hybridizing a primer such that. その後、蛍光色素等の標識を付与した塩基アナログ(伸長反応が停止する、例えばd Thereafter, base analogs (extension reaction a labeled applying is stopped, such as a fluorescent dye, for example, d
dNTP)を共存させ、プライマーの3'側から1塩基、変異に対応したものを伸長させる。 dNTPs) coexist, one base from the 3 'side of the primer, extending the one corresponding to the mutation. その後、洗浄を行い、標識からの信号を検出することにより、変異部分の塩基配列を同定する。 Thereafter, washing by detecting a signal from the label, to identify the nucleotide sequence of the mutant portion.

【0004】また、このプライマー伸長法を利用して、 [0004] In addition, by using this primer extension method,
マススペクトロスコピーによる検出を用いる方法も考案されている。 A method using detection by mass spectroscopy have been devised. Tangらは、PROBE(Primer Tang et al., PROBE (Primer
Oligo Base Extension)と呼ばれる方法を開発している(Proc.Natl.Aca Have developed a method called Oligo Base Extension) (Proc.Natl.Aca
d. d. Sci. Sci. USA,96:10016−1002 USA, 96: 10016-1002
0)。 0). この方法は、SNP部位を含む100bp程度の長さのDNAをPCRにより増幅し、脱塩により精製し、5'末にピオチン化又はチオール化等の化学修飾を行い、格子状にウェルを形成したシリコンチップに固定する。 This method, the length of the DNA of about 100bp containing the SNP site was amplified by PCR, and purified by desalting, 5 'perform chemical modifications such as Piochin reduction or thiolation the end to form a well in a grid pattern It is fixed to the silicon chip. 固定、洗浄後、チップ上で3種類のdNTPと1 Fixed, washed, and 3 kinds of dNTP on the chip 1
種類のddNTPを用いて、プライマー伸長反応を行う。 Using different ddNTPs, performing a primer extension reaction. 反応終了後、チップの洗浄を行い、TOF MAS After completion of the reaction, the cleaning of the chip, TOF MAS
S用マトリックス溶液添加して試料の結晶化を行い、最後のMALDI(Matrix Assisted L Matrix solution added to perform crystallization of the specimen S, the last MALDI (Matrix Assisted L
aser Desorption Ionizatio aser Desorption Ionizatio
n)マススペクトロスコピーによって、伸長により伸長した塩基種、すなわち1塩基変異部分の塩基種を同定するものである。 By n) mass spectroscopy, base species extended by extension, that is to identify the base type of single nucleotide mutation site. これらのプライマー伸長反応は、プライマーの3'末端側直近の塩基を直接検出するものであり、1塩基の変異検出に有効な方法である。 These primer extension reaction is used to detect the 3 'end nearest base primer directly, an effective method to mutation detection of single base. さらに、M In addition, M
ALDI TOFによる方法は、塩基種のラベルが不要であるという効果がある。 The method according ALDI TOF has the effect of base type of the label is not required. しかし、これらの技術は、プライマー伸長反応を行う上で標的核酸分子を何らかの固相表面上に固相し、さらには固相した核酸分子とプライマーのハイブリダイゼーションおよび伸長反応を行うために、その反応効率は著しく低下し、操作が煩雑である等の問題点がある。 However, these techniques, and the solid phase on any solid surface of the target nucleic acid molecule in performing a primer extension reaction, more in order to perform the hybridization and extension reactions of the nucleic acid molecules and primers solid phase, the reaction efficiency significantly decreased, there is a problem like the operation is complicated.

【0005】 [0005]

【発明が解決しようとする課題】本発明は、操作が簡便で、一塩基置換の型を短時間で判定することができる方法を提供することを目的とする。 [0008] The present invention, operation is simple, and an object thereof is to provide a method capable of determining a short time the type of single nucleotide substitutions.

【0006】 [0006]

【課題を解決するための手段】上記課題を解決するために、本発明は、一塩基多型の型を判定する方法であって、n位(nは1以上の整数)に一塩基多型部位を有する標的ポリヌクレオチドにプライマーがハイブリダイズしてなるポリヌクレオチドであって、前記プライマーの3'末端に位置するヌクレオチドが、前記標的ポリヌクレオチドの(n+1)位のヌクレオチドと対合するようにハイブリダイズしているポリヌクレオチドを調製する工程と;前記一塩基多型部位に存在し得るヌクレオチド種のうち検出すべきヌクレオチド種と相補的な修飾ヌクレオチドであって、追跡可能な標識で標識され、且つさらなる伸長反応が停止するように修飾された修飾ヌクレオチドを、前記工程で調製されたポリヌクレオチドと混合し、伸長反応を実施 In order to solve the above problems SUMMARY OF THE INVENTION The present invention is monobasic a polymorphic type determining method, a single nucleotide polymorphism in position n (n is an integer of 1 or more) a polynucleotide primer to a target polynucleotide is hybridized with the site, nucleotides located at the 3 'end of the primer, hybridizes to nucleotides pairing the target polynucleotide (n + 1) position process and to prepare a polynucleotide soybean; a complementary modified nucleotides and nucleotide species to be detected among the nucleotide species that may be present in the single-nucleotide polymorphism site, it is labeled with a tracer, and modified modified nucleotides as a further extension reaction is stopped by mixing a polynucleotide prepared in the above step, carried out extension reaction する工程と;前記標識の微小空間における位置変化を経時的に計測する工程と;蛍光相関分析法を用いて前工程の計測結果を解析して、検出すべきヌクレオチドと相補的な標識ヌクレオチドが、前記伸長反応工程において前記ポリヌクレオチドに取り込まれたか否かを決定することによって、一塩基多型の型を判定する工程と;を備えた方法を提供する。 Step of the; process and that over time measures the position change in the labeling of the minute space; by analyzing the measurement result before using fluorescence correlation spectroscopy process, the complementary labeled nucleotide as to be detected nucleotides, wherein by determining whether or not incorporated into the polynucleotide in the extension reaction step, a step of determining a type of single nucleotide polymorphisms; provides a method which includes a.

【0007】本明細書において、「一塩基多型」とは、 [0007] As used herein, the term "single nucleotide polymorphism"
一塩基変異に基づく遺伝的多型を意味する。 It means a genetic polymorphism that is based on single nucleotide.

【0008】 [0008]

【発明の実施の形態】本発明は、一塩基多型の型を判定する方法を提供する。 DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for determining the type of single nucleotide polymorphisms.

【0009】本明細書において、「一塩基多型の型を判定する」とは、一塩基変異が生じている部位(一塩基多型部位)に存在するヌクレオチドの種類を推定し、又は決定することをいう。 [0009] In this specification, "determines the type of single nucleotide polymorphism", estimates the type of nucleotide present at a site single nucleotide occurs (single nucleotide polymorphism site) or determined say that. 従って、本発明の方法を用いれば、例えば、一塩基多型部位にアデニン、グアニン、又はシトシンの何れかが存在することが知られているときに、当該部位のヌクレオチドをアデニンであると決定すること、又はアデニンでないと決定することができる。 Thus, using the method of the present invention, for example, when the adenine in a single nucleotide polymorphism site, guanine, or that any cytosines present are known to determine the site at nucleotide to be adenine it, or it may determine not to be adenine.

【0010】なお、一塩基多型部位を有する標的ポリヌクレオチドは、以下の実施例では一本鎖ポリヌクレオチドを使用しているが、特に一本鎖に限定されず、例えば二本鎖ポリヌクレオチドを使用してもよい。 [0010] Incidentally, the target polynucleotide having a single nucleotide polymorphic site, in the following embodiment uses a single-stranded polynucleotide is not particularly limited to the single-stranded, such as double-stranded polynucleotide it may also be used.

【0011】以下、実施例によって、本発明をさらに詳細に説明するが、以下の実施例は、いかなる意味においても、本発明の範囲を限定することを意図したものではない。 [0011] Hereinafter, examples will be described the present invention in more detail, the following examples in any way, not intended to limit the scope of the present invention.

【0012】[実施例1]本実施例では、図1を参照しながら、検出すべき一種類のヌクレオチド種と相補的な標識された修飾ヌクレオチドを用いて、一塩基多型部位に前記検出すべきヌクレオチド種が存在するか否かを判定する方法について説明する。 [0012] [Example 1] In this embodiment, with reference to FIG. 1, using a complementary labeled modified nucleotides and one type of nucleotide species to be detected, to the detection single nucleotide polymorphism site It described whether a method of determining whether to nucleotide species is present.

【0013】本実施例の方法を実施するには、まず、プライマー12が、標的一本鎖ポリヌクレオチド11中の一塩基多型部位13に隣接するようにハイブリダイズした部分的二本鎖ポリヌクレオチド14を調製する。 [0013] To implement the method of this embodiment, first, a primer 12 is hybridized partially double-stranded polynucleotide so as to be adjacent to the single nucleotide polymorphism site 13 in a target single-stranded polynucleotide 11 14 is prepared.

【0014】図1に示されているように、プライマー1 [0014] As shown in FIG. 1, the primer 1
2の3'末端に存在するヌクレオチドは、一塩基多型部位13に存在するヌクレオチド(図ではアデニン(A))の3'側に隣接するヌクレオチドとハイブリダイズしている。 2 of 3 'nucleotides present at the end, the single base nucleotide present in the polymorphic site 13 (in figure adenine (A)) 3' of are nucleotides hybridize adjacent to side.

【0015】部分的二本鎖ポリヌクレオチド14を調製するには、典型的には、標的一本鎖ポリヌクレオチド1 [0015] To prepare a partially double-stranded polynucleotide 14 is typically a target single-stranded polynucleotide 1
1にプライマー12をハイブリダイズさせればよい。 Primer 12 it is sufficient to hybridize to one.

【0016】部分的二本鎖ポリヌクレオチド14を調製した後、一塩基多型部位に存在し得るヌクレオチド種のうち検出すべきヌクレオチド種と相補的であり、且つ追跡可能な標識で標識された修飾ヌクレオチド15を部分的二本鎖ポリヌクレオチド3と混合する。 [0016] After preparation of partially double-stranded polynucleotide 14 is complementary to the single nucleotide polymorphism nucleotide species to be detected among the nucleotide species that may be present at the site, and tracking labels modified labeled with the nucleotide 15 is mixed with partially double stranded polynucleotide 3. 図1では、標識された修飾ヌクレオチド15の他に、標識されていない修飾ヌクレオチド16も示されているが、標識されていない修飾ヌクレオチド16は必ずしも加える必要はない。 In Figure 1, in addition to the labeled modified nucleotides 15, it has been modified nucleotides 16 unlabeled also shown, modified nucleotides 16 unlabeled need not necessarily be added.

【0017】前記標識は、追跡可能であれば任意の標識であり得るが、好ましい標識は、発光性の標識、とりわけ蛍光標識であり得る。 [0017] The label is may be tracked if any label, the preferred label, luminescent label can be especially a fluorescent label.

【0018】図1では、検出すべきヌクレオチド種がアデニンである場合が例示されているので、標識された修飾ヌクレオチドに含まれる核酸塩基はチミンとなっている。 [0018] In Figure 1, because if the nucleotide species to be detected is adenine is illustrated, the nucleic acid bases contained in the labeled modified nucleotides has a thymine.

【0019】標識された修飾ヌクレオチド15を加えた後には、伸長反応を行う。 [0019] After adding labeled modified nucleotides 15 performs extension reaction. 伸長反応を行うには、酵素、 To do extension reaction, the enzyme,
例えば、DNAポリメラーゼなどのポリメラーゼを使用すればよい。 For example, it may be used polymerase such as DNA polymerase.

【0020】標識された修飾ヌクレオチド15は、さらなる伸長反応が停止するように修飾されている。 The labeled modified nucleotides 15, further extension reactions are modified to stop. 「さらなる伸長反応が停止するように修飾された」とは、核酸の伸長反応において、修飾ヌクレオチド15自体はプライマーに付加されてプライマーを伸長させるが、引き続く伸長反応が起こらないように修飾されていることを意味する。 The "additional extension reaction was modified to stop", the extension reaction of nucleic acids, modified nucleotides 15 itself is to extend the primer is added to the primer is modified to subsequent extension reaction does not occur it means that. それ故、修飾ヌクレオチド15を用いれば、伸長反応は、一塩基だけ伸長しただけで停止する。 Therefore, the use of the modified nucleotide 15, the extension reaction is stopped only by extended by a single nucleotide. このような修飾ヌクレオチドは、核酸の塩基配列決定などの分野で多用されているジデオキシヌクレオチドであり得るが、これに限定されない。 Such modified nucleotides include, but may be dideoxy nucleotides have been widely used in fields such as sequencing of nucleic acids, but is not limited thereto.

【0021】伸長反応は、一塩基だけ伸長して停止するので、標的一本鎖ヌクレオチド11の一塩基多型部位に検出すべきヌクレオチド種が存在するときだけ、部分的二本鎖ポリヌクレオチド14を構成するプライマー12 The extension reaction will stop extends by one base, only when the nucleotide species to be detected single nucleotide polymorphic site of the target single-stranded nucleotide 11 is present, the partially double-stranded polynucleotide 14 primer 12 constituting
の3'末端に、標識された修飾ヌクレオチド15が付加される。 The 3 'end of a labeled modified nucleotides 15 are added. これに対して、標的一本鎖ヌクレオチド11の一塩基多型部位に検出すべきヌクレオチド種が存在しないときには、部分的二本鎖ポリヌクレオチド14に標識された修飾ヌクレオチド15は導入されない。 In contrast, when the nucleotide species to be detected single nucleotide polymorphic site of the target single-stranded nucleotide 11 is not present, a labeled modified nucleotides 15 partially double-stranded polynucleotide 14 is not introduced. 図1の場合、標的一本鎖ヌクレオチド11の中に検出すべきヌクレオチドであるアデニンが含まれているので、標識されたジデオキシチミジンが部分的二本鎖ポリヌクレオチド14に導入された様子が示されている。 For Figure 1, because it contains adenine nucleotide to be detected in a target single-stranded nucleotide 11, how the labeled dideoxy thymidine is introduced partially double-stranded polynucleotide 14 is shown ing.

【0022】伸長反応に続いて、部分的二本鎖ポリヌクレオチド14に取り込まれた、又は取り込まれなかった前記標識の微小空間における位置変化を経時的に計測する。 [0022] Following the extension reaction, incorporated into the partially double-stranded polynucleotide 14, over time to measure or unincorporated said label position change in a minute space.

【0023】本明細書において、「微小空間」とは、容積が10 −21 L(1nm四方の立方体の体積に相当する)〜10 −3 L、典型的には10 −18 L〜10 −9 In the present specification, the term "small space" volume (corresponding to a volume of 1nm square cube) 10 -21 L ~10 -3 L, typically 10 -18 L~10 -9
L、最も典型的には10 −15 L〜10 −12 Lの空間をいう。 L, most typically refers to the space of 10 -15 L~10 -12 L.

【0024】微小空間の形状は、球状、円錐状、立方体状、直方体状等任意の形状であり得る。 [0024] the micro space shaped, spherical, conical, cubic, can be any shape rectangular shape.

【0025】このような微小空間における標識の位置変化を計測するためには、典型的には、共焦点顕微鏡を使用する。 [0025] To measure the change in position of the label in such a minute space typically using a confocal microscope. 共焦点顕微鏡自体は、本分野で公知である。 Confocal microscopy itself are known in the art.

【0026】共焦点顕微鏡21を用いた検出は、図2に示されているように、 1. [0026] The detection using the confocal microscope 21, as shown in FIG. 2, 1. レーザー発生装置22からレーザー光を励起光として照射する; 2. Irradiating a laser beam as excitation light from the laser generator 22; 2. フィルター23を通過させた後、レーザー光を集光し、ダイクロイックミラー24によって試料中の一点にレーザー光を照射する; 3. After passing through the filter 23, the laser beam is condensed to irradiate the laser beam to a point in the sample by the dichroic mirror 24; 3. 試料中の蛍光物質をレーザー光で励起して発光させる; 4. A fluorescent substance in the sample emit light by excitation by laser light; 4. ピンホール25を通過させることにより、試料の焦点中の蛍光物質から発せられた蛍光のみを光増倍管(P By passing the pinhole 25, the photomultiplier only the fluorescence emitted from the fluorescent substance in the focal point of the sample (P
MT)26で増幅する; 5. MT) is amplified by 26; 5. データ処理装置27により、増幅した蛍光を解析し、表示装置28に結果を表示することによってなされる。 The data processor 27 analyzes the amplified fluorescence, is done by displaying the result on the display device 28.

【0027】共焦点顕微鏡の光源としては、例えば、アルゴンイオンレーザーを使用し得るが、蛍光物質の種類に応じて、波長の異なるクリプトンアルゴンイオンレーザー、ヘリウムネオンレーザー、ヘリウムカドミニウムレーザーも使用できる。 [0027] as a confocal microscope light source, for example, but may use an argon ion laser, depending on the type of fluorescent material, different krypton argon ion laser wavelength, a helium neon laser, helium-cadmium laser can also be used.

【0028】このような検出方法は、微小空間空間中に存在する標識から発せられる蛍光のみを検出するので、 [0028] Such detection methods, and detects only the fluorescence emitted from the label present in the micro space space,
バックグラウンドが極めて少なく、通常の蛍光検出に比べて著しく感度が高い。 Background is extremely small, significantly higher sensitivity than normal fluorescence detection.

【0029】「標識の位置変化の経時的な計測」は、一般的にはミリ秒〜分の単位、最も一般的には秒の単位で行われ、極めて短時間で終了し得る。 The "time measurement of position change of the label" units of the general milliseconds to minutes, most commonly performed in units of seconds, can be completed in a very short time.

【0030】計測が終了したら、計測結果を解析して、 [0030] When the measurement is completed, by analyzing the measurement result,
検出すべきヌクレオチドと相補的な標識ヌクレオチドが標的ポリヌクレオチドに導入されたか否かを決定する。 Complementary labeled nucleotide as to be detected nucleotide is determined whether it has been introduced into the target polynucleotide.

【0031】計測結果の解析は、蛍光相関分析法によって行うのが好ましい。 The measurement results analysis is preferably performed by fluorescence correlation spectroscopy. ここで、「蛍光相関分析法」とは、微小空間内に存在する平均数個(ある場合には一個)の蛍光物質が発する蛍光の強度を一定時間測定した後、ブラウン運動に由来する蛍光のゆらぎの自己相関関数をとり、該関数の分析によって、蛍光物質に関する種々のデータを取得する方法をいう。 Here, "fluorescence correlation analysis method" and, after measuring the intensity of fluorescence emitted by the fluorescent substance for a predetermined time (one in some cases) several average present in very small space, the fluorescence derived from Brownian motion taking the autocorrelation function of the fluctuation, by analysis of the function number, it refers to a method of obtaining various data relating to the fluorescent substance. 蛍光相関分析法(以下FCS(fluorescence correla Fluorescence correlation spectroscopy (hereinafter FCS (fluorescence correla
tion spectrometry)という)自体は公知であり、その詳細については、特表平11−502 tion spectrometry) hereinafter) are known per se, for the details, Hei 11-502
608号などを参照されたい。 See, such as 608 issue.

【0032】FCSを用いて、計測結果を解析するには、以下の操作を行う。 [0032] Using FCS, to analyze the measurement result, it performs the following operations.

【0033】 1. [0033] 1. 微小空間にレーザー光を照射する。 Irradiating a laser beam on the minute space. 2. 2. 該微小空間中に存在する蛍光物質から発せられる蛍光の強度を経時的に測定し、図3に示されているデータを取得する。 The intensity of the fluorescence emitted from the fluorescent substance existing in the fine small spaces was measured over time, to obtain the data shown in FIG. 3. 3. 異なる2時点の蛍光強度I(t)とI(t+τ)の積の期待値を計算し、自己相関関数G(τ)=<I The expected value of the product of the fluorescence intensity of the two different time points I (t) and I (t + τ) is calculated, the autocorrelation function G (τ) = <I
(t) I(t+τ)>を得る。 Get a (t) I (t + τ)>. 4. 4. 下式1: The following formula 1:

【数1】 [Number 1] (ここで、N:蛍光分子の平均数 τ small =wo 2/4small :サイズが小さい核酸の並進拡散時間 τ large =wo 2/4large :サイズが大きい核酸の並進拡散時間 y=繰り返し数が多い反復配列の割合 S=wo/zoである (なお、woは検出領域の径、2zoは領域長、D (Where, N: fluorescent molecules average number τ small = wo 2/4 D small of: small size translational diffusion time of the nucleic acid τ large = wo 2/4 D large: the translational diffusion time of the nucleic acid oversize y = repeatedly number is the ratio S = wo / zo of more repeats (Note, wo is the diameter of the detection area, 2Zo region length, D
smallとD largeは、それぞれサイズが小さい核酸及びサイズが大きい核酸の並進拡散定数である)) small and D large is the translational diffusion constant of a nucleic acid is large or small nucleic acids and size size, respectively))
を用いて、3. Using, 3. で得た自己相関関数を解析する。 In analyzing the autocorrelation function obtained.

【0034】FCSによるデータ解析には、Evote [0034] The data analysis by FCS, Evote
c BioSystems社から発売されているコンピュータープログラム「FCS」を使用できる。 c BioSystems Inc. computer has been released from the program the "FCS" can be used. 前記工程1〜4までの操作時間は、1つの試料当り10秒未満であり得る。 Operation time until the step 1-4, may be less than 10 seconds per one sample.

【0035】このような分析の概念は、図3によって、 The concept of such analysis, by Figure 3,
より明確となろう。 It will become more clear. すなわち、サイズが小さい場合にはブラウン運動の速度が大きいので、I(t)の周波数が大きい。 That is, since if the size is small speed of Brownian motion, the greater the frequency of the I (t). これに対して、サイズが大きい場合にはブラウン運動の速度が小さいので、I(t)の周波数が小さい。 In contrast, since the case of a large size velocity of the Brownian motion is small, the frequency of the I (t) is small.

【0036】従って、式(1)を用いれば、核酸のサイズ、各核酸の割合を推定し得る。 [0036] Thus, by using the equation (1) may estimate the size of the nucleic acid, the rate of each nucleic acid.

【0037】標的一本鎖ポリヌクレオチド11に標識された修飾ヌクレオチド15が取り込まれたときには、標識のブラウン運動の速度は小さくなるのに対して、標的一本鎖ポリヌクレオチドに修飾ヌクレオチド15が取り込まれないときには、標識のブラウン運動の速度は大きい。 [0037] When the labeled modified nucleotides 15 to a target single-stranded polynucleotide 11 is captured, whereas the rate of labeling of Brownian motion becomes smaller, modified nucleotides 15 incorporated into a single-stranded polynucleotide target when there is no, the speed of the labeling of the Brownian motion is large. それ故、本発明の方法に蛍光相関分析法を適用することにより、標的一本鎖ポリヌクレオチド中に修飾ヌクレオチド15が取り込まれたか否かが分かる。 Therefore, by applying the fluorescence correlation spectrometry method of the present invention, whether seen modified nucleotides 15 in the target single-stranded polynucleotide is incorporated.

【0038】図1のグラフのうち、曲線17は、標識されたジデオキシチミジンが取り込まれなかった場合の自己相関関数G(τ)の経時変化を示しており、曲線18 [0038] Among the graph of FIG. 1, curve 17 shows the change with time of the autocorrelation function G (tau) in the case where the labeled dideoxythymidine unincorporated, curve 18
は、標識されたジデオキシチミジンが取り込まれた場合の自己相関関数G(τ)の経時変化を示している。 Shows the time course of the autocorrelation function G (tau) in the case where the labeled dideoxy thymidine incorporated.

【0039】グラフから明らかなように、標識されたジデオキシチミジンが取り込まれた場合と取り込まれなかった場合とでは、曲線の形状が異なるので、蛍光相関分析法を用いれば、標識されたジデオキシチミジンが取り込まれたか否かを即時に決定することができる。 [0039] As apparent from the graph, in the case where the labeled dideoxythymidine unincorporated and when incorporated, the shape of the curve is different, the use of fluorescence correlation spectroscopy, is labeled dideoxythymidine it is possible to determine whether the captured immediately.

【0040】実際の測定では、標的一本鎖ポリヌクレオチド11と標識された修飾ヌクレオチド15は、一対一の割合で存在せずに、標識された修飾ヌクレオチド15 [0040] In actual measurement, a target single-stranded polynucleotide 11 and the labeled modified nucleotides 15, in the absence at a one-to-one ratio, labeled modified nucleotides 15
が標的一本鎖ポリヌクレオチド11よりも多いことが通常である。 It is usually but greater than a target single-stranded polynucleotide 11. そのため、曲線17と18の中間に、曲線1 Therefore, in the middle of the curve 17 and 18, curve 1
9のような形状の曲線が得られる場合が多い。 If curved shapes, such as 9 can be obtained in many cases.

【0041】標識された修飾ヌクレオチド15が標的一本鎖ポリヌクレオチド11に比べて過剰であると、標的一本鎖ポリヌクレオチド11に取り込まれた修飾ヌクレオチド15が極めて僅かとなる。 The labeled modified nucleotides 15 when an excessive compared to a target single-stranded polynucleotide 11, modified nucleotides 15 incorporated into a target single-stranded polynucleotide 11 becomes extremely small. このため、かかる場合に得られる曲線は、曲線17と区別できず、修飾ヌクレオチド15が標的一本鎖ポリヌクレオチド11に取り込まれなかったものと誤って判断されてしまうことになる。 Therefore, curve obtained in such a case can not be distinguished from the curves 17, modified nucleotides 15 so that would be determined incorrectly not to have been incorporated in a target single-stranded polynucleotide 11. それ故、標的一本鎖ポリヌクレオチド11に取り込まれなかった修飾ヌクレオチド15の量は、修飾ヌクレオチド15の全量の半分以下であることが望ましい。 Therefore, the amount of modified nucleotides 15 unincorporated target single-stranded polynucleotide 11 is preferably less than half of the total amount of modified nucleotides 15.

【0042】一方、標識された修飾ヌクレオチド15の量が少なすぎると、伸長反応の進行速度が極端に遅くなってしまうので、伸長反応が適切に進行し得る量の標識された修飾ヌクレオチド15を使用することが好ましい。 On the other hand, if the amount of labeled modified nucleotide 15 is too small, since the progression rate of the extension reaction becomes extremely slow, use amount of labeled modified nucleotides 15 extension reaction can proceed suitably it is preferable to.

【0043】以上のように、本実施例の方法を用いれば、一塩基多型部位に検出すべきヌクレオチド種が存在するか否かを簡易且つ迅速に判定することができる。 [0043] As described above, by using the method of this embodiment, it is possible to determine whether there is nucleotide species to be detected single nucleotide polymorphism site easily and quickly.

【0044】本実施例では、所定の部位が一塩基多型部位であることが明らかとなっている場合を例にとって説明を行ったが、本実施例の各工程は、ある部位が一塩基多型部位であるか否かが不明な場合に、当該部位に一塩基多型が存在するか否かを調べるためにも使用できる。 [0044] In this example, has been described a case where it predetermined site is a single nucleotide polymorphism site has revealed an example, each step of this embodiment, a site is single nucleotide polymorphisms If whether a type sites are unknown, it can also be used to investigate whether single nucleotide polymorphisms on the site is present.
すなわち、本発明は、一本鎖標的ポリヌクレオチド中の所定部位に一塩基多型が存在するか否かを調べる方法も提供する。 That is, the present invention also provides a method of examining whether a single nucleotide polymorphism at a predetermined site in the single stranded target polynucleotide is present.

【0045】所定の部位が一塩基多型部位であることが不明な場合には、当該部位に存在することが知られているヌクレオチド種とは異なるヌクレオチド種と相補的な修飾ヌクレオチドを用いて伸長反応を実施すればよい。 [0045] When a predetermined site it is not known a single-nucleotide polymorphism site, extended with a complementary modified nucleotides and different nucleotide species from the nucleotide species to be present at the site it is known the reaction may be carried out.
当該部位に存在することが知られているヌクレオチド種とは異なるヌクレオチド種と相補的な修飾ヌクレオチドがプライマーに付加されれば、前記所定の部位が一塩基多型部位であると推定される。 If complementary to modified nucleotides and different nucleotide species from the nucleotide species to be present at the site is known is added to the primer, the predetermined site is presumed to be the single nucleotide polymorphism site.

【0046】[実施例2]本実施例では、図4を参照しながら、一塩基多型部位に存在し得る複数のヌクレオチド種のうち、一塩基多型部位に何れのヌクレオチド種が存在するかを決定する方法について説明する。 [0046] In EXAMPLE 2 This Example, with reference to FIG. 4, one base of a plurality of nucleotide species that may be present in the polymorphic site, there are any nucleotide species to a single nucleotide polymorphism site a description will be given of a method of determining the.

【0047】本実施例の方法を実施するには、まず、プライマー42が、標的一本鎖ポリヌクレオチド41中の一塩基多型部位43に隣接するようにハイブリダイズした部分的二本鎖ポリヌクレオチド44を調製する。 [0047] To implement the method of this embodiment, first, a primer 42 is hybridized partially double-stranded polynucleotide so as to be adjacent to the single nucleotide polymorphism site 43 in a target single-stranded polynucleotide 41 44 is prepared. 部分的二本鎖ポリヌクレオチド43の調製方法は、実施例1 Process for the preparation of partially double-stranded polynucleotide 43, Example 1
に記載したとおりである。 It is as described.

【0048】図4の例では、一塩基多型部位43には、 In the example of FIG. 4, the single nucleotide polymorphism site 43,
アデニン(A)又はグアニン(G)が存在し得る。 Adenine (A) or guanine (G) may be present.

【0049】部分的二本鎖ポリヌクレオチド44を調製した後、一塩基多型部位に存在し得る各ヌクレオチド種と相補的な標識ヌクレオチド45及び46を、部分的二本鎖ポリヌクレオチド43と混合する。 [0049] After the partially double-stranded polynucleotide 44 were prepared, one base may be present in the polymorphic site and the nucleotide species is complementary labeled nucleotide 45 and 46, mixed with partially double-stranded polynucleotide 43 . 図4の例では、 In the example of FIG. 4,
標識ヌクレオチド45はジデオキシチミジンであり、標識ヌクレオチド46は、ジデオキシグアノシンである。 Labeled nucleotide 45 is a dideoxythymidine, labeled nucleotide 46 is dideoxyguanosine.
両標識ヌクレオチドを区別するために、標識ヌクレオチド45と標識ヌクレオチド46には異なる物質を標識することが好ましい。 To distinguish the two labeled nucleotides, it is preferred to label the different substances in the labeled nucleotide 45 and labeled nucleotides 46.

【0050】標的一本鎖ポリヌクレオチド41中の一塩基多型部位43がアデニンであれば、プライマー42の末端には標識ヌクレオチド45が付加される。 The single nucleotide polymorphism site 43 in a target single-stranded polynucleotide 41 if adenine, the end of the primer 42 is labeled nucleotide 45 is added. 標的一本鎖ポリヌクレオチド41中の一塩基多型部位43がシトシンであれば、プライマー42の末端には標識ヌクレオチド46が付加される。 If single nucleotide polymorphism site 43 in a target single-stranded polynucleotide 41 cytosine, is labeled nucleotide 46 is added to the end of the primer 42.

【0051】伸長反応後に、標識ヌクレオチド中の標識の微小空間における位置変化を経時的に計測する。 [0051] After extension reaction, over time to measure the position change in the labeling of small space in labeled nucleotides. 計測方法は、実施例1に記載したとおりであり、標識ヌクレオチドが取り込まれなかった場合には曲線47が得られ、標識ヌクレオチドが取り込まれた場合には曲線48 Measurement method is as described in Example 1, the curve 47 is obtained when the labeled nucleotides not incorporated, the curve when the labeled nucleotide incorporated is 48
が得られる。 It is obtained.

【0052】計測後、蛍光相関分光法により計測結果を解析する。 [0052] After the measurement, analyzing the measurement result by fluorescence correlation spectroscopy. 各プライマーが異なる蛍光標識で修飾されていれば、測定の際に各蛍光標識に適したフィルター等を用いることによって、何れのプライマーが標的ポリヌクレオチドに結合したかを決定できる。 If each primer is modified with different fluorescent labels, by using a filter or the like suitable for each fluorescent label in the measurement, it can be determined either primer is bound to the target polynucleotide. 蛍光相関分光法を用いて、所定のプライマーが標的ポリヌクレオチドに結合したか否かを決定する方法は、実施例1において前述したとおりである。 Using fluorescence correlation spectroscopy, a method of determining whether a predetermined primer is bound to the target polynucleotide, are as described above in Example 1.

【0053】複数種類のプライマーを用いる本実施例の方法によれば、一塩基多型部位に存在するヌクレオチドの種類を正確に決定することができる。 [0053] According to the method of multiple types of the embodiment using primers, it is possible to accurately determine the type of nucleotide present at a single nucleotide polymorphic site.

【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS

【図1】実施例1の方法を示す模式図。 Figure 1 is a schematic view showing the method of Example 1.

【図2】共焦点顕微鏡の構成を示す図。 Figure 2 is a diagram showing a configuration of a confocal microscope.

【図3】蛍光相関分光法の測定に用いるデータを示す図。 FIG. 3 shows the data used for the measurement of fluorescence correlation spectroscopy.

【図4】実施例2の方法を示す模式図。 Figure 4 is a schematic diagram showing a method of Example 2.

【符号の説明】 DESCRIPTION OF SYMBOLS

11 標的一本鎖ポリヌクレオチド 12 プライマー 13 一塩基多型部位 14 部分的二本鎖ポリヌクレオチド 15 標識された修飾ヌクレオチド 16 修飾されていない修飾ヌクレオチド 17 曲線 18 曲線 19 曲線 21 共焦点顕微鏡 22 レーザー発生装置 23 フィルター 24 ダイクロイックミラー 25 ピンホール 26 光倍増管 27 データ処理装置 28 表示装置 41 標的一本鎖ポリヌクレオチド 42 プライマー 43 一塩基多型部位 44 部分的二本鎖ポリヌクレオチド 45 標識ヌクレオチド 46 標識ヌクレオチド 47 曲線 48 曲線 11 a target single-stranded polynucleotide 12 primer 13 single nucleotide polymorphism site 14 partially double-stranded polynucleotide 15 labeled modified nucleotides 16 unmodified modified nucleotides 17 Curve 18 Curve 19 Curve 21 confocal microscope 22 laser generator 23 filter 24 dichroic mirror 25 pinhole 26 photomultiplier 27 data processing apparatus 28 a display device 41 a target single-stranded polynucleotide 42 primer 43 single nucleotide polymorphism site 44 partially double-stranded polynucleotide 45 labeled nucleotides 46 labeled nucleotides 47 curve 48 curve

Claims (1)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】 一塩基多型の型を判定する方法であって、 n位(nは1以上の整数)に一塩基多型部位を有する標的ポリヌクレオチドにプライマーがハイブリダイズしてなるポリヌクレオチドであって、前記プライマーの3' 1. A method for determining the type of single nucleotide polymorphisms, n-position (n is an integer of 1 or more) polynucleotides primers to a target polynucleotide having a single nucleotide polymorphism site is hybridized a is, 3 of the primers'
    末端に位置するヌクレオチドが、前記標的ポリヌクレオチドの(n+1)位のヌクレオチドと対合するようにハイブリダイズしているポリヌクレオチドを調製する工程と;前記一塩基多型部位に存在し得るヌクレオチド種のうち検出すべきヌクレオチド種と相補的な修飾ヌクレオチドであって、追跡可能な標識で標識され、且つさらなる伸長反応が停止するように修飾された修飾ヌクレオチドを、前記工程で調製されたポリヌクレオチドと混合し、伸長反応を実施する工程と;前記標識の微小空間における位置変化を経時的に計測する工程と;蛍光相関分析法を用いて前工程の計測結果を解析して、検出すべきヌクレオチドと相補的な標識ヌクレオチドが、前記伸長反応工程において前記ポリヌクレオチドに取り込まれたか否かを決定するこ Nucleotide located at the terminal, the target polynucleotide (n + 1) position of a nucleotide and preparing a hybridizing polynucleotide to mate; nucleotide species that may be present in the single-nucleotide polymorphism site of a complementary modified nucleotides and nucleotide species to be detected, it is labeled with a tracer, and the modified modified nucleotides as a further extension reaction is stopped, a polynucleotide prepared in the above step mixing and a step of carrying out the extension reaction; step and that over time measures the position change in the labeling of the minute space; by analyzing the measurement result before using fluorescence correlation spectroscopy process, complementary to the to be detected nucleotides labeling nucleotides, determining child whether or not incorporated into the polynucleotide in the extension reaction step によって、一塩基多型の型を判定する工程と;を備えた方法。 By a step of determining a type of single nucleotide polymorphism; method provided with.
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