JPH01145000A - Determination of mutation on nucleic acid base sequence - Google Patents
Determination of mutation on nucleic acid base sequenceInfo
- Publication number
- JPH01145000A JPH01145000A JP62303316A JP30331687A JPH01145000A JP H01145000 A JPH01145000 A JP H01145000A JP 62303316 A JP62303316 A JP 62303316A JP 30331687 A JP30331687 A JP 30331687A JP H01145000 A JPH01145000 A JP H01145000A
- Authority
- JP
- Japan
- Prior art keywords
- nucleic acid
- base sequence
- mutation
- acid sample
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 72
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 71
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 71
- 230000035772 mutation Effects 0.000 title claims abstract description 25
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims abstract description 26
- 239000002853 nucleic acid probe Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 19
- 150000005621 tetraalkylammonium salts Chemical class 0.000 claims abstract description 10
- 239000012266 salt solution Substances 0.000 claims abstract description 6
- 238000009396 hybridization Methods 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 39
- 238000010494 dissociation reaction Methods 0.000 abstract description 5
- 230000005593 dissociations Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract 1
- 230000000295 complement effect Effects 0.000 description 7
- 238000010828 elution Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 208000026350 Inborn Genetic disease Diseases 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 208000016361 genetic disease Diseases 0.000 description 3
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
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- 229920002678 cellulose Polymers 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
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- NOIRDLRUNWIUMX-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;6-amino-1h-pyrimidin-2-one Chemical compound NC=1C=CNC(=O)N=1.O=C1NC(N)=NC2=C1NC=N2 NOIRDLRUNWIUMX-UHFFFAOYSA-N 0.000 description 1
- FFKUHGONCHRHPE-UHFFFAOYSA-N 5-methyl-1h-pyrimidine-2,4-dione;7h-purin-6-amine Chemical compound CC1=CNC(=O)NC1=O.NC1=NC=NC2=C1NC=N2 FFKUHGONCHRHPE-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
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- 238000010521 absorption reaction Methods 0.000 description 1
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- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
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- 238000004925 denaturation Methods 0.000 description 1
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- 229920001059 synthetic polymer Polymers 0.000 description 1
- 125000005207 tetraalkylammonium group Chemical group 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical compound C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は核酸上で起った突然変異等の塩基配列の変異を
検出する方法に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for detecting mutations in base sequences such as mutations occurring in nucleic acids.
（従来技術とその問題点） 近年、遺伝子工学の進歩に伴ない、動物、植物。(Prior art and its problems) In recent years, with advances in genetic engineering, animals and plants.
細Ｕ、ウィルス等の生物の遺伝情報が明らかになりつつ
ある。特に人間の遺伝子に対する解析は急速に進んでお
り、遺伝病、癌等においては核酸の塩基配列のレベルで
明らかになりつつある。その結果ある種の遺伝病では、
核酸に塩基配列の僅か一部分の変異、即ち点突然変異に
因ることも知られている０以上のように遺伝病等の診断
は、核酸の塩基配列を調べ正常な塩基配列と比較するこ
とにより可能である。従来の方法によれば２例えばまず
核酸試料を細胞から抽出し制限酵素により切断し、を気
泳動法等の方法で核酸の断片の大きさに従って分離し１
分離した該断片をサザンブロッティング法によりフィル
ターに固定化し、ラジオアイソトープで標識された核酸
プローブとハイブリダイズさせ、そのパターンにより変
異の有無を判定する方法があげられる。！！な、他の方
法として１例えば核酸試料を直接フィルターに固定化し
。The genetic information of organisms such as microorganisms and viruses is becoming clearer. In particular, the analysis of human genes is progressing rapidly, and genetic diseases, cancers, etc. are being revealed at the level of nucleic acid base sequences. As a result, in certain genetic diseases,
Diagnosis of genetic diseases such as 0 or more, which are known to be caused by mutations in a small portion of the base sequence of nucleic acids, that is, point mutations, can be made by examining the base sequence of the nucleic acid and comparing it with the normal base sequence. It is possible. According to conventional methods, for example, a nucleic acid sample is first extracted from cells, cut with restriction enzymes, and separated according to the size of nucleic acid fragments using a method such as pneumophoresis.
An example of this method is to immobilize the separated fragments on a filter by Southern blotting, hybridize with a radioisotope-labeled nucleic acid probe, and determine the presence or absence of a mutation based on the pattern. ! ! Another method, for example, is to directly immobilize a nucleic acid sample on a filter.
これにラジオアイソトープで標識された核酸プローブを
ハイブリダイズさせ、ハイブリッドの安定性を測定して
変異の有無を判定する方法、あるいは、核酸試料と核酸
プロニブ°をハイブリダイズさせ、これをホルムアミド
勾配をつけたアクリルアミドゲル電気泳動にかけ、ハイ
ブリッドの変性点をオートラジオグラフにより測定して
変異の有無を判定する方法等があげられる。しかしなが
ら。A method in which a nucleic acid probe labeled with a radioisotope is hybridized to this, and the stability of the hybrid is measured to determine the presence or absence of a mutation.Alternatively, a nucleic acid sample is hybridized with a nucleic acid pronib°, and then a formamide gradient is applied. For example, the presence or absence of a mutation can be determined by subjecting the hybrid to acrylamide gel electrophoresis and measuring the denaturation point of the hybrid using an autoradiograph. however.
例えば制限酵素を用いる方法では操作が複雑で時間がか
かり、また核酸塩基配列の変異が必ずしも制＠＃素によ
る切断パターンとして現れない等の問題点がある。また
、核酸試料と核酸１０−プのハイブリッドの安定性の変
化を調べて変異の有無を判定する方法においては、核酸
試料中の変異の有無の検出に先立って対照を定めて、該
対照の安定性について詳細な検討が必要である。この理
由は核酸試料と核酸１０−プがハイブリッドを形成する
ための塩基間の水素結合、即ちアデニン−チミンまなは
ウラシル（Ａ−ＴまたはＵ）、グアニン−シトシン（Ｇ
−Ｃ）において、Ｇ−Ｃ結合はＡ−Ｔ（またはＡ−Ｕ）
結合に較べ強力であり。For example, methods using restriction enzymes have problems such as complicated and time-consuming operations, and mutations in the nucleic acid base sequence do not necessarily appear as a cleavage pattern due to the restriction enzyme. In addition, in the method of determining the presence or absence of a mutation by examining changes in the stability of a hybrid between a nucleic acid sample and a nucleic acid 10-p, a control is established prior to detecting the presence or absence of a mutation in the nucleic acid sample, and the stability of the control is determined. Detailed consideration of gender is required. The reason for this is the hydrogen bonds between the bases for hybridization between the nucleic acid sample and the nucleic acid 10-p, i.e., adenine-thymine, uracil (AT or U), guanine-cytosine (G
-C), the G-C bond is A-T (or A-U)
It is stronger than a bond.
従ってＧ−Ｃ結合の割合によって該ハイブリッドの安定
性が変化する事による。Therefore, the stability of the hybrid changes depending on the ratio of G--C bonds.
（問題点を解決するための手段）
本発明者らは従来技術にみちれる問題点を解決すべく鋭
意研究を行なった結果、簡単な操作により迅速に実施可
能でしかも核酸試料塩基配列中の変異の有無を判定する
ための対照を必要としない方法を完成させた。即ち本発
明は核酸プローブをもちいて核酸塩基配列における変異
を検出する方法に於いて。(Means for Solving the Problems) The present inventors have conducted intensive research to solve the problems that are present in the prior art, and have found that the present inventors have found a method that can be carried out quickly with simple operations, and that can be used to reduce mutations in the base sequence of a nucleic acid sample. We have completed a method that does not require a control to determine the presence or absence of . That is, the present invention relates to a method for detecting mutations in a nucleic acid base sequence using a nucleic acid probe.
■ 担体に固定化した核酸１０−プに対し核酸試料をテ
トラアルキルアンモニウム塩溶液中でハイブリダイズさ
せ。(2) A nucleic acid sample is hybridized to the nucleic acid 10-p immobilized on a carrier in a tetraalkylammonium salt solution.
■ ハイブリダイズしなかった核酸試料を除去した後。■ After removing unhybridized nucleic acid samples.
■ ハイブリッド形成時の温度から０℃〜１００℃の範
囲に温度を上昇させて核酸試料を解離させ。(2) Dissociate the nucleic acid sample by increasing the temperature from the temperature at the time of hybridization to a range of 0°C to 100°C.
■ 解離した核酸試料を測定する
ことを特徴とする核酸塩基配列における突然変異の測定
法に関するものである。(2) This invention relates to a method for measuring mutations in nucleic acid base sequences, which is characterized by measuring dissociated nucleic acid samples.
以下本発明の詳細な説明する。The present invention will be explained in detail below.
本発明では、核酸試料への核酸プローブの特異性を得る
ために、１０塩基以上の長さを有する核酸プローブを用
いることが望ましい、また９例えば核酸試料と対応する
核酸プローブのハイブリッドが１００塩基対以上となる
場合においては、核酸試料中の変異による該ハイブリッ
ド中のミスマツチが少数であると、該ミスマツチに起因
するハイブリッドの安定性の変化は微妙となり測定しに
くくなる。従って、用いる核酸プローブは１０〜１００
塩基、更に好ましくは１５〜８０塩基程度のものがよい
、但し、この様な場合においてもハイブリッド中のミス
マツチが複数存在する場合。In the present invention, in order to obtain specificity of the nucleic acid probe to the nucleic acid sample, it is preferable to use a nucleic acid probe having a length of 10 bases or more; In such cases, if the number of mismatches in the hybrid due to mutations in the nucleic acid sample is small, changes in the stability of the hybrid due to the mismatches will be subtle and difficult to measure. Therefore, the number of nucleic acid probes used is 10 to 100.
A base, more preferably about 15 to 80 bases, is preferred; however, even in such a case, if there are multiple mismatches in the hybrid.
さらには核酸試料中の変異が複数個の塩基の欠失あるい
は挿入等によるときはこの限りではない。Furthermore, this does not apply when the mutation in the nucleic acid sample is due to the deletion or insertion of multiple bases.
核酸試料としては、動物細胞例えば白血球細胞。Nucleic acid samples include animal cells such as white blood cells.
腎細胞、肝細胞等また。細菌、ウィルス等の微生物、さ
らには植物細胞等から抽出した核酸を用いることが出来
る。核酸１０−プは、核酸試料中の変異の有無を調べた
い部分とハイブリダイズ可能な状態で固定化されていれ
ば良く、この様に固定するため、末端固定が好ましい、
担体としては。Kidney cells, liver cells, etc. Also. Nucleic acids extracted from microorganisms such as bacteria and viruses, and even plant cells can be used. The nucleic acid 10-p only needs to be immobilized in a state in which it can hybridize with the part of the nucleic acid sample in which the presence or absence of mutations is to be investigated.
As a carrier.
天然あるいは合成ポリマー等、水溶液に不溶性で通常の
生化学反応に適用される温度範囲で安定であるものであ
ればなんら制限はない、固定化の方法としては２例えば
セルロースを担体とした時は。There are no restrictions as long as it is insoluble in aqueous solutions and stable in the temperature range applicable to ordinary biochemical reactions, such as natural or synthetic polymers.For example, when cellulose is used as a carrier, there are two methods of immobilization.
核酸プローブの５′末端の燐酸基を介して隣酸エステル
の形で共有結合固定することが出来る。また核酸プロー
ブの末端に脂肪族アミノ基を有したような誘導体であれ
ば、アミノ基に対して反応性のある基を導入した担体に
対して１例えばアミド結合あるいはウレタン型結合等の
ような形で共有結合させることも出来る。It can be covalently immobilized in the form of a phosphoric acid ester via the 5'-end phosphoric acid group of the nucleic acid probe. In addition, if the nucleic acid probe is a derivative having an aliphatic amino group at the end, it may be attached to a carrier into which a group reactive with amino groups is introduced, such as an amide bond or a urethane bond. It is also possible to make a covalent bond.
本発明では、核酸試料と核酸プローブ間のハイブリッド
における塩基対組成に由来する安定性の違いを排除する
ために、全工程をテトラアルキルアンモニウム塩の存在
下で行なう、テトラアルキルアンモニウム塩はＡ−Ｔ
（あるいはＡ−Ｕ）結合及びＧ−Ｃ結合の結合強度を均
一にする。従ってハイブリッドの安定性は該ハイブリッ
ドの塩基対数にのみ依存する。このことはハイブリッド
の解離を引き起こす条件、即ちハイブリッドの安定性を
その塩基対数から容易に推測することを可能とするもの
であり１強いては核酸試料塩基配列の変異の有無を判定
するための対照を必要としないことを京味する。テトラ
アルキルアンモニウム塩は２．０〜３．５モル／リット
ル好ましくは２．４〜３．０モル／リットルの濃度が好
ましい、この濃度範囲以外では先に述べたような効果が
低下する。また、テトラアルキルアンモニウムのアルキ
ル鎖は、炭素数１〜２のものが好ましく炭素数３以上の
テトラアルキルアンモニウム塩では先に述べた様な効果
が低下する。核酸試料と核酸プローブをハイブリダイズ
させるには１通常類られた方法１例えば４０℃〜７０℃
の温度下で接触させ後に温度を徐々に低下させるなどし
て行なえばよい。In the present invention, all steps are performed in the presence of a tetraalkylammonium salt in order to eliminate differences in stability resulting from the base pair composition in the hybrid between a nucleic acid sample and a nucleic acid probe.
(or make the bond strength of A-U bond and G-C bond uniform). Therefore, the stability of a hybrid depends only on the number of base pairs of the hybrid. This makes it possible to easily estimate the conditions that cause hybrid dissociation, that is, the stability of the hybrid, from the number of base pairs. Enjoy what you don't need. The concentration of the tetraalkylammonium salt is preferably 2.0 to 3.5 mol/liter, preferably 2.4 to 3.0 mol/liter; outside this concentration range, the above-mentioned effects will be reduced. Further, the alkyl chain of the tetraalkylammonium preferably has 1 to 2 carbon atoms, and a tetraalkylammonium salt having 3 or more carbon atoms will reduce the above-mentioned effects. To hybridize a nucleic acid sample and a nucleic acid probe, 1. Common methods 1. For example, 40°C to 70°C
This may be carried out by bringing the two into contact at a temperature of about 100 ml, and then gradually lowering the temperature.
ハイブリッドを形成しなかった核酸試料は洗浄により除
去し、ハイブリッドを形成した核酸試料は引き続き該温
度を上昇させることにより再び解離させる。この時核酸
試料と核酸プローブ間にＡ−Ｔ （Ａ−Ｕ）、Ｇ−Ｃ結
合以外のミスマツチした部分が存在するハイブリッドで
は、完全に相補的に結合したハイブリッドに比べて安定
性が低いため比較的低い温度で核酸試料の解離が起こる
。完全相補的にハイブリダイズした核酸であっても９０
℃〜１００℃では解離しているので温度は核酸試料と核
酸プローブがハイブリッドを形成した時の温度を基準と
して０℃〜１００℃のＲ囲に上昇させればよい、解離し
てきた核酸試料は１例えば紫外域の吸光度、！！た操作
に先立って該核酸試料をラジオアイソトープあるいは蛍
光物質等の標識を施した場合には、それら標識を測定す
ることにより行なえばよい、また溶出してきた核酸試料
をポストラベルし測定してもよい０以上説明した様な操
作を迅速、簡便にかつ正確に行ない得る本発明の実施の
−ＷＸａｔとして核酸プローブを固定化した担体をカラ
ムに充填しテトラアルキルアンモニウム塩溶液を移相と
して用いたカラム形式を挙げることができる。この場合
には担体としてカラム充填の容易なビーズ状、あるいは
粒子状のものを用いることが好ましい０本形式において
は、テトラアルキルアンモニウム塩溶液の温度を随時上
昇させる事でハイブリッドの安定性に起因する核酸試料
の溶出を連続的に測定することが出来る。Non-hybridized nucleic acid samples are removed by washing, and hybridized nucleic acid samples are subsequently dissociated again by increasing the temperature. At this time, hybrids in which there are mismatched parts other than A-T (A-U) and G-C bonds between the nucleic acid sample and the nucleic acid probe have lower stability than hybrids that bind in a completely complementary manner. Dissociation of nucleic acid samples occurs at relatively low temperatures. Even for perfectly complementary hybridized nucleic acids, 90
Since dissociation occurs between ℃ and 100℃, the temperature should be raised to an R range of 0℃ to 100℃ based on the temperature at which the nucleic acid sample and the nucleic acid probe form a hybrid. For example, absorbance in the ultraviolet region! ! If the nucleic acid sample is labeled with a radioisotope or a fluorescent substance prior to the procedure, the measurement may be performed by measuring those labels, or the eluted nucleic acid sample may be post-labeled and measured. A column format in which a carrier on which a nucleic acid probe is immobilized as WXat is packed in a column and a tetraalkylammonium salt solution is used as a phase shifter, in which the above-described operations can be carried out quickly, simply, and accurately. can be mentioned. In this case, it is preferable to use a bead-like or particle-like carrier that can be easily packed into a column. Elution of nucleic acid samples can be measured continuously.
（発明の効果） テトラアルキルアンモニウム塩の存在により。(Effect of the invention) Due to the presence of tetraalkylammonium salts.
ハイブリッド中のＡ−Ｔ （Ａ−Ｕ）、Ｇ−Ｃ結合の結
合力を均一にすることが可能となる。従って。It becomes possible to make the bonding strength of AT (A-U) and G-C bonds in the hybrid uniform. Therefore.
本発明では、核酸試料と対応する核酸プローブとのハイ
ブリッド中のＧ−ＣＭ成を考にせず、該ハイブリッドの
長さ、つまり塩基対数のみを安定性の測定時に考慮すれ
ばよい、即ちある塩基対数の完全相補的なハイブリッド
が核酸プローブと核酸試料に解離する温度はテトラアル
キルアンモニウム塩溶液中では、塩基組成に左むされる
ことなく。In the present invention, it is only necessary to consider the length of the hybrid, that is, the number of base pairs, when measuring stability, without considering the G-CM composition in the hybrid between the nucleic acid sample and the corresponding nucleic acid probe. The temperature at which a fully complementary hybrid dissociates into a nucleic acid probe and a nucleic acid sample in a tetraalkylammonium salt solution is independent of base composition.
同一温度で生じる。即ち、核酸試料と核酸プローブにミ
スマツチの存在するハイブリッドからの核酸プローブが
解離する温度は完全相補的なハイブリッドから、核酸試
料が解離する温度に比べ低温である。この安定性の変化
、即ちハイブリッドからの核酸試料の解離温度の変化を
測定することで。Occurs at the same temperature. That is, the temperature at which a nucleic acid probe from a hybrid in which there is a mismatch between the nucleic acid sample and the nucleic acid probe dissociates is lower than the temperature at which the nucleic acid sample dissociates from a perfectly complementary hybrid. By measuring changes in this stability, i.e. changes in the dissociation temperature of the nucleic acid sample from the hybrid.
核酸試料中の変異を検出する事が出来る０本発明では、
上記の様に核酸試料中の変異の有無を測定する方法にお
いて、従来の制限酵素を用いた方法番こ比べ、より正確
に塩基配列の変異を把握する事が出来る。In the present invention, mutations in nucleic acid samples can be detected.
In the method for measuring the presence or absence of mutations in a nucleic acid sample as described above, mutations in base sequences can be determined more accurately than conventional methods using restriction enzymes.
また、更には核酸試料と核酸プローブ間のハイブリッド
中の塩基組成の影響を排除出来るため、検出のたびごと
にハイブリッドが完全相補的である場合の安定性を知る
ための対照を必要としない。Furthermore, since the influence of the base composition in the hybrid between the nucleic acid sample and the nucleic acid probe can be eliminated, there is no need for a control to determine the stability of the hybrid when it is completely complementary each time it is detected.
（実施例）
以下の実施例により本発明のさらに詳細な説明を行なう
が２本発明はこれら実施例に限定されるものではない。(Examples) The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.
（実施ｇ４１）
「核酸１０−ブ固定化ゲルの調製」
ＤＮＡ合成装置により下記に示した塩基配列の５１ 末
端にアミノ基を有するオリゴヌクレオチド誘導体を合成
した。これをカルボニルジイミダゾールで活性化したセ
ルロースと反応させることにより核酸プローブを固定化
したゲルを調製した。(Example g41) "Preparation of gel immobilized with 10-nucleic acid" An oligonucleotide derivative having an amino group at the 51-terminus of the base sequence shown below was synthesized using a DNA synthesizer. By reacting this with cellulose activated with carbonyldiimidazole, a gel with immobilized nucleic acid probes was prepared.
５’ ８８２− ＡＧＧ ＴＧＡ ＡＴＴ Ｔ
ＣＴＴＡＡ ＡＣＡ ＧＣＴ ３’
続いて、前述の核酸１０−プと完全相補的な塩基配列を
持つ２１Ｊｌ＃■、及び５′側から１１番目に１ミスマ
ツチを有する２１量体■の計２種類のオリゴヌクレオチ
ドを、 ＤＮＡ合成装置により合成した。塩基配列は
以下の通り。5' 882- AGG TGA ATT T
CTTAA ACA GCT 3' Next, a total of two types of oligonucleotides, 21Jl#■ having a base sequence completely complementary to the aforementioned nucleic acid 10-p, and 21-mer ■ having a 1 mismatch at the 11th position from the 5' side. was synthesized using a DNA synthesizer. The base sequence is as follows.
■ ３’ ＴＣＣＡＣＴ ＴＡＡ ＡＧＡ Ａ
ＴＴ ＴＧＴ ＣＧＡ ５’
■ ３’ ＴＣＣＡＣＴ ＴＡＡ ＡＣＡ Ａ
ＴＴ ＴＧＴ ＣＧＡ ５’
温度グラジェント槽内に核酸プローブを固定化したカラ
ムをセットし、 ３１４テトラメチルアンモニウム、Ｍ
ｔｍ溶液（１５ｓＨＮａＣｌ、１．５ｓＨＮａ−Ｃ１ｔ
ｒａｔｅ、ｐＨ７，０）で平衡化し、槽内の温度を５０
℃に保ちつつ、パルプより試料核酸を注入しカラム内で
ハイブリダイズさせた。■ 3' TCCACT TAA AGA A
TT TGT CGA 5' ■ 3' TCCACT TAA ACA A
TT TGT CGA 5' Set a column immobilized with a nucleic acid probe in a temperature gradient tank, and add 314 tetramethylammonium, M
tm solution (15sHNaCl, 1.5sHNa-Clt
Equilibrate at a pH of 7.0) and lower the temperature inside the tank to 50.
While maintaining the temperature at °C, a sample nucleic acid was injected from the pulp and hybridized within the column.
この間、温度を４０℃まで徐々に下げハイブリッド形成
を促進させ、３０分後にＦｌｏｗを開始しく１００μＩ
／ｍｉｎ、）ハイブリダイズしなかった試料を洗い出し
た。５０分に温度上昇を開始しカラム内でハイブリダイ
ズしている試料を溶出させ、溶出してきた核酸を紫外吸
収＜２６０ｎｍ）でモニターし検出した０図１は完全相
補２１量体■及び中央に１ミスマツチをもつ２１１体■
の混合試料を前述のシステムを用い分離した例である。During this time, the temperature was gradually lowered to 40°C to promote hybridization, and after 30 minutes, 100μI was applied to start the flow.
/min,) samples that did not hybridize were washed out. At 50 minutes, the temperature started to rise and the sample hybridized in the column was eluted, and the eluted nucleic acids were monitored and detected using ultraviolet absorption (<260 nm). 211 bodies with mismatches ■
This is an example of separating a mixed sample using the above-mentioned system.
それぞれの溶出時間は６７分及び８２分でであった。結
果を図１に示す。The respective elution times were 67 minutes and 82 minutes. The results are shown in Figure 1.
図１は実施例１の測定結果を示すものである。
ｉ！１軸は吸光度及び温度、横軸は時間である。
図中のＡＮＮＥＡＬＩＮＧは核！！５２試料をカラム内
でハイブリダイズさせている時間で、この間は移動相の
流れはない、 ＦＬＯＷ Ｓ丁ＡＲＴで移動相を流し始
め。
５ＥＰＡＲＡＴＩＮＧで核酸試料の分離を開始する。ｉ
＆初の振り切れているピークはハイブリッドを形成しな
かった過剰の核酸試料で２番目のピークはｌミスマツチ
を有する核酸試料、３番目のピークが完全相補的な核酸
試料の溶出をそれぞれ現している。FIG. 1 shows the measurement results of Example 1. i! The first axis is absorbance and temperature, and the horizontal axis is time. ANNEALING in the diagram is nuclear! ! 52 This is the time during which the sample is hybridized in the column. During this time, there is no flow of mobile phase. Start flowing the mobile phase with FLOW START. Start separating the nucleic acid sample with 5EPARATING. i
& The first off-center peak represents the excess nucleic acid sample that did not form a hybrid, the second peak represents the elution of the nucleic acid sample with a mismatch, and the third peak represents the elution of the completely complementary nucleic acid sample.
Claims (2)
異を検出する方法において、 ［１］担体に固定化した核酸試料に対応した核酸プロー
ブと核酸試料をテトラアルキルアンモニウム塩溶液存在
下でハイブリダイズさせ ［２］ハイブリダイズしなかった核酸試料を除去した後 ［３］ハイブリッド形成時の温度から０℃〜１００℃の
範囲に上昇させて核酸試料を解離させ、［４］解離した
核酸試料を測定する ことを特徴とする核酸塩基配列における突然変異の測定
法(1) In a method for detecting mutations in a nucleic acid base sequence using a nucleic acid probe, [1] A nucleic acid probe corresponding to a nucleic acid sample immobilized on a carrier and a nucleic acid sample are hybridized in the presence of a tetraalkylammonium salt solution. [2] After removing the unhybridized nucleic acid sample, [3] dissociating the nucleic acid sample by raising the temperature to a range of 0°C to 100°C from the temperature at the time of hybridization, and [4] measuring the dissociated nucleic acid sample. A method for measuring mutations in a nucleic acid base sequence, characterized by
、テトラアルキルアンモニウム塩溶液を移相として用い
ることを特徴とする特許請求の範囲第（１）項記載の方
法(2) The method according to claim (1), characterized in that a carrier on which a nucleic acid probe is immobilized is packed into a column, and a tetraalkylammonium salt solution is used as a phase shifter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62303316A JPH01145000A (en) | 1987-12-02 | 1987-12-02 | Determination of mutation on nucleic acid base sequence |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62303316A JPH01145000A (en) | 1987-12-02 | 1987-12-02 | Determination of mutation on nucleic acid base sequence |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01145000A true JPH01145000A (en) | 1989-06-07 |
| JPH0563160B2 JPH0563160B2 (en) | 1993-09-09 |
Family
ID=17919496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62303316A Granted JPH01145000A (en) | 1987-12-02 | 1987-12-02 | Determination of mutation on nucleic acid base sequence |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01145000A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2750504A1 (en) * | 1996-06-27 | 1998-01-02 | Appligene Oncor | Analysis of nucleic acids captured on immobilised oligo:nucleotide(s) |
| JPH10239300A (en) * | 1997-02-28 | 1998-09-11 | Kagaku Gijutsu Shinko Jigyodan | Full automation gene analysis system |
| EP1072960B1 (en) * | 1999-07-09 | 2005-10-05 | Seiko Epson Corporation | Process cartridge of an image forming apparatus, comprising a charging roller and a cleaning unit movable into contact with the charger by a motor driver |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01108999A (en) * | 1987-10-23 | 1989-04-26 | Tosoh Corp | Method for detecting mutation in the nucleic acid base sequence |
-
1987
- 1987-12-02 JP JP62303316A patent/JPH01145000A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01108999A (en) * | 1987-10-23 | 1989-04-26 | Tosoh Corp | Method for detecting mutation in the nucleic acid base sequence |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2750504A1 (en) * | 1996-06-27 | 1998-01-02 | Appligene Oncor | Analysis of nucleic acids captured on immobilised oligo:nucleotide(s) |
| JPH10239300A (en) * | 1997-02-28 | 1998-09-11 | Kagaku Gijutsu Shinko Jigyodan | Full automation gene analysis system |
| EP1072960B1 (en) * | 1999-07-09 | 2005-10-05 | Seiko Epson Corporation | Process cartridge of an image forming apparatus, comprising a charging roller and a cleaning unit movable into contact with the charger by a motor driver |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0563160B2 (en) | 1993-09-09 |
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