リチャードW.ベリンジャーとDavid L.アッシュ、技術支援及び撮影のためのアプリケーションの科学者、サーモサイエンティフィックの光度計の製品、。

1。光度計2000C分光光度計を用いて微量核酸の定量<ol><li>開始するには、下側の光学面にきれいな純水の2〜3μLをピペッティングすることにより微量分光光度計のサンプル保持システムの上限と下限の光学面をきれいに。 </li><li>レバーアームを閉じて、上側の台座は、脱イオン水と接触することを保証する。レバーアームを持ち上げ、きれいな、乾いた、糸くずの出ないラボペーパーで光学面の両方を拭き取ってください。 </li><li> NanoDropソフトウェアを開いて、核酸のアプリケーションを選択します。下側の光学面に緩衝液1μLを分注してブランク測定を行うために、少量の、校正済みのピペットを使用してください。レバーアームを下げ、核酸のアプリケーションで&quot;ブランク&quot;を選択します。 </li><li>一度ブランク測定が完了すると、きれいな、乾いた、糸くずの出ないラボペーパーで光学面の両方をきれいに。 </li><li>測定されるサンプルの適切な定数を選択してください。 <table border="1"><tbody><tr><td> サンプルの種類 </td><td> オプションを選択します。 </td><td> 濃度を計算するために使用される定数 </td></tr><tr><td> dsDNAを</td><td> DNA - 50 </td><td> 50 </td></tr><tr><td> ssDNAを</td><td> DNA - 33 </td><td> 33 </td></tr><tr><td> RNA </td><td> RNA - 40 </td><td> 40 </td></tr><tr><td>オリゴ</td><td>カスタム</td><td> 15から150 </td></tr></tbody></table> 表1。光度計微量分光光度計、TrayCellマイクロキュベットを使用する従来のキュベットベースの分光光度計、およびPicoGreenアッセイと組み合わせて微量光度計のfluorospectrometerを使用して直接A280吸光度測定用の代表的な核酸の濃度範囲。 </li><li>下の光学台に核酸サンプルの1μLを分注し、レバーアームを閉じます。測定が独立したボリュームなので、サンプルはのみ行われる測定の2つの光学面の間のギャップを埋める必要があります。 </li><li>アプリケーションソフトの&quot;測定&quot;を選択します。ソフトウェアは自動的に核酸の濃度および純度の比率を計算します。サンプルの測定に続いて、スペクトル出力を確認してください。 </li><li>ソフトウェアは自動的に核酸の濃度および純度の比率を計算します。サンプルの測定に続いて、サンプルの品質を評価するためのスペクトル画像を確認してください。 </li><li>典型的な核酸のサンプルは、非常に特徴的なプロファイルを持つことになります。 <img src="/files/ftp_upload/2565/2565fig1.jpg" alt="図1" /> 図1代表的な核酸のサンプルは、非常に特徴的なプロファイルを持つことになります。 </li><li>特定の核酸の分離技術に関連する汚染物質の一般的な情報源は、フェノール/トリゾールと列の抽出などがあります。フェノール/トリゾール抽出の場合には、残留試薬の汚染は、220から240 nmの間の異常なスペクトルによってだけでなく、260〜280 nm領域の変化によって示されるかもしれません。逆に、列の抽出からの残留グアニジンは、230 nm付近のピークと230 nmから約240 nmの谷のシフトに貢献するかもしれない。 <img src="/files/ftp_upload/2565/2565fig2.jpg" alt="図2" /> （2）試料BとCのピークと谷の変化、汚染物質が核酸サンプルのスペクトルに影響を与えることができる方法を示す試料と比較して図 。 </li><li>正確にサンプルの品質を評価するために、280分の260または230分の260の比率は全体のスペクトルの質と組み合わせて分析する必要があります。純粋な核酸は、典型的にはそれぞれ、〜1.8の280分の260の比率とDNAとRNAのための〜2.0の280分の260比が得られる。この比率は、pHとブランクと試料の測定を行うために使用される緩衝液のイオン強度に依存しています。基本的な解決策は、0.2から0.3での比率をオーバー表す一方、酸性溶液は、0.2から0.3での比率を過小表します。大幅に異なる純度の比率は、280 nm付近で強く吸収されるタンパク質、フェノールやその他の汚染物質の存在を示すことがあります。 230分の260の純度比は1.8〜2.2の範囲で一般的に&quot;純粋な&quot;核酸の値を持つDNAの純度の第2の測定です。期待値よりも著しく低い純度の比率は、使用される分離技術はさらなる最適化を必要とする可能性を示している可能性があります。 </li><li>光度計分光光度計は、直接の吸光度を用いてタンパク質やタンパク質比色定量法によってを定量化するために使用することができます。適切な列の形成と再現性を確保するために、2μlの試料をタンパク質サンプルのためにお勧めします。微量タンパク質の濃度測定光度計分光光度計を使用して、次のJoveのビデオを参照してください<a href="http://www.jove.com/index/Details.stp?ID=1610">http://www.jove.com/index/Details.stp?ID=1610を</a> </li></ol><ol><li>光度計3300を用いた高感度微量核酸の定量を実証するために、PicoGreen蛍光キットが使用されます。開始するには、室温にキットの標準と、すべての核酸サンプルを平衡化してください。蛍光試料は光に敏感であり、黄色またはアルミラップチューブに保管する必要があります。 </li><li>必要とされるPicoGreen実用的なソリューションのためだけでなく、測定するすべての標準およびサンプルに十分な1 × TEバッファーを準備します。製造業者のプロトコルごとにヌクレアーゼフリー水で提供される20倍のTEバッファーを希釈することにより1xTEバッファを準備します。反応容量は、200μLからわずか10 ulの範囲で指定できます。この例では、10μLの反応ボリュームが用意されます。 </li><li>ヌクレアーゼフリーバイアルまたはチューブの2倍の最終的な所望の濃度で希釈した二本鎖DNAの基準を準備する。ヌクレアーゼフリーアンバーまたは箔で覆われたチューブラベルの付いた個々に希釈した二本鎖DNAの基準の各5μLを転送する。 </li><li>適切に標識されたヌクレアーゼフリーのアンバーや箔で覆われた管への関心の各二本鎖DNAの試料のアリコート5μL。 </li><li>製造業者のプロトコルに従ってPicoGreenワーキング溶液を調製。興味の標準またはサンプル（この例では5μL）のいずれかを含む各チューブにPicoGreenワーキング溶液に等量を転送する。 </li><li>リファレンスソリューションとして機能する負の制御を、準備するために1 × TEバッファーとPicoGreenワーキング溶液の等容量を兼ね備えています。 </li><li>穏やかなピペッティングでよく、各反応管の内容物を混合し、室温で5分間反応をインキュベートする。蛍光シグナルは温度によって影響されるすべてのスタンダードとサンプルは測定前に同じ温度に平衡化する必要があります。 </li><li>楽器を準備し、測定を実行するには、最初のサンプル保持システムの下限と上限の表面を清掃してください。下側の光学面上に脱イオン水の2〜3μLピペットは、近くにしてレバーアームを開いて、きれいな、糸くずの出ない、ラボ用ワイプで上下測定を汚点。表面はlintは励起源の存在下で蛍光を示すことと、測定に干渉することができるように進む前に糸くずのないことを確認します。 </li><li>測定器ソフトウェアを開いて、&quot;核酸&quot;アプリケーションを選択し、&quot;PicoGreen - dsDNAを&quot;オプションを選択します。 </li><li>楽器をブランクに、下の台座に1 × TEの2μLを追加し、アームを閉じて、測定器ソフトウェアの分野では&quot;ブランク&quot;を選択します。ブランクが完了すると、サンプル保持システムの両面から空のソリューションをオフ汚点。 </li><li>穏やかなピペッティングによる参照の解決を混ぜる。下の台座の上に低保持のヒント、ピペット2μLを使用し、アームを閉じてください。 </li><li>測定タイプの&quot;リファレンス&quot;を選択し、目的の単位を選択します。測定サイクルを開始する&quot;測定&quot;をクリック。 </li><li>測定サイクルが完了すると、腕を開いて、徹底的にサンプル保持システムの表面をオフに汚点。 5つまでの測定は、各レプリケートのための新鮮なアリコートを使用して、参照の複製。 </li><li>標準曲線を構築するために追加の標準のプロセスを繰り返します。最大7つの基準に使用することができます。ソフトウェアは、5にそれぞれの標準のための複製を格納するために設計されています。標準の複製サンプルの濃度を自動的に決定されるから標準曲線を生成するために平均化されています。 </li><li>標準曲線が完了すると、、&quot;サンプル&quot;タブを選択し、それぞれのサンプルのID情報を入力して、それぞれの未知試料を測定する。 </li></ol>

<ol>
	<li>Wilfinger, W. W., Mackey, K., Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+and+Ionic+Strength+on+the+Spectrophotometric+Assessment+of+Nucleic+Acid+Purity.">Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity.</a> BioTechniques. 22, 474-481 (1997).</li><li>Voolstra, C., Jungnickel, A., Borrmann, L., Kirchner, R., Huber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Quantification+of+Nucleic+Acids:+LabelGuard+enables+photometric+quantification+of+submicroliter+samples+using+a+standard+photometer.">Spectrophotometric Quantification of Nucleic Acids: LabelGuard enables photometric quantification of submicroliter samples using a standard photometer.</a> Implen Applications Note. , (2006).</li><li>Jr, I. n. g. l. e., D, J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spectrochemical analysis. , XV-590 (1988).</li><li>Van Lancker, M., Gheyssens, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+four+frequently+used+assays+for+quantitative+determination+of+DNA.">A comparison of four frequently used assays for quantitative determination of DNA.</a> Anal. Lett. 19, 615-623 (1986).</li></ol>

フィリップデジャルダンとデブラコンクリンは、この資料に使用される楽器を作り出すサーモサイエンティフィック㈱エルプロダクツ株式会社によって雇用されている。

ここで紹介する定量プロトコルは、最も広く受け入れられている微量のシステムに基づいています。このようなシステムは、より大きなサンプルの量と封じ込め装置の使用に依存している伝統的な核酸の定量方法論、実用的な代替案を提供しています。 光度計微量技術は、液柱を形成するために、測定される試料の表面張力の特性に依存しているサンプル保持システムを採用しています。それは、サンプルが適切な列形成のための上限と下限の光学測定面に接触することが不可欠です。任意の時点での測定結果が再現が不正確かどうかと思われる場合は、それが最も可能性が高いサンプルの不均一性や液柱の破損の結果です。 洗剤やイソプロピルアルコールは、彼らが台座の測定面を無条件可能性があるため、洗浄剤を推奨されていません。無条件の台座には、サンプルの液滴の平坦化、その結果、台座の疎水性表面の特性が危険にさらされている場合に発生します。無条件の台座は、測定中に液柱の破損につながることができます。 <img src="/files/ftp_upload/2565/2565fig3.jpg" alt="図3" /> 図3。無条件の表面は、水滴の平坦化によって特徴付けられる。 Uncondtioned台座の表面は、光度計台座コンディショニング化合物（PR - 1、サーモサイエンティフィック㈱エルプロダクツ）を使用して再調整することができます。化合物を再調整の薄層が〜30秒間乾燥させ、上部と下部台座の表面に適用され、その後きれいな、乾いた、糸くずの出ないラボペーパーを使用してオフこすった。が正常に再調整状態は底部の台座の表面に適用した場合、最大ビーズ水滴によって特徴付けられる。 <img src="/files/ftp_upload/2565/2565fig4.jpg" alt="図4" /> 図は、4。再生された表面は、水滴のビーズアップによって特徴付けられる。 微量定量システムが大幅にサンプルの消費を削減し、より伝統的な定量化のシステムに比べて劇的に濃度範囲を増加させる。微量定量は、しばしばそのような針生検とレーザーキャプチャーマイクロダイセクションなどの限られた細胞集団が関与する状況を可能にする技術となっていますが、この手法の効率性と使いやすさは、それにも伝統的な核酸の定量方法に広く受け入れられている代替きましたと、サンプルが豊富です。

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<td>NanoDrop 2000c</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>ND-2000C</td>
<td>&nbsp;</td>
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<td>NanoDrop 3300</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
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nanodrop microvolume quantitation of nucleic acids

生体分子のアッセイは、継続的に多くの場合、微量の定量を行うことができるものについては核酸の定量のための従来のキュベットベースの計測器の使用を排除し、材料の徐々に少量を使用することを開発されています。 光度計微量サンプル保持システム（サーモサイエンティフィックの光度計の製品）などのキュベットやキャピラリーのような伝統的な封じ込め装置の独立したサンプルの微量をキャプチャして保持する光ファイバ技術と自然の表面張力の特性を組み合わせることで機能します。さらに、システムは本質的に希釈を行うために必要がなくなり、核酸濃度測定値の広い範囲につながる短いパスの長さを、採用しています。分光分析に必要なサンプルの量を減らすことはまた、多くの分子のワークフロー全体を通じて追加の品質管理の手順を含めることを容易に、効率を高め、最終的に下流の結果が大きな自信につながる。 限られた大量の高感度蛍光分析の必要性はまた、最近の実験の進歩で浮上している。同じ微量サンプル保持技術を用いて、蛍光の測定は、蛍光アッセイのボリュームの要件が大幅に削減できるように、材料の2μLを実行することができる。 10μL以下のようなmicroreactionsは、専用の微量fluorospectrometerを使用可能になりました。 二つの微量核酸の定量のプロトコルは、従来のキュベットベースのプロトコルへの実用的な代替案として、統合されたサンプル保持システムを使用することが実証されます。最初に、微量分光光度計を使用して直接A260吸光度法が説明されています。これは、微量fluorospectrometerで低次元ボリューム蛍光反応を可能にする蛍光ベースの方法のデモが続いている。これらの新しい技術は、1〜pg /μLのサンプルの最小限の消費量15,000 ng /μLのに至るまでの核酸濃度の評価を可能にします。

Watch this Scientific Journal Video about NanoDrop Microvolume Quantitation of Nucleic Acids at JoVE.com

NanoDrop Microvolume Quantitation of Nucleic Acids

従来の核酸の定量方法論への実践的かつ効率的な代替案として光度計微量システムの使用は、2つの微量核酸の定量プロトコルのデモンストレーションを通して説明されています。

核酸の光度計微量定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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nanodrop-microvolume-quantitation-of-nucleic-acids

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Biology

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ビデオ: NanoDrop Microvolume Quantitation of Nucleic Acids

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

光度計2000C分光光度計を使った微量タンパク質の濃度測定

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

生物学

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

組織サンプルのγH2AX病巣の定量

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

マイクロボリュームバイオスペックナノ分光光度計を用いて核酸およびタンパク質の濃度測定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

電気鉛フリー、シーケンシャル核酸およびタンパク質の分離

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

3DNAを有する核酸構造の分析と構築

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

多価不飽和脂肪酸の食事補給<em&gt;線虫（Caenorhabditis elegans）</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

メチルトランスフェラーゼと補因子類似体核酸およびタンパク質の配列特異的標識化

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

ハイスループットシークエンシングによりマイクロRNA定量のための低分子RNA分子の高効率ライゲーション

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮細胞接着性の定量<em&gt;インビトロ</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

デジタル核酸定量アッセイの簡単な一括読み出し

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...