JP2968282B2 - Method and kit for preparing single-stranded DNA - Google Patents
Method and kit for preparing single-stranded DNAInfo
- Publication number
- JP2968282B2 JP2968282B2 JP17552189A JP17552189A JP2968282B2 JP 2968282 B2 JP2968282 B2 JP 2968282B2 JP 17552189 A JP17552189 A JP 17552189A JP 17552189 A JP17552189 A JP 17552189A JP 2968282 B2 JP2968282 B2 JP 2968282B2
- Authority
- JP
- Japan
- Prior art keywords
- dna
- exonuclease
- stranded dna
- kit
- double
- 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 - Fee Related
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は遺伝子工学に利用される１本鎖DNAの調製方
法及びキットに関する。The present invention relates to a method and a kit for preparing single-stranded DNA used for genetic engineering.
従来２本鎖のDNAを１本鎖にする方法は、例えば２本
鎖DNAの溶液を90℃程度に加熱した後氷上で急冷する方
法や、0.2N NaOH溶液中にて60℃３〜５分間処理後、中
和してエタノール沈殿を行い真空乾燥を行う方法等があ
る。Conventional methods for converting double-stranded DNA into single-stranded DNA include, for example, a method in which a double-stranded DNA solution is heated to about 90 ° C. and then rapidly cooled on ice, or a method in which the solution is heated to 60 ° C. for 3 to 5 minutes in a 0.2N NaOH solution. After the treatment, there is a method of neutralization, ethanol precipitation and vacuum drying.
更に、２本鎖DNAを認識し、１本鎖になるようにDNAを
分解する５′→３′エキソヌクレアーゼ、３′→５′エ
キソヌクレアーゼを用いる方法もある。Furthermore, there is a method using a 5 ′ → 3 ′ exonuclease which recognizes double-stranded DNA and degrades the DNA so as to become single-stranded, and 3 ′ → 5 ′ exonuclease.
しかしながら、前者の方法は単にDNA鎖間の水素結合
を弱めるにすぎず、処理前のDNA鎖は２本とも存在して
いるため、操作方法によっては部分的に２本鎖に戻るこ
とがあり得る。このような部分的に２本鎖になったDNA
を例えばシークエンスに供すると、DNAポリメラーゼに
よるDNA鎖伸長反応が阻害を受け、良好なシークエンス
結果が得られないこともある。同様に、後者の酵素を用
いる方法は、そのDNAの両端からDNAを分解することが多
く、片ストランドだけの１本鎖を完全な形で残すことが
できないばかりでなく、酵素処理後の産物は不必要なも
う一方のストランドのDNAも残ってきてしまう。However, the former method merely weakens the hydrogen bonds between the DNA strands, and since both DNA strands exist before the treatment, there is a possibility that the DNA strands may partially return to double strands depending on the operation method. . Such partially double-stranded DNA
For example, when subjected to sequencing, the DNA chain elongation reaction by DNA polymerase is inhibited, and good sequencing results may not be obtained. Similarly, in the latter method using an enzyme, the DNA is often degraded from both ends of the DNA, so that not only a single strand of one strand cannot be completely left, but also the product after the enzyme treatment is Unnecessary DNA of the other strand also remains.
本発明の目的は、２本鎖DNAを１本鎖にする際に、他
方のDNA鎖がその後の操作に全く影響を与えないよう
に、不要な方のDNA鎖を完全に分解してしまう方法、及
びそれに用いるキットを提供することにある。An object of the present invention is to provide a method for completely decomposing unnecessary DNA strands when converting double-stranded DNA into single-stranded DNA so that the other DNA strand has no effect on subsequent operations. , And a kit for use therein.
本発明を概説すれば、本発明の第１の発明は１本鎖DN
Aを調製する方法に関する発明であり、２本鎖DNAの５′
末端の一方だけをエキソヌクレアーゼによる分解を受け
る形状にし、他方の５′末端又はリン酸結合部位をエキ
ソヌクレアーゼによる分解を受けない形状にした後、そ
のDNAに５′末端に作用するエキソヌクレアーゼを作用
させ、一方のDNA鎖だけを分解することを特徴とする。In summary, the first invention of the present invention is a single-stranded DN
The invention relates to a method for preparing A, and comprises 5 ′
After making only one of the ends into a shape that is subjected to degradation by exonuclease and the other 5 ′ end or a phosphate binding site into a shape that is not subjected to degradation by exonuclease, the DNA is exposed to an exonuclease that acts on the 5 ′ end. And decompose only one of the DNA strands.
本発明の第２の発明は、上記第１の発明の方法を用い
て１本鎖DNAを調製するためのキットに関する発明であ
って、少なくとも下記のＡ及びＢの試薬： A:5′末端に作用するエキソヌクレアーゼ B:Aのエキソヌクレアーゼに対応する濃縮バッファー を含むことを特徴とする。The second invention of the present invention relates to a kit for preparing a single-stranded DNA using the method of the first invention, and comprises at least the following reagents A and B: A: It is characterized by containing a concentration buffer corresponding to the exonuclease of exonuclease B: A that acts.
上記エキソヌクレアーゼの例としては、λ−エキソヌ
クレアーゼ、T3−エキソヌクレアーゼ、T7−エキソヌク
レアーゼ等が挙げられる。Examples of the exonuclease, lambda-exonuclease, T 3 - exonuclease, T 7 - exonuclease, and the like.
本発明者らは、λ−エキソヌクレアーゼが２本鎖DNA
の両５′末端からDNAを分解することに着目し、一方の
５′末端のみをリン酸がついた形状にし、他方の５′末
端を様々な形態に変化させたDNAをλ−エキソヌクレア
ーゼの基質として反応を行ったところ、反応生成物が通
常の約1/2倍の分子量を持つことを見出した。The present inventors have found that λ-exonuclease is a double-stranded DNA
Noting that the DNA is degraded from both 5 'ends, only one 5' end is formed into a form with phosphoric acid, and the other 5 'end is changed into various forms, and the DNA is converted into λ-exonuclease. When the reaction was carried out as a substrate, it was found that the reaction product had a molecular weight about 1/2 times that of a normal product.
５′末端の形態の変化の例としては、例えば５′末端
にリン酸がついていないもの、DNA鎖のリン酸結合の部
分にＳ原子が結合したもの、DNAの塩基に色素分子を結
合したもの、及び５′末端にDMTが結合したもの等があ
り、これらの形態変化を起こさせた５′末端はλ−エキ
ソヌクレアーゼによる分解を受けないことを確認した。Examples of changes in the form at the 5 'end include those having no phosphate at the 5' end, those having an S atom bonded to a phosphate bond of a DNA chain, those having a dye molecule bonded to a base of DNA. And 5'-terminal to which DMT was bound, and it was confirmed that the 5'-terminal having undergone these morphological changes was not degraded by λ-exonuclease.
また、λ−エキソヌクレアーゼと同じ反応機構を持つ
T3−エキソヌクレアーゼやT7−エキソヌクレアーゼを用
いて同様の実験を行ったところ、λ−エキソヌクレアー
ゼを用いた時と同様に、形態変化を起こさせた側の５′
末端からの分解は起こらなかった。Also has the same reaction mechanism as λ-exonuclease
When a similar experiment was performed using T 3 -exonuclease or T 7 -exonuclease, the 5 ′ on the side that caused the morphological change was similar to the case using λ-exonuclease.
No degradation from the ends occurred.
これら５′末端が変化したDNAを調製する際には、あ
らかじめプライマーの５′末端を変化させておいてから
テンプレートDNAにアニーリングさせ、DNAポリメラーゼ
で２本鎖にする方法が一般的である。In preparing these DNAs in which the 5 'end has been changed, it is common to change the 5' end of the primer in advance, then anneal to the template DNA, and make it double-stranded with a DNA polymerase.
最近、DNA増幅システムが開発され、DNAの増幅が多用
されている。このシステムでは１種類のテンプレートDN
Aに２種類のプライマーを混合するが、この一方のプラ
イマーのみを前述のごとく５′末端にリン酸がついたも
のを用い、他方の５′末端をλ−エキソヌクレアーゼに
よる分解を受けない形態に変化させておくとよい。Recently, a DNA amplification system has been developed, and DNA amplification is frequently used. In this system, one type of template DN
A is mixed with two kinds of primers. Only one of these primers has a phosphoric acid at the 5 'end as described above, and the other 5' end is in a form not subject to degradation by λ-exonuclease. It is good to change it.
以上のように、あらかじめDNA鎖を修飾しておくこと
により、エキソヌクレアーゼを作用させることによって
容易に１本鎖DNAを調製することが可能である。As described above, by modifying the DNA strand in advance, it is possible to easily prepare a single-stranded DNA by allowing exonuclease to act.
本発明方法に用いるエキソヌクレアーゼ、及びそのエ
キソヌクレアーゼ用の濃縮バッファーをそろえてキット
としておくことで本発明方法における１本鎖DNAの調製
を簡便に行うことができる。By preparing an exonuclease to be used in the method of the present invention and a concentrated buffer for the exonuclease to prepare a kit, single-stranded DNA can be easily prepared in the method of the present invention.
なお、本発明のキットには、上記の試薬以外に、クロ
ーニング用ベクター、及びそのクローニングサイトを挟
む２種類のプライマー、更にキナーゼ、及びそれ用のバ
ッファー等を加えてもよい。The kit of the present invention may contain, in addition to the above reagents, a cloning vector, two types of primers sandwiching the cloning site, a kinase, a buffer for the same, and the like.
以下、本発明を実施例により更に詳細に説明するが、
本発明はこれら実施例に限定されない。Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to these examples.
実施例１ ２本鎖DNAの５′末端から分解して１本鎖DNAを調製す
るキット（表１）を作製した。Example 1 A kit (Table 1) for preparing a single-stranded DNA by degrading from the 5 'end of a double-stranded DNA was prepared.
実施例２ （１） ５′末端を修飾した２本鎖DNAから１本鎖DNAの
調製 331塩基が入ったM13ssDNAをテンプレートとし、M13プ
ライマー（Primer）RV（宝酒造社製）の５′末端にアル
カリホスファターゼとATPを用いてリン酸を結合させた
ものと下記５種のプライマーの組合せで、ジーン アン
プ キット（gene Amp kit）（パーキン エルマー シ
ータス社製）DNA増幅システムを用いてDNAの増幅を行っ
た。 Example 2 (1) Preparation of single-stranded DNA from double-stranded DNA modified at 5 'end M13ssDNA containing 331 bases was used as a template, and alkali was added to the 5' end of M13 primer (Primer) RV (Takara Shuzo). DNA was amplified using a gene amp kit (Perkin-Elmer Cetus) DNA amplification system using a combination of phosphoric acid bound using phosphatase and ATP and the following five primers. .
プライマー 1.M13プライマーM4（宝酒造社製）５′末端のリン酸が
ない。Primer 1. M13 primer M4 (Takara Shuzo) No phosphate at 5 'end.
2.M13タムラ（Tamra）プライマー（アプライド バイオ
システムズ社製）５′末端の塩基に蛍光色素が結合した
もの。2. M13 Tamra primer (manufactured by Applied Biosystems) with a fluorescent dye bound to the 5'-terminal base.
3.M13プライマー（有機合成社製）５′末端の一つ内側
のリン酸結合部分に蛍光色素が結合したもの。3. M13 primer (manufactured by Organic Synthesis Co., Ltd.) with a fluorescent dye bound to the phosphate binding portion inside one of the 5 'ends.
4.M13プライマーM4のリン酸結合部分にＳ原子が結合し
たもの。（倉敷紡績社製、注文生産） 5.M13プライマーM4の５′末端にDMT基が結合したもの。4. M13 primer with S atom bonded to the phosphate binding portion of M4. (Made to order by Kurashiki Spinning Co., Ltd.) 5. M13 primer M4 with DMT group bound to the 5 'end.
増幅したDNA溶液をフェノール処理し、DNAをエタノー
ル沈殿後乾燥させ、各DNAサンプルに表１のキット１回
分の試薬及び15μの水を添加混合し、37℃で２時間反
応させた。The amplified DNA solution was treated with phenol, the DNA was precipitated with ethanol, and dried. Each DNA sample was mixed with the reagent of one kit shown in Table 1 and 15 µ of water, and reacted at 37 ° C for 2 hours.
上記実験の結果、増幅直後のDNAの分子量は、５種の
組合せですべて同じであったが、λ−エキソヌクレアー
ゼ処理後のDNAの分子量は、増幅直後の分子量の約1/2を
示した。As a result of the above experiment, the molecular weight of the DNA immediately after amplification was the same in all five combinations, but the molecular weight of the DNA after treatment with λ-exonuclease was about 1/2 of the molecular weight immediately after amplification.
またλ−エキソヌクレアーゼ処理後の５種のサンプル
をフェノール処理し、DNAをエタノール沈殿後乾燥させ
た。Five samples after the λ-exonuclease treatment were treated with phenol, and the DNA was precipitated with ethanol and dried.
このDNAを、アルカリ変性又は熱変性することなく、M
13プライマーRV（宝酒造社製）をプライマーにしてアニ
ーリングし、ジデオキシシークエンスを行ったところ、
１〜５のプライマーを用いたものはすべて同じシークエ
ンス結果が得られた。The DNA is treated with M without denaturation with alkali or heat.
13 Annealing using primer RV (Takara Shuzo) as a primer and dideoxy sequencing,
All of the primers using primers 1 to 5 gave the same sequence results.
つまり、あらかじめ２本鎖DNAの一方の５′末端をλ
−エキソヌクレアーゼ耐性になるようにし、他方の末端
をこれら酵素によって分解を受けるような形にしておく
ことによって、単なる酵素反応により２本鎖DNAを１本
鎖DNAに変えることができた。That is, one 5 'end of the double-stranded DNA is
By making it exonuclease resistant and leaving the other end degradable by these enzymes, double-stranded DNA could be converted to single-stranded DNA by a simple enzymatic reaction.
本発明により２本鎖DNAから完全な１本鎖DNAの調製が
可能となり、従来、避けられなかった不必要な方の相補
鎖による影響を受けない系を確立することができた。According to the present invention, complete single-stranded DNA can be prepared from double-stranded DNA, and a system not affected by the unnecessary complementary strand, which has been inevitable in the past, could be established.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 加藤 郁之進 滋賀県大津市瀬田３丁目４番１号 寳酒 造株式会社中央研究所内 (56)参考文献 米国特許4521509（ＵＳ，Ａ) (58)調査した分野(Int.Cl.6，ＤＢ名) C12N 15/00 - 15/90 C12Q 1/00 - 1/70 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＷＰＩ（ＤＩＡＬＯＧ)────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu-shi, Shiga Takara Shuzo Co., Ltd. Central Research Laboratory (56) Reference US Patent 4521509 (US, A) (58) 6) Surveyed field (Int. Cl. 6 , DB name) C12N 15/00-15/90 C12Q 1/00-1/70 BIOSIS (DIALOG) WPI (DIALOG)
Claims (2)
ヌクレアーゼによる分解を受ける形状にし、他方の５′
末端又はリン酸結合部位をエキソヌクレアーゼによる分
解を受けない形状にした後、そのDNAに、５′末端に作
用するエキソヌクレアーゼを作用させ、一方のDNA鎖だ
けを分解することを特徴とする１本鎖DNAの調製方法。(1) Only one of the 5 'ends of a double-stranded DNA is formed into a shape which is subjected to degradation by exonuclease, and the other 5' end is formed.
After forming the terminal or phosphate binding site into a shape that is not susceptible to degradation by exonuclease, the DNA is subjected to an exonuclease acting on the 5 'end to degrade only one of the DNA strands. Method for preparing strand DNA.
調製するためのキットであって、少なくとも下記のＡ及
びＢの試薬： A:5′末端に作用するエキソヌクレアーゼ B:Aのエキソヌクレアーゼに対応する濃縮バッファー を含むことを特徴とする１本鎖DNA調製用キット。2. A kit for preparing single-stranded DNA using the method according to claim 1, wherein at least the following reagents A and B: A: Exonuclease B: A acting on the 5 'end A kit for preparing a single-stranded DNA, comprising a concentration buffer corresponding to the exonuclease of (1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17552189A JP2968282B2 (en) | 1989-07-10 | 1989-07-10 | Method and kit for preparing single-stranded DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17552189A JP2968282B2 (en) | 1989-07-10 | 1989-07-10 | Method and kit for preparing single-stranded DNA |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0343084A JPH0343084A (en) | 1991-02-25 |
| JP2968282B2 true JP2968282B2 (en) | 1999-10-25 |
Family
ID=15997515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17552189A Expired - Fee Related JP2968282B2 (en) | 1989-07-10 | 1989-07-10 | Method and kit for preparing single-stranded DNA |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2968282B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6270901B2 (en) | 2016-04-21 | 2018-01-31 | 三菱電機株式会社 | FMCW radar equipment |
-
1989
- 1989-07-10 JP JP17552189A patent/JP2968282B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0343084A (en) | 1991-02-25 |
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