Name: dna polymerase activity assay using near-infrared fluorescent labeled dna visualized by acrylamide gel electrophoresis
Uploaded: 2017-10-06T15:00:00.000+00:00
Duration: 7 min 38 s
Description: Watch this Scientific Journal Video about DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis at JoVE.com

この仕事はだった科学の進歩 (コットレル大学学者賞 #22548) の研究株式会社トライリンク バイオ (ResearchReward グラント #G139) でサポートされています。

<ol>
	<li>Ong, J. L., Loakes, D., Jaroslawski, S., Too, K., Holliger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+DNA+polymerase,+RNA+polymerase+and+reverse+transcriptase+activity+in+a+single+polypeptide.">Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.</a> J Mol Biol. 361 (3), 537-550 (2006).</li><li>Chen, T., Romesberg, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+polymerase+evolution.">Directed polymerase evolution.</a> FEBS letters. 588 (2), 219-229 (2014).</li><li>Leconte, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+DNA+polymerases+for+next-generation+sequencing.">Directed evolution of DNA polymerases for next-generation sequencing.</a> Angew Chem Int Ed Engl. 49 (34), 5921-5924 (2010).</li><li>Xia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+novel+polymerase+activities:+mutation+of+a+DNA+polymerase+into+an+efficient+RNA+polymerase.">Directed evolution of novel polymerase activities: mutation of a DNA polymerase into an efficient RNA polymerase.</a> Proc Natl Acad Sci U S A. 99 (10), 6597-6602 (2002).</li><li>Chen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+thermophilic+DNA+polymerases+for+the+recognition+and+amplification+of+C2'-modified+DNA.">Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.</a> Nat Chem. 8 (6), 556-562 (2016).</li><li>Thirunavukarasu, D., Chen, T., Liu, Z., Hongdilokkul, N., Romesberg, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+2'-Fluoro-Modified+Aptamers+with+Optimized+Properties.">Selection of 2'-Fluoro-Modified Aptamers with Optimized Properties.</a> J Am Chem Soc. 139 (8), 2892-2895 (2017).</li><li>Taylor, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalysts+from+synthetic+genetic+polymers.">Catalysts from synthetic genetic polymers.</a> Nature. 518 (7539), 427-430 (2015).</li><li>Alves Ferreira-Bravo, I., Cozens, C., Holliger, P., DeStefano, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+2'-deoxy-2'-fluoroarabinonucleotide+(FANA)+aptamers+that+bind+HIV-1+reverse+transcriptase+with+picomolar+affinity.">Selection of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity.</a> Nucleic Acids Res. 43 (20), 9587-9599 (2015).</li><li>Schultz, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taq+DNA+Polymerase+Mutants+and+2'-Modified+Sugar+Recognition.">Taq DNA Polymerase Mutants and 2'-Modified Sugar Recognition.</a> Biochemistry. 54 (38), 5999-6008 (2015).</li><li>Rosenblum, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+discovery+of+new+combinations+of+mutant+DNA+polymerases+and+modified+DNA+substrates.">Design and discovery of new combinations of mutant DNA polymerases and modified DNA substrates.</a> Chembiochem. 18 (8), 816-823 (2017).</li><li>Creighton, S., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+kinetic+analysis+of+DNA+polymerase+fidelity+in+the+presence+of+proofreading+using+bacteriophage+T4+DNA+polymerase.">Gel kinetic analysis of DNA polymerase fidelity in the presence of proofreading using bacteriophage T4 DNA polymerase.</a> J Biol Chem. 270 (9), 4759-4774 (1995).</li><li>Joyce, C. M., Benkovic, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+fidelity:+kinetics,+structure,+and+checkpoints.">DNA polymerase fidelity: kinetics, structure, and checkpoints.</a> Biochemistry. 43 (45), 14317-14324 (2004).</li><li>Leconte, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization,+and+optimization+of+an+unnatural+base+pair+for+expansion+of+the+genetic+alphabet.">Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet.</a> J Am Chem Soc. 130 (7), 2336-2343 (2008).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proc Natl Acad Sci U S A. 74 (12), 5463-5467 (1977).</li><li>Karger, B. L., Guttman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Sequencing+by+Capillary+Electrophoresis.">DNA Sequencing by Capillary Electrophoresis.</a> Electrophoresis. 30 (Suppl 1), S196-S202 (2009).</li><li>Lawyer, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+expression,+purification,+and+enzymatic+characterization+of+full-length+Thermus+aquaticus+DNA+polymerase+and+a+truncated+form+deficient+in+5'+to+3'+exonuclease+activity.">High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.</a> PCR Methods Appl. 2 (4), 275-287 (1993).</li><li>Lawyer, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+expression+in+Escherichia+coli+of+the+DNA+polymerase+gene+from+Thermus+aquaticus.">Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.</a> J Biol Chem. 264 (11), 6427-6437 (1989).</li><li>Carroll, S. S., Cowart, M., Benkovic, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutant+of+DNA+polymerase+I+(Klenow+fragment)+with+reduced+fidelity.">A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity.</a> Biochemistry. 30 (3), 804-813 (1991).</li><li>Shendure, J., Ji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+DNA+sequencing.">Next-generation DNA sequencing.</a> Nat Biotechnol. 26 (10), 1135-1145 (2008).</li><li>Larsen, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+strategy+for+expanding+polymerase+function+by+droplet+microfluidics.">A general strategy for expanding polymerase function by droplet microfluidics.</a> Nat Commun. 7, 11235 (2016).</li><li>Cozens, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Synthesis+of+Nucleic+Acids+with+Defined+Regioisomeric+2'-5'+Linkages.">Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2'-5' Linkages.</a> Angew Chem Int Ed Engl. 54 (51), 15570-15573 (2015).</li></ol>

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ここでは、DNA ポリメラーゼを介した DNA の合成を特徴付けるアッセイを説明しました。近赤外線標識 DNA プライマーを使用し変性ポリアクリルアミドゲル電気泳動を使用して、異なるサイズのオリゴヌクレオチドを解決する、我々 は合成の正確な測定を有効にするオリゴヌクレオチドの単一のヌクレオチドの解像度を取得できます。これらのアプローチは、酵素 (セクション 2.1) の全体の活?...

DNA ポリメラーゼは、正確かつ効率的な DNA 合成を行い、ゲノムの整合性の維持に不可欠な。エラーすることがなく何百もの 1 秒あたりのヌクレオチドを合成する能力も分子生物学とバイオ テクノロジーの DNA ポリメラーゼの不可欠なツールになります。ただし、これらのプロパティも M DNA 基板上へのアプリケーションを制限します。一般的に言えば、自然の DNA ポリメラーゼを合成できない多くの可能性のある貴重な DNA の M 基板可能性が高いため生体内で非標準基板の使用に対する高選択的圧力に。多くのグループが進化の M DNA 合成1,2,3,4、5のことができる突然変異の DNA ポリメラーゼを生成する方法を開発しています。これらの努力は、DNA6,7,8のバイオ テクノロジーの有用性を拡大しています。
M-DNA を合成する DNA ポリメラーゼの変異体の能力を評価する私たち9,10, その他11,12,13通常使用 DNA の生体外の測定本稿で説明するポリメラーゼ活性。これらの実験で DNA ポリメラーゼが二重標識プライマー/テンプレートとヌクレオシド三リン酸基板; 共同培養製品は、ゲルの電気泳動によって評価されます。実験の具体的な質問、突然変異の DNA ポリメラーゼ、変更されたプライマー、によって修正されたテンプレートまたは変更されたヌクレオシドの三リン酸塩使用できます、変異体酵素活動の体系的な生化学的評価を有効にします。
歴史的には、これらの試金は DNA 合成; を追跡する 5' 放射性ラベルに頼ってきた最も一般的に、 32P、 33P が使用されています。通常、ラベル付けには、T4 ポリヌクレオチド キナーゼ11を使用して実現されます。ただし、有限の存続期間、放射性ラベル及びその安全な処分のコストが比較的高いため当社グループは代わりに分類される DNA 合成 5' 近赤外蛍光を使用します。比較的低コストの近赤外ゲルの撮像素子を使用して、同様の検出限界放射性ラベル (未発表結果) を使用して事前研究を観察しています。過去の観測9、正常に再現しております、我々 はない前に測定した速度定数 (結果は未発表) で大規模な定量的な違いを観察しています。
DNA のサイズとしたがって、DNA 合成の範囲を分析するには、我々 はサンガー シーケンス14キャピラリー電気泳動15の出現の前に、のもともと開発されたポリアクリルアミドゲルの電気泳動方法に依存します。分離や移動の距離は、分子量の測定として使用できます。大判、ポリアクリルアミドゲル垂直は単一のヌクレオチド分解能、さまざまな長さのオリゴヌクレオチドを DNA の定量的観測を実現できます。
総称して、これらの実験は、ポリメラーゼ特性に対する堅牢な方法です。時間のため反応、準備とケアの敏感な性質は再現性のある結果を達成するために必要です。さらに、アクリルアミド ゲル多数 DNA 変更、他の反応単一ヌクレオチド分解能でと同様、DNA 合成を測定する非常に効果的な方法ですが、技術的に困難な場合ができます。ここのプロトコルがうまくいけば、ユーザーは最も一般的なミスを回避しながらこれらの実験を実行するを有効にします。

<table><tbody><tr><td>Tris HCl</td><td>Promega</td><td>H5123</td><td></td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5131</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>Acetylated BSA</td><td>Promega</td><td>PR-R3961</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td></td></tr><tr><td>Dithiothreitol (i.e. DTT)</td><td>Research Products International</td><td>D11000</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (i.e. EDTA) (0.5M solution)</td><td>Sigma</td><td>03690-100mL</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Formamide</td><td>Acros</td><td>AC42374-5000</td><td></td></tr><tr><td>Orange G</td><td>Sigma Aldrich</td><td>O3756</td><td></td></tr><tr><td>Bromophenol blue</td><td>Fisher Scientific</td><td>50-701-6973</td><td></td></tr><tr><td>dNTPs</td><td>Fisher Scientific</td><td>FERR0191</td><td></td></tr><tr><td>M-dNTPs (riboNTPs)</td><td>Fisher Scientific</td><td>45-001-341 (343, 345, 347)</td><td></td></tr><tr><td>M-dNTPs (all other modified NTPs)</td><td>TriLink Biotechnologies</td><td>assorted</td><td></td></tr><tr><td>primer 1</td><td>IDT DNA / TriLink Biotechnologies</td><td>Custom Syntheses</td><td>We use the IR700 dye which can be purchased as a custom synthesis. We typically purchase the oligonucleotides HPLC purified. Sequence is 5&amp;rsquo;-dTAATACGACTCACTATAGGGAGA</td></tr><tr><td>template 1</td><td>IDT DNA / TriLink Biotechnologies</td><td>Custom Syntheses</td><td>We typically purchase the oligonucleotides HPLC purified. Sequence is 5&amp;rsquo;-dCGCTAGGACGGCATTGGATCAGTCTCCCTATAGTGAGTCGTATTA</td></tr><tr><td>Acrylamide</td><td>Research Products International</td><td>A11405</td><td>38.67% acrylamide and 1.33% bis-acrylamide</td></tr><tr><td>Tris/Borate/EDTA (TBE) solid</td><td>Research Products International</td><td>T22020</td><td></td></tr><tr><td>Urea ultrapure</td><td>Research Products International</td><td>U20200</td><td></td></tr><tr><td>Gel tape</td><td>CBS Scientific</td><td>GT-72-10</td><td></td></tr><tr><td>Large white spring clamp polypropylene</td><td>CBS Scientific</td><td>GPC-0001</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Fisher Scientific</td><td>BP179</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Fisher Scientific</td><td>BP15020</td><td></td></tr><tr><td>0.75 mm spacers</td><td>CBS Scientific</td><td>SGS-20-0740A</td><td></td></tr><tr><td>33x42 Notched Glass Plate Set</td><td>CBS Scientific</td><td>SGP33-040A</td><td></td></tr><tr><td>Wedge plate separator</td><td>CBS Scientific</td><td>WPS-100</td><td></td></tr><tr><td>Comb for gel electrophoresis</td><td>CBS Scientific</td><td>SG33-0734</td><td></td></tr><tr><td>Gel electrophoresis rig</td><td>CBS Scientific</td><td>SG-400-33</td><td></td></tr><tr><td>ultrapure water</td><td></td><td></td><td>we use a Milli-Q system from Millipore</td></tr><tr><td>DNA polymerases</td><td></td><td></td><td>we prepare these in our laboratory using published protocols.</td></tr></tbody></table>

dna polymerase activity assay using near-infrared fluorescent labeled dna visualized by acrylamide gel electrophoresis

酵素、堅牢な定量的メソッドがネイティブと組み換え酵素の特性評価のため必要です。DNA ポリメラーゼの DNA 合成は、体外DNA 合成法によるポリアクリルアミドゲル電気泳動が続くと言えます。この試金の目標は、変更された DNA (DNA) と自然な DNA の合成を定量化することです。これらのアプローチは単一のヌクレオチド分解能、酵素オリゴヌクレオチド合成時の個々 のステップの観察とオリゴヌクレオチドを解決する場合に特に便利です。これらのメソッドは、DNA 合成の個々 のステップ、DNA 合成と DNA 結合親和性の誤り率の定常状態速度定数の測定などの生化学的および生物物理学的プロパティの配列の評価に適用されています。使用して、変更されたヌクレオシドの三リン酸塩に限らず、(NTP) は、M-DNA および/または突然変異 DNA のポリメラーゼ、ペアを効果的に評価できる基板 DNA ポリメラーゼの相対的なユーティリティを含むコンポーネントを変更しました。ここでは、我々 は非伝統的なプライマー DNA の分類の近赤外蛍光標識 DNA などの戦略に合わせて行う必要があります変更を含め、自身を分析を詳しく説明します。また、注ぐアクリルアミドゲルの重要な技術的なステップの詳細な実行して、することがあります技術的に挑戦的です。

全体的な活動 (セクション 2.1、図 1で説明) と定常状態速度論 (セクション 2.1、図 2の結論のノートで説明します) の質的評価の成功ポリアクリルアミド ゲルの分析が表示されます。失敗ポリアクリルアミド ゲル分析している (図 3)。
市販のはしごや分子?...

Watch this Scientific Journal Video about DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis at JoVE.com

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

アクリルアミドゲル電気泳動法で可視化した近赤外蛍光標識 DNA を用いた DNA ポリメラーゼ活性測定法

このプロトコルでは、電気泳動、ゲル画像を用いた近赤外蛍光に分類された DNA に変化の観察を通じて変更された DNA の DNA ポリメラーゼの合成の特性について説明します。アクリルアミドゲルはサイズに応じて異なる速度で移行短い核酸の分離の高分解能イメージングに使用されます。

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

dna-polymerase-activity-assay-using-near-infrared-fluorescent-labeled

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

14.1K ビュー.  W.M. Keck Science Department of Claremont Mckenna, Pitzer, and Scripps College. このプロトコルでは、電気泳動、ゲル画像を用いた近赤外蛍光に分類された DNA に変化の観察を通じて変更された DNA の DNA ポリメラーゼの合成の特性について説明します。アクリルアミドゲルはサイズに応じて異なる速度で移行短い核酸の分離の高分解能イメージングに使用されます。

ビデオ: DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

赤外蛍光色素で標識されたオリゴヌクレオチドを使用したゲルシフトアッセイのために最適化されたプロトコル

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

生化学

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

DNA 染色青い光曝露による塩の沈殿物に基づく法

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

小ヌクレオチド基質のポリヌクレオチドリン酸化を測定する非放射性アッセイ

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

ナノルシフェラーゼレポーターアッセイによるDNA二本鎖切断修復の定量(英語)

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

著者スポットライト:DNA損傷応答を標的とする新規抗がん剤の開発

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

ハイスループットで定量的な発光ベースのレポーターアッセイを用いたDNA二本鎖切断修復活性の評価

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

がん研究

<meta charset="utf8"></head><body>Precision DNAzyme ナノマシンによる識別型RNAおよびDNA分析

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>著者スポットライト:DNAナノセンサーの進歩 – 分子検出における感度と選択性の課題への対処

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - 核酸認識用ナノマシン

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

合成オリゴヌクレオチドを用いたDNA酵素のアッセイ

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

著者スポットライト:Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

アクリルアミドゲル電気泳動法で可視化した近赤外蛍光標識 DNA を用いた DNA ポリメラーゼ活性測定法

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赤外蛍光色素で標識されたオリゴヌクレオチドを使用したゲルシフトアッセイのために最適化されたプロトコル

DNA 染色青い光曝露による塩の沈殿物に基づく法

小ヌクレオチド基質のポリヌクレオチドリン酸化を測定する非放射性アッセイ

ハイスループットで定量的な発光ベースのレポーターアッセイを用いたDNA二本鎖切断修復活性の評価

ハイスループットで定量的な発光ベースのレポーターアッセイを用いたDNA二本鎖切断修復活性の評価

ハイスループットで定量的な発光ベースのレポーターアッセイを用いたDNA二本鎖切断修復活性の評価

DNAzyme 10-23 - 核酸認識用ナノマシン

DNAzyme 10-23 - 核酸認識用ナノマシン

DNAzyme 10-23 - 核酸認識用ナノマシン

修飾合成オリゴヌクレオチドを用いた核酸代謝酵素のアッセイ