JP3679536B2 - Thermostable DNA synthase - Google Patents
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- JP3679536B2 JP3679536B2 JP01924897A JP1924897A JP3679536B2 JP 3679536 B2 JP3679536 B2 JP 3679536B2 JP 01924897 A JP01924897 A JP 01924897A JP 1924897 A JP1924897 A JP 1924897A JP 3679536 B2 JP3679536 B2 JP 3679536B2
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Description
【０００１】
【発明の属する技術分野】
この発明は、ＤＮＡ鎖の試験管内での合成や増幅、塩基配列の決定等に用いる新規な耐熱性ＤＮＡ合成酵素と、この酵素をコードするＤＮＡ配列、並びにこのＤＮＡ合成酵素の製造方法に関するものである。
【０００２】
【従来の技術とその課題】
ＤＮＡ合成酵素（ＤＮＡ polymerase）は１本鎖ＤＮＡに相補的なＤＮＡ鎖の合成を触媒する酵素の総称である。ＤＮＡの塩基配列決定や試験管内でのＤＮＡ増幅などには必須の酵素であるが、特にＰＣＲ(Polymerase chain reaction) においては、その一連の反応サイクルを自動化する上で「耐熱性ＤＮＡ合成酵素」は不可欠である。
【０００３】
このような耐熱性ＤＮＡ合成酵素としてはＴaq、Ｐfu、ＫOD等が知られており、それぞれの特性に応じて使い分けられている。しかしながら、これら既存の耐熱性ＤＮＡ合成酵素を用いたＰＣＲ等のＤＮＡ合成の場合には、鋳型となるＤＮＡ鎖によっては、合成されるＤＮＡ鎖の伸長が途中で停止してしまい、鋳型ＤＮＡ鎖の全領域の増幅や、その塩基配列の決定が困難もしくは不可能となるという問題を有していた。また、合成停止による不完全なＤＮＡ断片がＰＣＲ産物中に混入する場合には、目的とする増幅断片を精製しなけらばならないという不都合も存在した。
【０００４】
この発明は、以上のとおりの事情に鑑みてなされたものであって、ＤＮＡ鎖をＰＣＲ等により合成、増幅するに際して、合成鎖の伸長を途中で停止させることなく、鋳型ＤＮＡ鎖の全長を効率よく増幅することのできる新規な耐熱性ＤＮＡ合成酵素を提供することを目的としている。
またこの発明は、この耐熱性ＤＮＡ合成酵素をコードするＤＮＡ配列と、このＤＮＡ配列の発現産物として耐熱性ＤＮＡ合成酵素を製造する方法を提供することを目的としてもいる。
【０００５】
【課題を解決するための手段】
この発明は、上記の課題を解決するものとして、配列番号１のアミノ酸配列からなる耐熱性ＤＮＡ合成酵素（請求項１）を提供する。
またこの発明は、配列番号１のアミノ酸配列をコードするＤＮＡ（請求項２）と、このＤＮＡを含むクローニングベクター（請求項３）を提供する。このようなクローニングベクターとしては、大腸菌ＨＭＳ174（ＤＥ3）/pＤＰ320（FERM P-16052）が保有する組換え体プラスミドpＤＰ320（請求項４）をも提供する。
【０００６】
さらにまた、この発明は、上記請求項２のＤＮＡを含む発現ベクターにより形質転換した細胞を培養し、培地中に産生された目的酵素を単離・精製することを特徴とする耐熱性ＤＮＡ合成酵素の製造方法（請求項５）を提供する。
【０００７】
【発明の実施の形態】
この発明の耐熱性ＤＮＡ合成酵素は、具体的には、ピロコッカスフリオサス（Pyrococcus furiosus）由来のＰfuＤＮＡ合成酵素を公知の変異遺伝子作成法 (Strategies, vol 9, p3-4,1996) によって遺伝子工学的に改変した酵素である（以下、この発明の耐熱性ＤＮＡ合成酵素を「改変型ＰfuＤＮＡ合成酵素」と記載することがある）。この酵素の作成は以下のとおりに行なった。すなわち、ＰfuＤＮＡ合成酵素の遺伝子は塩基配列が公知であるため、その両端に相補的なオリゴヌクレオチドを合成し、これをプライマーとして、上記細菌のゲノムＤＮＡを鋳型とするＰＣＲ法によりＰfuＤＮＡ合成酵素の遺伝子を調製した。この遺伝子ＤＮＡ断片をベクターにクローニングし、上記文献に記載の方法により変異させた。遺伝子の変異は、ＰfuＤＮＡ合成酵素のアミノ酸配列の一部がＫODＤＮＡ合成酵素のアミノ酸配列に置き変わるように塩基を置換させた。ＰfuＤＮＡ合成酵素とＫODＤＮＡ合成酵素は、アミノ酸配列が約８０％相同であり、ＰＣＲの際に同様の合成停止を生じさせるが（図１）、ＫODＤＮＡ合成酵素の伸長速度はＰfuＤＮＡ合成酵素のそれの６〜10倍である。そこで、ＰfuＤＮＡ合成酵素のアミノ酸残基をＫODＤＮＡ合成酵素のアミノ酸残基に置換することによって、合成鎖の伸長停止が改善され、しかも伸長速度の速い酵素が得られる可能性があるからである。そして、このようにして変異させた遺伝子を大腸菌で発現させ、その発現産物を回収し、精製することによってこの発明の改変型ＰfuＤＮＡ合成酵素を得た。
【０００８】
実際には、この発明の発明者等は、上記の方法により数多くの改変型ＰfuＤＮＡ合成酵素を作成し、それぞれについてＤＮＡ合成実験をおこない、伸長したＤＮＡ鎖を電気泳動的に解析することによって、従来酵素に比べて合成鎖の伸長停止が著しく改善されたＤＮＡ合成酵素を得、この発明を完成させた。
このＤＮＡ合成酵素は、配列番号１に示したアミノ酸配列を有しており、このアミノ酸配列は、従来公知のＰfuＤＮＡ合成酵素のアミノ酸配列のうち、表１に示すアミノ酸残基が置換された新規な配列である。そして、この新規酵素を用いてＰＣＲ等のＤＮＡ合成を行なった場合には、下記の実施例に示すように、従来のＤＮＡ合成酵素を用いた場合に生じる合成停止がほぼ完全に解消される。もちろん従来酵素によって効率よく増幅されるＤＮＡ鎖は同様に効率良く増幅することができる。
【０００９】
【表１】
また、この改変型ＰfuＤＮＡ合成酵素をコードするＤＮＡ配列としては、上記の酵素作成過程で得られたＰfuＤＮＡ合成酵素遺伝子の変異遺伝子を例示することができる。この変異遺伝子は組換え体プラスミドpＤＰ320にクローニングされており、このpＤＰ320は大腸菌ＨＭＳ174(ＤＥ3)に導入され、工業技術院生命工学工業技術研究所に寄託されている (寄託番号FERM P- 16052)。ただし、この発明のＤＮＡ合成酵素はそのアミノ酸配列が配列番号１であることを必要かつ充分な要件とするものであり、そのような酵素をコードする遺伝子は、配列番号１の各アミノ酸残基に対応する塩基コドンをつなぎ合わせたＤＮＡ配列として適宜にデザインすることができる。
【００１１】
以下、実施例を示し、この発明のＤＮＡ合成酵素についてさらに詳細かつ具体的に説明するが、この発明は以下の例に限定されるものではない。
【００１２】
【実施例】
実施例１：改変型ＰfuＤＮＡ合成酵素遺伝子の作成
（１）ＰfuＤＮＡ合成酵素遺伝子のクローニング
ＰfuＤＮＡ合成酵素遺伝子の塩基配列（Nucleic Acids Research, vol.21, p259-265, 1993) に従ってＰＣＲプライマーを合成し、ピロコッカスフリオサス (P. furiosus)のゲノムＤＮＡを鋳型とするＰＣＲによって目的遺伝子を増幅し、これを大腸菌用の発現ベクターにクローニングした。詳細は以下のとおりである。
【００１３】
P.furiosus DSM3638を上記文献に記載された方法で培養した。先ず、文献記載の培地を調製し、高温加圧滅菌ののち、蜜素ガスを吹き込み、植菌して95℃で15時間静置培養した。200mlの培養掖から遠心分離により約0.5mgの菌体を得た。集菌体を緩衝液Ａ（10mMトリス−ＨＣＬ，pH8.0, 1mMＥＤＴＡ, 100mM Ｎacl ）に懸濁し、10% ＳＤＳを１ml加え、撹拌の後、プロテイナーゼＫを0.5mg 加えて55℃で60分反応させた。反応液を順次フェノ一ル抽出、フェノ一ル／クロロホルム抽出、クロロホルム抽出し、エタノールを加えてＤＮＡを不溶化し、回収した。得られたＤＮＡを１mlのＴＥバッファー（10mMトリス−ＨＣｌ，pH8.0，１mMＥＤＴＡ）に溶解し、0.5mgのＲＮase Ａを加えて37℃で60分反応させたのち、再度フェノール抽出、フェノ一ル／クロロホルム抽出、クロロホルム抽出し、エタノ一ル沈殿でＤＮＡを回収してＴＥバッファ−に溶解させ、約0.3mgのＤＮＡを得た。
【００１４】
次いで、目的のＤＮＡ合成酵素遺伝子をＰＣＲ増幅するために、既知の配列データをもとに配列番号２および３に示す２種のプライマ−ＤＮＡを合成した。すなわち、フォアードプライマー配列中には目的遺伝子の開始コドンＡＴＧおよび制限酵素ＮcoＩ配列(5'-CCATGG-3')を導入し、リバースプライマーは終止コドンの下流の適当な位置に結合するように設計した。ＰＣＲは、P.furiosusＤＮＡ２μgとプライマー各10pmolを用い、ＬＡＴaq（宝酒造）と添付のバッファー条件で、50μlの反応系で行った。サイクル条件は、酵素を加える前に93℃／３分を行い、94℃／0.5 分、55℃／0.5 分、72℃／1.0 分を３０サイクルした。増幅したＤＮＡ断片を精製し、ＮcoＩで処理した後、同じくＮcoＩで切断後に平滑末端化し、さらにＮcoＩ処理した発現ベクターｐＥＴ15−ｂのＴ７プロモーター下流に組み込んだ。この発現ベクターをｐＤＰＷＴ100とし、挿入遣伝子の塩基配列を確認した。
（２）ＰfuＤＮＡ合成酵素遺伝子の改変
クローン化したＰfuＤＮＡ合成酵素遺伝子を組み込んだ発現ベクターｐＤＰＷＴ100に対して、期待する変異を含んだオリゴヌクレオチド（配列番号４および５）とプロメガ社の突然変異導入キットを用い、公知の方法 (Strategies, vol 9, p3-4,1996) に従って改変型ＰfuＤＮＡ合成酵素の遺伝子を、発現ベクターｐＤＰＷＴ100上で作成し、発現ベクターｐＤＰ320 を構築した。なお、この改変型遺伝子の塩基配列を決定することにより、改変型ＰfuＤＮＡ合成酵素のアミノ酸配列（配列番号１）を確認した。
実施例２：改変型ＰｆｕＤＮＡ合成酵素の大腸菌での発現と精製
実施例１（２）で作成した改変型ＰｆｕＤＮＡ合成酵素の遺伝子を次のとおりに大腸菌で発現させ、精製した。
【００１５】
実施例１（２）で作成した改変型ＰｆｕＤＮＡ合成酵素遺伝子をもつ発現ベクターｐＤＰ320 を大腸菌ＨＭＳ173(ＤＥ３）株に導入し、終濃度0.1mMのＩＰＴＧを含んだＬＢ培地で14時間培養し、酵素を大腸菌体内に発現誘導した。遠心して菌体を集めた後、150mM Ｔris/ＨＣl(pH7.5)、2mM ＥＤＴＡ、0.24mM ＡＰＭＳＦおよび0.2%のＴween20を含む緩衝液で超音波処理を行いながら、改変型ＰｆｕＤＮＡ合成酵素を抽出した。この粗抽出液を80℃、15分の熱処理を行うことで大腸菌由来のＤＮＡ合成酵素を失活させると共に、この発明のＤＮＡ合成酵素の部分精製を行なった。部分精製画分は50mMＴris/ＨＣl(pH7.5)、１mMＥＤＴＡ、0.2%Ｔween20、7mM 2-mercaptoethanol および10% glycerolの緩衝液に対し透析した。この段階で改変型ＰfuＤＮＡ合成酵素に特異的なＤＮＡ合成活性を検出した。
実施例３：改変型ＰfuＤＮＡ合成酵素によるプライマー伸長反応
実施例２で部分精製した改変型ＰfuＤＮＡ合成酵素を用い、鋳型ＤＮＡに相補的なＤＮＡ鎖のプライマー伸長反応を試験した。
【００１６】
20mMＴris/ＨＣl(pH8.0)、２mM ＭgＣl2、50μg/mlＢＳＡ、0.1%Ｔriton Ｘ-100、１mMの各cold dNTPs(0.1mM for dCTP)、[α-32P] dCTPの10μCiとＭ13(-21)のプライマーをアニールさせた0.63μgのpBLUESCRIPT プラスミドを含む反応液20μlに、上記の部分精製酵素画分１μgを入れ、75℃で１分および３分間反応させた。伸長したＤＮＡ鎖を８M ureaを含んだポリアクリルアミドゲル電気泳動で分離した後、イメージアナライザ−によりそのパターンを解析した。また、対照として、従来の野性型ＰfuＤＮＡ合成酵素を用いて、同様のＤＮＡ合成を行なった。
【００１７】
結果は図２に示したとおりである。従来の野性型ＰfuＤＮＡ合成酵素を用いた場合には、合成停止による不完全はＤＮＡ鎖の存在を示すバンドが少なくとも１０個観察されたが、この発明の改変型ＰfuＤＮＡ合成酵素によるＤＮＡ合成では、これらのバンドは消失した。一方、1000ベース近傍の良く伸長したＤＮＡ鎖の蓄積には差は見られなかった。
【００１８】
【発明の効果】
以上詳しく説明したとおり、この発明によって、ＤＮＡ鎖をＰＣＲ等によって増幅するに際して、合成鎖の伸長を途中で停止させることなく、鋳型ＤＮＡ鎖の全長を効率よく合成、増幅することのできる新規な耐熱性合成酵素が提供される。これによって、ＤＮＡ鎖の試験管内での合成や増幅、塩基配列の決定等を簡便かつ高精度で行なうことが可能となる。
【００１９】
【配列表】
配列番号：１
配列の長さ：775
配列の型：アミノ酸
配列の種類：タンパク質
配列
配列の長さ：35
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列
配列の長さ：35
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列
配列の長さ：66
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列
配列の長さ：66
配列の型：核酸
鎖の数：１本鎖
トポロジー：直鎖状
配列の種類：他の核酸（合成ＤＮＡ）
配列
【図面の簡単な説明】
【図１】従来のＰfuＤＮＡ合成酵素とＫODＤＮＡ合成酵素のプライマー伸長活性を示す電気泳動の結果である。
【図２】従来のＰfuＤＮＡ合成酵素（野性型）とこの発明の改変型ＰfuＤＮＡ合成酵素のプライマー伸長活性を示す電気泳動の結果である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel thermostable DNA synthase used for synthesis and amplification of DNA strands in a test tube, determination of a base sequence, etc., a DNA sequence encoding the enzyme, and a method for producing the DNA synthase is there.
[0002]
[Prior art and its problems]
DNA polymerase is a general term for enzymes that catalyze the synthesis of a DNA strand complementary to a single-stranded DNA. It is an indispensable enzyme for determining the base sequence of DNA and amplifying DNA in a test tube. Especially in PCR (Polymerase chain reaction), the “thermostable DNA synthase” It is essential.
[0003]
As such thermostable DNA synthase, Taq, Pfu, KOD, etc. are known, and they are properly used according to their characteristics. However, in the case of DNA synthesis such as PCR using these existing thermostable DNA synthetases, depending on the DNA strand used as a template, the elongation of the synthesized DNA strand stops halfway, and the template DNA strand There was a problem that it was difficult or impossible to amplify the entire region and to determine its base sequence. In addition, when an incomplete DNA fragment due to synthesis termination is mixed in the PCR product, there is also a disadvantage that the target amplified fragment has to be purified.
[0004]
The present invention has been made in view of the circumstances as described above. When a DNA strand is synthesized and amplified by PCR or the like, the entire length of the template DNA strand can be efficiently obtained without stopping the elongation of the synthetic strand. It is an object to provide a novel thermostable DNA synthase that can be amplified well.
Another object of the present invention is to provide a DNA sequence encoding the thermostable DNA synthase and a method for producing a thermostable DNA synthase as an expression product of the DNA sequence.
[0005]
[Means for Solving the Problems]
The present invention, as to solve the above problems, to provide a heat-resistant DNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 (claim 1).
The present invention also provides a DNA encoding the amino acid sequence of SEQ ID NO: 1 (Claim 2) and a cloning vector (Claim 3) containing this DNA . As such a cloning vector, a recombinant plasmid pDP320 (claim 4) possessed by E. coli HMS174 (DE3) / pDP320 (FERM P-16052) is also provided.
[0006]
Furthermore, the present invention provides a thermostable DNA synthase characterized by culturing cells transformed with the expression vector containing the DNA of claim 2 and isolating and purifying the target enzyme produced in the medium. The manufacturing method of (5) is provided.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Specifically, the thermostable DNA synthase of the present invention is obtained by genetic engineering of Pfu DNA synthase derived from Pyrococcus furiosus by a known mutant gene preparation method (Strategies, vol 9, p3-4, 1996). (Hereinafter, the thermostable DNA synthase of the present invention may be referred to as “modified Pfu DNA synthase”). This enzyme was prepared as follows. That is, since the base sequence of the gene of Pfu DNA synthase is known, a complementary oligonucleotide is synthesized at both ends, and the gene of Pfu DNA synthase is obtained by PCR using the bacterial genomic DNA as a template using this as a primer. Was prepared. This gene DNA fragment was cloned into a vector and mutated by the method described in the above literature. In the gene mutation, the base was substituted so that a part of the amino acid sequence of Pfu DNA synthase was replaced with the amino acid sequence of KOD DNA synthase. Pfu DNA synthase and KOD DNA synthase are about 80% homologous in amino acid sequence, and cause similar synthesis termination during PCR (FIG. 1), but the extension rate of KOD DNA synthase is 6 times that of Pfu DNA synthase. ~ 10 times. Therefore, by substituting the amino acid residue of PfuDNA synthetase with the amino acid residue of KODDNA synthetase, the elongation stop of the synthetic chain may be improved and an enzyme having a high elongation rate may be obtained. The gene thus mutated was expressed in E. coli, and the expression product was recovered and purified to obtain the modified Pfu DNA synthase of the present invention.
[0008]
In practice, the inventors of the present invention have prepared a number of modified Pfu DNA synthases by the above-described method, conducted DNA synthesis experiments for each, and electrophoretically analyzed the extended DNA strands. A DNA synthesizing enzyme having a significantly improved extension stop of the synthetic strand compared to the enzyme was obtained and the present invention was completed.
This DNA synthase has the amino acid sequence shown in SEQ ID NO: 1, and this amino acid sequence is a novel amino acid residue shown in Table 1 in which amino acid residues shown in Table 1 are substituted. Is an array. When DNA synthesis such as PCR is carried out using this novel enzyme, the synthesis stop that occurs when conventional DNA synthase is used is almost completely eliminated as shown in the following examples. Of course, a DNA strand that is efficiently amplified by conventional enzymes can be similarly amplified efficiently.
[0009]
[Table 1]
Examples of the DNA sequence encoding this modified Pfu DNA synthase include mutant genes of the Pfu DNA synthase gene obtained in the above-described enzyme preparation process. This mutated gene has been cloned into a recombinant plasmid pDP320, which was introduced into E. coli HMS174 (DE3) and deposited with the Institute of Biotechnology, Industrial Technology Institute (deposit number FERM P-16052). However, the DNA synthase of the present invention requires and sufficiently requires that its amino acid sequence is SEQ ID NO: 1, and the gene encoding such an enzyme is present at each amino acid residue of SEQ ID NO: 1. It can be appropriately designed as a DNA sequence in which corresponding base codons are connected.
[0011]
EXAMPLES Hereinafter, examples will be shown and the DNA synthase of the present invention will be described in more detail and specifically, but the present invention is not limited to the following examples.
[0012]
【Example】
Example 1: Preparation of modified Pfu DNA synthase gene (1) Cloning of Pfu DNA synthase gene PCR primers were synthesized according to the base sequence of Pfu DNA synthase gene (Nucleic Acids Research, vol. 21, p259-265, 1993). The target gene was amplified by PCR using the genomic DNA of P. furiosus as a template and cloned into an expression vector for E. coli. Details are as follows.
[0013]
P. furiosus DSM3638 was cultured by the method described in the above literature. First, a medium described in the literature was prepared, and after high-temperature autoclaving, infused with nitrous gas, inoculated and cultured at 95 ° C. for 15 hours. About 0.5 mg of cells were obtained from 200 ml of culture cake by centrifugation. The collected cells are suspended in buffer A (10 mM Tris-HCL, pH 8.0, 1 mM EDTA, 100 mM NaCl), 1 ml of 10% SDS is added, and after stirring, 0.5 mg of proteinase K is added and reacted at 55 ° C. for 60 minutes. I let you. The reaction solution was sequentially extracted with phenol, phenol / chloroform and chloroform, and ethanol was added to insolubilize and collect the DNA. The obtained DNA was dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), 0.5 mg of RNase A was added and reacted at 37 ° C. for 60 minutes, followed by phenol extraction and phenol again. / Chloroform extraction, chloroform extraction, DNA was recovered by ethanol precipitation and dissolved in TE buffer to obtain about 0.3 mg of DNA.
[0014]
Subsequently, in order to PCR-amplify the target DNA synthase gene, two kinds of primer DNAs shown in SEQ ID NOs: 2 and 3 were synthesized based on the known sequence data. That is, the start codon ATG of the target gene and the restriction enzyme NcoI sequence (5′-CCATGG-3 ′) were introduced into the forward primer sequence, and the reverse primer was designed to bind to an appropriate position downstream of the stop codon. . PCR was performed in a 50 μl reaction system using 2 μg of P. furiosus DNA and 10 pmol of each primer under the conditions of LATaq (Takara Shuzo) and the attached buffer. Cycle conditions were 93 ° C / 3 minutes before adding the enzyme, and 30 cycles of 94 ° C / 0.5 minutes, 55 ° C / 0.5 minutes, 72 ° C / 1.0 minutes were performed. The amplified DNA fragment was purified, treated with NcoI, cut with NcoI, blunt-ended, and further inserted downstream of the T7 promoter of the NcoI-treated expression vector pET15-b. This expression vector was designated as pDPWT100, and the base sequence of the insertion gene was confirmed.
(2) Modification of Pfu DNA synthase gene Oligonucleotides (SEQ ID NOs: 4 and 5) containing the expected mutation and Promega's mutation introduction kit for the expression vector pDPWT100 incorporating the cloned Pfu DNA synthase gene The modified Pfu DNA synthase gene was prepared on the expression vector pDPWT100 according to a known method (Strategies, vol 9, p3-4, 1996), and the expression vector pDP320 was constructed. The amino acid sequence of the modified Pfu DNA synthase (SEQ ID NO: 1) was confirmed by determining the base sequence of this modified gene.
Example 2: Expression and purification of modified Pfu DNA synthase in Escherichia coli The modified Pfu DNA synthase gene prepared in Example 1 (2) was expressed in E. coli and purified as follows.
[0015]
The expression vector pDP320 having the modified Pfu DNA synthase gene prepared in Example 1 (2) was introduced into E. coli HMS173 (DE3) strain and cultured in LB medium containing IPTG at a final concentration of 0.1 mM for 14 hours. Expression was induced in E. coli. After collecting the cells by centrifugation, the modified Pfu DNA synthase was extracted while sonicating with a buffer containing 150 mM Tris / HCl (pH 7.5), 2 mM EDTA, 0.24 mM APMSF and 0.2% Tween20. . The crude extract was heat-treated at 80 ° C. for 15 minutes to inactivate E. coli-derived DNA synthase and partially purify the DNA synthase of the present invention. The partially purified fraction was dialyzed against a buffer of 50 mM Tris / HCl (pH 7.5), 1 mM EDTA, 0.2% Tween 20, 7 mM 2-mercaptoethanol and 10% glycerol. At this stage, DNA synthesis activity specific to the modified Pfu DNA synthase was detected.
Example 3: Primer extension reaction with modified Pfu DNA synthase Using the modified Pfu DNA synthase partially purified in Example 2, the primer extension reaction of a DNA strand complementary to the template DNA was tested.
[0016]
20 mM Tris / HCl (pH 8.0), 2 mM MgCl 2 , 50 μg / ml BSA, 0.1% Triton X-100, 1 mM cold dNTPs (0.1 mM for dCTP), [α- 32 P] dCTP 10 μCi and M13 (-21 1 μg of the above partially purified enzyme fraction was added to 20 μl of a reaction solution containing 0.63 μg of pBLUESCRIPT plasmid annealed with the primer (1) and reacted at 75 ° C. for 1 minute and 3 minutes. The extended DNA strand was separated by polyacrylamide gel electrophoresis containing 8M urea, and the pattern was analyzed by an image analyzer. As a control, the same DNA synthesis was performed using a conventional wild-type Pfu DNA synthase.
[0017]
The results are as shown in FIG. In the case of using the conventional wild type Pfu DNA synthase, at least 10 bands indicating the presence of DNA strands were observed incomplete due to the termination of synthesis, but in the DNA synthesis using the modified Pfu DNA synthase of the present invention, these bands were observed. The band disappeared. On the other hand, there was no difference in the accumulation of well-extended DNA strands near 1000 bases.
[0018]
【The invention's effect】
As described above in detail, according to the present invention, when a DNA strand is amplified by PCR or the like, it is possible to efficiently synthesize and amplify the entire length of a template DNA strand without stopping the elongation of the synthetic strand. A sex synthase is provided. This makes it possible to perform synthesis and amplification of DNA strands in a test tube, determination of a base sequence, and the like simply and with high accuracy.
[0019]
[Sequence Listing]
SEQ ID NO: 1
Array length: 775
Sequence type: Amino acid sequence type: Protein sequence
Sequence length: 35
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Other nucleic acid (synthetic DNA)
Array
Sequence length: 35
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Other nucleic acid (synthetic DNA)
Array
Sequence length: 66
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Other nucleic acid (synthetic DNA)
Array
Sequence length: 66
Sequence type: Number of nucleic acid strands: Single strand topology: Type of linear sequence: Other nucleic acid (synthetic DNA)
Array
[Brief description of the drawings]
FIG. 1 is a result of electrophoresis showing primer extension activity of conventional Pfu DNA synthase and KOD DNA synthase.
FIG. 2 is a result of electrophoresis showing primer extension activity of a conventional Pfu DNA synthase (wild type) and a modified Pfu DNA synthase of the present invention.
Claims (5)
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP01924897A JP3679536B2 (en) | 1997-01-31 | 1997-01-31 | Thermostable DNA synthase |
| DE69841602T DE69841602D1 (en) | 1997-01-31 | 1998-02-02 | Thermophilic DNA polymerase with modified function |
| EP05005854A EP1564288B1 (en) | 1997-01-31 | 1998-02-02 | Function-modified thermophilic DNA polymerase |
| EP98901095A EP1013759B1 (en) | 1997-01-31 | 1998-02-02 | Method and apparatus for predicting protein function site, method for improving protein function, and function-improved protein |
| DE69836971T DE69836971T2 (en) | 1997-01-31 | 1998-02-02 | METHOD AND APPARATUS FOR PREDICTING FUNCTIONAL PROTEINDOMAINS, METHOD FOR IMPROVING THE PROTEIN FUNCTION, AND FUNCTIONALLY IMPROVED PROTEIN |
| PCT/JP1998/000430 WO1998033900A1 (en) | 1997-01-31 | 1998-02-02 | Method and apparatus for predicting protein function site, method for improving protein function, and function-improved protein |
| US10/345,205 US7231301B2 (en) | 1997-01-31 | 2003-01-16 | Method and a system for predicting protein functional site, a method for improving protein function, and a function-modified protein |
| US11/806,448 US7820423B2 (en) | 1997-01-31 | 2007-05-31 | Method and a system for predicting protein functional site, a method for improving protein function, and a function-modified protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP01924897A JP3679536B2 (en) | 1997-01-31 | 1997-01-31 | Thermostable DNA synthase |
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| Publication Number | Publication Date |
|---|---|
| JPH10210979A JPH10210979A (en) | 1998-08-11 |
| JP3679536B2 true JP3679536B2 (en) | 2005-08-03 |
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| JP01924897A Expired - Lifetime JP3679536B2 (en) | 1997-01-31 | 1997-01-31 | Thermostable DNA synthase |
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| JP2017510286A (en) * | 2014-04-11 | 2017-04-13 | キング アブドラ ユニバーシティ オブ サイエンス アンド テクノロジー | DNA polymerases derived from red sea saltwater pool organisms |
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