JP3079554B2 - Synthetic DNA purification method - Google Patents
Synthetic DNA purification methodInfo
- Publication number
- JP3079554B2 JP3079554B2 JP02274363A JP27436390A JP3079554B2 JP 3079554 B2 JP3079554 B2 JP 3079554B2 JP 02274363 A JP02274363 A JP 02274363A JP 27436390 A JP27436390 A JP 27436390A JP 3079554 B2 JP3079554 B2 JP 3079554B2
- Authority
- JP
- Japan
- Prior art keywords
- dna
- synthetic dna
- gel filtration
- purification method
- present
- 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.)
- Expired - Lifetime
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は合成DNAの簡易な精製法に関する。The present invention relates to a simple method for purifying synthetic DNA.
DNAの合成は、従来より機械化され自動合成されてい
るが、オキシム処理により担体よりDNAを切り出し、さ
らにアンモニア処理により保護基を脱離した後は、担
体の濾別、濾液の減圧濃縮、エーテル抽出（３
回）、水相の減圧濃縮、アセトニトリル150mM TEAB
０％、10％、25％、50％４種の溶液の準備、SEPPAK
処理０％1ml×５本、25％1ml×７本、25％1ml×５本を
分取して紫外吸収スペクトルの測定、HPLCでトリチル
体DNAのみ分取、減圧濃縮、酢酸処理、エーテル
抽出（３回）、減圧濃縮、という計11の工程により粗
精製を行ない、その後HPLCによる精製、分取がなされて
いる。Conventionally, DNA synthesis has been mechanized and automated.However, after DNA is cut out from the carrier by oxime treatment and the protecting group is removed by ammonia treatment, the carrier is separated by filtration, the filtrate is concentrated under reduced pressure, and ether is extracted. (3
Times), vacuum concentration of the aqueous phase, acetonitrile 150 mM TEAB
Preparation of 4 kinds of 0%, 10%, 25% and 50% solutions, SEPPAK
5% of the treated 0% 1ml x 5, 25% 1ml x 7 and 25% 1ml x 5 were measured for ultraviolet absorption spectrum, only the trityl DNA was separated by HPLC, concentrated under reduced pressure, acetic acid treatment, ether extraction ( The crude purification is performed by a total of 11 steps of 3) and concentration under reduced pressure, and then purification and fractionation by HPLC are performed.
しかしながら、このような複雑な工程で処理を行ない
粗精製を行なうと、手間（時間）がかかると同時に操作
段階が多いので、その都度サンプルのロスや他の物質と
のコンタミネーションの確率が高くなる。そのため、当
業界においては解決すべき問題点として指摘されている
が、未だ工程数が少なくて簡易かつ容易な合成DNAの精
製法は知られていないのが実情である。However, when the crude purification is performed by performing the treatment in such a complicated process, it takes time (time) and many operation steps, so that the probability of loss of the sample and contamination with other substances increases each time. . Therefore, it has been pointed out as a problem to be solved in the art, but a simple and easy method for purifying synthetic DNA with a small number of steps has not yet been known.
従って、本発明の目的は合成DNAの簡易な精製法を提
供することにある。Accordingly, an object of the present invention is to provide a simple method for purifying synthetic DNA.
〔課題を解決するための手段〕 本発明者は、前記課題を解決するために鋭意検討した
結果、従来からの精製処理工程（前記の11工程）に替え
てディスポーザブルのゲル濾過型カラムを用いることに
より、１工程で粗精製が行なえることを見い出し、本発
明を完成するに到った。[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventor has found that a conventional disposable gel filtration column is used instead of the purification process (the above 11 processes). As a result, it was found that the crude purification could be performed in one step, and the present invention was completed.
即ち、本発明の要旨は、合成DNAを担体より切り出し
かつ保護基を脱離した後、ゲル濾過法を用いることを特
徴とする合成DNAの精製法に関する。That is, the gist of the present invention relates to a method for purifying a synthetic DNA, which comprises using a gel filtration method after cutting out the synthetic DNA from a carrier and removing a protecting group.
本発明の方法において、担体からの合成DNAの切り出
しは、通常合成機により自動的にあるいはオキシム溶液
を用いて常法により行なうことができる。また、保護基
の脱離は、オキシム処理後に減圧濃縮し、濃アンモニウ
ム水を用いて反応させる公知の方法により行なうことが
できる。In the method of the present invention, the cutting of synthetic DNA from the carrier can be usually performed automatically by a synthesizer or by a conventional method using an oxime solution. The elimination of the protecting group can be carried out by a known method in which oxime treatment is followed by concentration under reduced pressure and reaction using concentrated ammonium water.
本発明の方法における精製は、このように公知の方法
により処理されて担体より切り出され、かつ脱保護され
た合成DNAをゲル濾過にアプライすることにある。即
ち、DNAの合成後、次の実験に使えるように保護基をは
ずすが、この操作に使用する試薬および脱離した保護基
を目的物であるDNAと分離しなければならない。この分
離操作として、ゲル濾過法を用いると、分子の大きさに
よって分けることが出来、前記の試薬や保護基等の不純
物は小さいのでゲルにトラップされるが、目的物である
DNAは大きいのでゲルを素通りしてフラクションに回収
される。従って、溶出されたフラクション中には高濃度
にDNAが回収される。The purification in the method of the present invention consists in applying the synthetic DNA which has been treated by a known method, cut out from the carrier and deprotected, to gel filtration. That is, after the synthesis of DNA, the protecting group is removed so that it can be used in the next experiment, but the reagent used for this operation and the removed protecting group must be separated from the target DNA. If gel filtration is used as the separation operation, the separation can be performed according to the size of the molecule, and the impurities such as the reagents and the protecting groups are trapped in the gel because the impurities are small.
Because the DNA is large, it passes through the gel and is collected in fractions. Therefore, a high concentration of DNA is recovered in the eluted fraction.
本発明におけるゲル濾過処理は、通常のゲル濾過処理
と変わるところはなく公知の方法が適用できるが、操作
を簡素化するにはディスポーザブルカラムを使用するこ
とができ、溶出条件は減菌水で数回に分けて分取する。The gel filtration treatment in the present invention is not different from ordinary gel filtration treatment, and a known method can be applied.However, to simplify the operation, a disposable column can be used, and the elution conditions are several times with sterile water. Dispense in batches.
ディスポーザブルカラムを用いた場合の処理は通常、
次のようになされる。Processing using a disposable column is usually
This is done as follows.
まず、DNAの自動合成機により合成されたDNAの溶液を
減菌水に溶解し、NAPカラム、PDカラム等のゲル濾過用
カラムに入れ、DNA溶液をカラムに浸透させた後、減菌
水を加えては溶出し、濾液をフラクションとして分取す
る。フラクションは、通常５つ程度とし、溶出後各フラ
クションについて300〜220nmでの吸収スペクトルを測定
してDNAの分取を確認する。First, a solution of DNA synthesized by an automatic DNA synthesizer is dissolved in sterilized water, put into a gel filtration column such as a NAP column or a PD column, and the DNA solution is allowed to permeate the column. In addition, it elutes and the filtrate is collected as a fraction. The number of fractions is usually about five, and after elution, the fractionation of DNA is confirmed by measuring the absorption spectrum of each fraction at 300 to 220 nm.
DNAは、通常フラクションの２本目から４本目までの
間に溶出し粗精製されるが、DNAの存在が確認されたフ
ラクションについて、HPLCによりさらに精製することが
できる。DNA is generally eluted and roughly purified between the second and fourth fractions, but the fraction in which the presence of DNA has been confirmed can be further purified by HPLC.
本発明の精製方法が適用される合成DNAの大きさは、
特に制限されるものではない。The size of the synthetic DNA to which the purification method of the present invention is applied,
There is no particular limitation.
合成されたDNAの糖鎖５′端の保護機であるジメトキ
シトリチル基（DMTr基）は、合成機によっては自動的に
酸処理によりはずし、OH基とするが、本発明のゲル濾過
カラムはDNAのDMTr体、OH体には関係なく同様の結果が
得られる。従って、ゲル濾過カラムにアプライする際に
は、DMTr体、OH体のいずれでもよい。The dimethoxytrityl group (DMTr group), which is a protector at the 5 ′ end of the sugar chain of the synthesized DNA, is automatically removed by an acid treatment depending on the synthesizer, and is converted to an OH group. Similar results are obtained irrespective of the DMTr form and the OH form. Therefore, when applying to a gel filtration column, either a DMTr form or an OH form may be used.
以下、実施例により本発明に更に詳しく説明するが、
本発明はこれらの実施例により何ら限定されるものでは
ない。Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited by these examples.
実施例１ 11merのDNAを（株）島津製作所製のDNA自動合成装置
（GSS−１）により合成した。合成されたDNAは、該合成
機により自動的に糖鎖５′端の保護基であるジメトキシ
トリチル基（DMTr基）が酸処理によりはずされ、OH基と
した試料が得られた。この試料についてオキシム処理を
50℃５時間行ない担体よりDNAを切り出し、さらに濃ア
ンモニア処理を50℃５時間行ない脱保護を行ない、次い
でそのまま、ゲル濾過型カラム（NAP−10:ファルマシア
社製）にアプライして粗精製を行なった。溶出は減菌水
0.4mlを加えて行ない、フラクションは５本に分けて溶
出し、紫外吸収スペクトル（300〜220nm）を測定し、DN
Aの溶出を確認した。第１図で示されるように、DNAはフ
ラクションの２本目から４本目までの間で溶出してい
た。Example 1 An 11-mer DNA was synthesized using an automatic DNA synthesizer (GSS-1) manufactured by Shimadzu Corporation. From the synthesized DNA, a dimethoxytrityl group (DMTr group), which is a protecting group at the 5 'end of the sugar chain, was automatically removed by an acid treatment by the synthesizer, and a sample was obtained in which the OH group was obtained. Oxime treatment of this sample
The DNA was cut out from the carrier at 50 ° C. for 5 hours, treated with concentrated ammonia for 5 hours at 50 ° C. for deprotection, and then applied as it was to a gel filtration column (NAP-10: Pharmacia) for crude purification. Was. Elution is sterile water
0.4 ml was added, fractions were eluted in 5 parts, and the ultraviolet absorption spectrum (300-220 nm) was measured.
Elution of A was confirmed. As shown in FIG. 1, DNA was eluted between the second and fourth fractions.
本発明の精製方法により、合成DNAの精製に関する操
作が１工程になり、手間、時間が省け、簡素化できる。
さらに、ゲル濾過カラムには充填剤をパックするため、
フィルターがカラムの上下についているが、このことに
より合成DNAと担体の分離を同時にすることができる
し、HPLCにかける前にプレフィルトレーションする手間
も省けるので便利である。According to the purification method of the present invention, the operation relating to the purification of the synthetic DNA is performed in one step, so that labor, time and labor can be saved and simplified.
Furthermore, to pack the packing material in the gel filtration column,
Filters are provided above and below the column, which makes it possible to simultaneously separate the synthetic DNA and the carrier, and is convenient because the need for prefiltration before HPLC is eliminated.
第１図は、NAP−10処理後の合成DNA（11mer）の紫外吸
収スペクトルを示した図である。FIG. 1 is a view showing an ultraviolet absorption spectrum of a synthetic DNA (11mer) after NAP-10 treatment.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭58−42969（ＪＰ，Ａ) 特開 昭61−72797（ＪＰ，Ａ) 特開 昭61−52288（ＪＰ，Ａ) 有機合成化学、第42巻、第５号、 （1984）、第429−436頁 Ｊｏｕｒｎａｌ ｏｆ Ｃｈｒｏｍａ ｔｏｇｒａｐｈｙ，Ｖｏｌ．320（1985) ｐ．440−444 (58)調査した分野(Int.Cl.7，ＤＢ名) C07H 21/00 - 21/04 C07H 1/06 C12N 15/10 ＣＡＰＬＵＳ（ＳＴＮ)──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-58-42969 (JP, A) JP-A-61-72797 (JP, A) JP-A-61-52288 (JP, A) Synthetic Organic Chemistry Vol. 42, No. 5, (1984), pp. 429-436, Journal of Chromatography, Vol. 320 (1985) p. 440-444 (58) Field surveyed (Int. Cl. 7 , DB name) C07H 21/00-21/04 C07H 1/06 C12N 15/10 CAPLUS (STN)
Claims (1)
脱離した後、ディスポーザブルのゲル濾過型カラムによ
り精製することを特徴とする合成DNAの精製法。1. A method for purifying a synthetic DNA, comprising cutting out the synthetic DNA from a carrier, removing a protecting group, and purifying the synthetic DNA with a disposable gel filtration column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP02274363A JP3079554B2 (en) | 1990-10-12 | 1990-10-12 | Synthetic DNA purification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP02274363A JP3079554B2 (en) | 1990-10-12 | 1990-10-12 | Synthetic DNA purification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04149188A JPH04149188A (en) | 1992-05-22 |
| JP3079554B2 true JP3079554B2 (en) | 2000-08-21 |
Family
ID=17540619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP02274363A Expired - Lifetime JP3079554B2 (en) | 1990-10-12 | 1990-10-12 | Synthetic DNA purification method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3079554B2 (en) |
-
1990
- 1990-10-12 JP JP02274363A patent/JP3079554B2/en not_active Expired - Lifetime
Non-Patent Citations (2)
| Title |
|---|
| Ｊｏｕｒｎａｌ ｏｆ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ，Ｖｏｌ．３２０（１９８５）ｐ．４４０−４４４ |
| 有機合成化学、第４２巻、第５号、（１９８４）、第４２９−４３６頁 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04149188A (en) | 1992-05-22 |
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