S. Lusvarghi、J. Sztuba-Solinska、KJ Purzycka、JWラウシュとSFJル·グライスは、国立癌研究所、国立衛生研究所、米国の学内研究プログラムによってサポートされています。

<ol>
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利害の衝突は宣言されていない。

我々はここで高スループットSHAPEの詳細なプロトコルがあるどの規模のRNAのための一塩基の解像度に二次構造の決定を可能にする手法を提示する。また、二次構造の予測アルゴリズムを実験SHAPEデータを結合するだけではどちらの方法で可能であるよりも高精度にRNA二次元モデルの生成を容易にする。蛍光標識されたプライマーと自動CEの組み合わせは、単一の実験で長いRNA配列の解決を促進?...

スプライシング、翻訳、ウイルス複製および癌の調節に関与する触媒と非コーディングRNAの機能を理解するために、RNAの構造の詳細な知識が必要である1,2。残念なことに、RNA折りたたみの正確な予測は手ごわい課題を提示。クラシックプロービングエージェントは実験当たり100-150ヌクレオチドに限定され、このような毒性など多くの欠点から不完全なヌクレオチドカバレッジおよび/またはスループットに苦しむ。肉眼二次構造予測アルゴリズムは、効果的に精力的に類似した構造を区別することができないことに起因する不正確さに起因し、同様に不利である。特に大規模なRNAは、また、それらのコンフォメーションの柔軟性と、これらの技術に必要な高純度の試料を大量に起因するX線結晶構造解析と核磁気共鳴（NMR）分光法、などの3D構造決定の方法にしばしば難治性である。 HIGHスループットのSHAPEは、単一ヌクレオチド解像度で大RNAの構造をプロービングする効果的な、単純なアプローチを提供することによってこれらの問題の多くを解決する。また、SHAPEに使用される試薬は、取り扱いが安全で容易であり、試薬をプロービング他のほとんどの化学物質とは対照的に、すべての4つのリボヌクレオチドと反応する。これらの試薬 ​​はまた、 インビボ可能コンテキスト （複数可）3 でその中にRNAを精査すること、細胞膜を貫通することができる。もともと週実験室で開発された4、SHAPEはRNAの多種多様〜9キロバイトHIV-1 RNAゲノムの5の完全な二次構造の決定が最も顕著な例を分析するために使用されている。 SHAPEを使用して他の注目すべき成果はビリオン関連HIV-1 RNAの3中のタンパク質結合部位を同定するだけでなく、感染ウイロイド6、人間の長い非コードRNA 7、酵母リボソーム8、リボスイッチ9の構造の解明を含む。 WHイルSHAPEプロトコルの元と高スループットの変化が他の場所で10-12公開されている、本研究は、蛍光オリゴヌクレオチドを用いてハイスループットSHAPEによるRNAの二次構造の決定の詳細な説明は、ベックマン·コールターCEQ 8000遺伝分析装置を提供し、 SHAPEfinderとRNAStructure（V5.3）ソフトウェア。以前に未発表の技術的な詳細とトラブルシューティングに関するアドバイスも含まれています。 SHAPEのバリエーション SHAPEとそのバリエーションの本質は選択的に修正のサイトでかさばる付加物を生産し、2&#39;-水酸基（2&#39;-OH）リボースグループをアシル求電子無水物に水溶液中のRNAの露出です。この化学反応は、ベースが対になっている間、一本鎖ヌクレオチドは、これらの試薬による求電子攻撃を助長するコンホメーションを採用することが、より傾向があるように、地元のRNA構造力学を尋問の手段として、またはアーキテクチャコンストラクタainedヌクレオチドは10以下または非反応性である。付加体形成の部位が修飾されたRNA（ &quot;（+）&quot;プライマー伸長反応）上の特定の部位にハイブリダイズし、蛍光または放射性標識されたプライマーから開始する逆転写によって検出される。逆転写酵素（RT）でアシル化リボヌクレオチドを​​通過しなかった場合には、cDNA産物のプールは、その長さ修飾部位と一致製造される。制御&quot;（ - ）&quot;試薬にさらされていないRNAを用いたプライマー伸長反応は、構造、非特異的なRNA鎖の破損等を RNAにそのDNA合成の早期終了（ すなわち 、 &quot;止まり&quot;）が原因ように制御されるかもしれない。化学修飾によって生成された一時停止と区別する。最後に、同じプライマーから開始する2ジデオキシ配列決定反応は電気泳動後のRNA一次配列と反応ヌクレオチドを​​相関させるためのマーカーとして使用される。 SHAPの元のアプリケーションで（ - ）、および2つ配列決定反応E、同一の32 P-末端標識プライマー（+）のために利用される。これらの反応の生成物は、5〜8％ポリアクリルアミドスラブゲルに隣接するウェルにロードし、ポリアクリルアミドゲル電気泳動（PAGE; 図1）変性により分画している。従来のSHAPEによって生成されたゲル画像の定量的分析は、SAFA、半自動フット解析ソフトウェア13を用いて行うことができる。 対照的に、高スループットSHAPEは、蛍光標識プライマーおよび自動化されたキャピラリー電気泳動を用いる。具体的には、調査中のRNAのそれぞれの地域のために、一般的な配列を有する4 DNAプライマーのセットが、異なる5 &#39;蛍光標識は、合成または購入する必要があります。これらの異なる標識オリゴヌクレオチドは、プライム2 SHAPE反応と2配列決定反応、プールし、分留/自動キャピラリー電気泳動（CE）によって検出されたそのうちの製品に役立つ。 WHEREASのRNAの100〜150ヌクレオチドの反応性プロフィールは、独創的なアプローチを使用して、4つの一連の反応から得ることができる、高スループットSHAPEは、単一のプールされた試料300から600 3ヌクレオチドの分解能を可能にする。できるだけ多くの96としてサンプルが12連続したCEの実行（ 図2）の過程で分別のために準備ができますが、同時に、分別することがあり、最大の反応8セットまで。また、SHAPEfinderソフトウェアは、プロセスに開発され、CEQや他の遺伝的分析装置から出てくるデータを分析し、より自動化されており、SAFA 13または他のゲル解析パッケージよりもはるかに少ないユーザーの介入を必要とする。 より高度な高スループットの方法論は、最近取得した次世代シーケンシング技術と一緒に構造特異的酵素ではなく、アルキル化試薬を使用PARS（RNA構造の並列解析）14とFragの-SEQ（断片シークエンシング）15、などの浮上しているINFORMATIORNA構造に関するnである。これらの技術の魅力にもかかわらず、プロービングヌクレアーゼに固有の多くの制限はまだ16のままです。これらの問題は、次世代のシーケンシングは、従来の形状で実施したのと同様の化学修飾およびRNAの逆転写によって先行されるSHAPEシークエンシング（配列SHAPE-）17プロトコルで回避することができる。これらのメソッドは、RNAの構造決定の未来を表すことが、次世代シーケンシングは非常に高価であり、多くの研究室が利用できないままであることを覚えておくことが重要です。 形状データ解析遺伝分析装置に生成されたデータは、移動時間のインデックスに対してプロットされているキャピラリ検出器に流れる試料の蛍光強度（複数可）を含み、電気泳動の形で提示される。このプロットは、4つの蛍光チャネルに対応する重複のトレースの形をとるsが異なる蛍光を検出するために使用され、各トレースは、個々のcDNAの配列決定または製品に対応するピークで構成される。電気泳動データは、タブ区切りのテキストファイルとして遺伝アナライザからエクスポートしShapeFinder変換と解析ソフトウェア18にインポートされます。 ShapeFinderは、最初の移行時間とピークのボリュームを正確それぞれ、反応生成物のアイデンティティと数量を反映することを保証するためにデータを数学的な一連の変換を実行するために使用される。ピークは次いで、位置合わせて一体化し、その結果、一次RNA配列と一緒にまとめられている。 RNAの当該セグメントの &quot;反応性プロフィールは、&quot;（+）RNAヌクレオチドそれぞれに関連付けられた値からの制御値を減算し、後述するように、データを正規化することによって得られる。このプロファイルは、正規化された反応性valを変換しRNAstructure（V5.3）ソフトウェア19,20、にインポートされRNAの二次構造の折りたたみア​​ルゴリズムに組み込まれている擬似エネルギー制約へのUE。このようにアルゴリズムをプロービングや製本化学物質を組み合わせることで大幅にどちらかの方法だけでは12,21に比べて構造予測の精度を向上させます。 RNAstructureの出力（V5.3）は、最低エネルギーの画像を含むRNA二次構造テキストドットブラケット表記でSHAPE反応性プロファイル（S）だけでなく、同​​じ構造を色分け。後者はその後、そのようなヴァルナ22およびPseudoViewer 23としてRNA二次構造のグラフィカル表示に専用のソフトウェアにエクスポートすることもできる。 <img alt="図1" fo:content-width="4.8in" fo:src="/files/ftp_upload/50243/50243fig1highres.jpg" src="/files/ftp_upload/50243/50243fig1.jpg" /> 図1。 SHAPE 4,10経由RNAの構造決定のフローチャート（A）RNA MAYは、生物学的試料から、またはインビトロ転写によって得られる。（B）ソースに応じて、RNAは、折り畳まれたまたは他の方法で処理され、SHAPE試薬で修飾されている。（C）逆転写反応は、蛍光または放射性標識プライマーを用いた（D）cDNA産物であるどちらキャピラリーまたはスラブゲルベースの電気泳動経由画し。（E）フラグメント分析。（F）のRNA構造予測。 <a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">より大きい数字を表示するには、ここをクリックしてください。</a> <img alt="図2" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> 図2。 CEベースのSHAPEの高スループットの文字が急速複数RNAの解析、および/ ​​または、同じRNAの複数のセグメントを許可します。（） </st栄&gt;は（緑、青と赤のカラーコード化）（B）RNAの切片を蛍光プライマーの異なるセット（黒矢印）（C）を用いて独立して、プローブされたRNAは、300〜600ヌクレオチドの区間に分割することができる方法のセットを表し反応は〜3キロバイトRNA1のための完全なカバレッジを提供し、それぞれのウェルA1、B1、C1、 等にプールされ、ロードされています。 RNAは2、3、4等からの反応生成物は、同様に連続した電気泳動の実行で分画に調製することができる。 <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">より大きい数字を表示するには、ここをクリックしてください。</a>

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>N-methylisatoic anhydride (NMIA)</td>
			<td>Life technologies</td>
			<td>M25</td>
			<td>Dissolve in anhydrous DMSO</td>
		</tr>
		<tr>
			<td>1-methyl-t-nitroisatoic anhydride (1M7)</td>
			<td>see ref. 22</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transcriptase</td>
			<td>Life technologies</td>
			<td>18080044</td>
			<td>10,000 units</td>
		</tr>
		<tr>
			<td>Thermo sequenase cycle sequencing kit</td>
			<td>Affymetrix</td>
			<td>78500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials provided by the user</td>
		</tr>
		<tr>
			<td>RNA of interest</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6 pmol per reaction (the limit of detection will be determined by the instrument)</td>
		</tr>
		<tr>
			<td>Sets of four 5' labeled primers (Cy5, Cy5.5, WellRed D2 and WellRed D1/Licor IR800)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primers are complementary to the RNA and are used in reverse transcription and sequencing reactions. The listed fluorophores are optimal for the Beckman Coulter 8000 CEQ. Primers may be purchased or synthesized in house.</td>
		</tr>
		<tr>
			<td>DNA template</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>DNA is used for sequencing reactions, and must contain the sequence of the RNA being studied - including any 3'terminal extension, if present. Where applicable, it is often convenient to use the RNA transcription template.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Buffers</td>
		</tr>
		<tr>
			<td>10x RNA renaturation buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>100 mM Tris-HCl pH 8.0, 1 M KCl, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>5X RNA folding buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>200 mM Tris-HCl pH 8.0, 25 mM MgCl2, 2.5 mM EDTA, 650 mM KCl. (This buffer might be changed depending on the case (e.g. pH, EDTA, Mg, RNase inhibitor)</td>
		</tr>
		<tr>
			<td>2.5X RT mix</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4 &mu;l 5X buffer, 1 &mu;l 100 mM DTT, 1.5 &mu;l water,1 &mu;l 10 mM dNTPs, 0.5 &mu;l SuperScript III. Note that the 5X buffer and 100 mM DTT are provided with purchase of SuperScript III (Invitrogen).</td>
		</tr>
		<tr>
			<td>GenomeLab Sample Loading Solution (Beckman Coulter)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Attention: Avoid multiple freeze-thaw cycles</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Capillary electrophoresis</td>
			<td>Beckman</td>
			<td>CEQ8000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>varies</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

rna secondary structure prediction using high-throughput shape

生物学的プロセスに関与してRNAの機能を理解することは、RNA構造の徹底的な知識が必要です。この終わりに向かって、またはSHAPE &quot;プライマー伸長により解析のハイスループット選択2 &#39;ヒドロキシルアシル化&quot;と呼ばれる方法論は、一塩基分解能を持つRNAの二次構造を予測することができます。このアプローチは、優先的に水溶液中でのRNAの一本鎖またはフレキシブル領域をアシル化する化学プローブエージェントを利用しています。化学修飾部位が修飾されたRNAの逆転写によって検出され、この反応の生成物は、自動化されたキャピラリー電気泳動（CE）によって分画されている。逆転写酵素は、SHAPE試薬によって変更それらのRNAヌクレオチドで一時停止しているため、得られたcDNAライブラリーは、間接的に、単一の折られたRNAの文脈で足止めされているそれらのリボヌクレオチドを​​マッピング。 ShapeFinderソフトウェアを使用して、自動化されたCEによって生成された電気泳動図はNUに加工と変換される自身がRNAStructure（V5.3）予測アルゴリズムで使用される擬似エネルギー制約に変換されcleotide反応テーブル。 シリコ RNAの二次構造予測でプロービングSHAPEを組み合わせることにより得られる二次元RNA構造は単独の方法を用いて得られた構造体よりもはるかに正確であることが見出されている。

RNAは存在下でHIV-1回転応答エレメント（RRE）および3 &#39;末端構造カセット4は 、それが 37℃で加熱、冷却、およびインキュベーションによって折られた後に、in vitro転写により線状プラスミドから調製した°Cを含有するのMgCl 2。 RNAはNMIAに露出した後、3 &#39;末端構造カセットにハイブリダイズした5&#39;-末端標識DNAプライマーから逆転写。得られたcDNAライブラリ...

Watch this Scientific Journal Video about RNA Secondary Structure Prediction Using High-throughput SHAPE at JoVE.com

RNA Secondary Structure Prediction Using High-throughput SHAPE

プライマー伸長により解析し、高スループット選択2 &#39;ヒドロキシルアシル化（SHAPE）数百から一塩基の解像度で数千ヌクレオチドRNAの構造を決定するための技術が、逆転写、キャピラリー電気泳動および二次構造予測ソフトウェアをプロービングする新規化学物質を利用しています。

ハイスループットSHAPEを使用したRNA二次構造予測

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

rna-secondary-structure-prediction-using-high-throughput-shape

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

31.2K ビュー.  Frederick National Laboratory for Cancer Research. プライマー伸長により解析し、高スループット選択2 'ヒドロキシルアシル化（SHAPE）数百から一塩基の解像度で数千ヌクレオチドRNAの構造を決定するための技術が、逆転写、キャピラリー電気泳動および二次構造予測ソフトウェアをプロービングする新規化学物質を利用しています。

ビデオ: RNA Secondary Structure Prediction Using High-throughput SHAPE

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

生物学

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

コンピュータベースのタンパク質の構造と機能の予測のためのプロトコル

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

自動化を使用して染色体のハイスループット物理マッピング<em&gt;その場で</em&gt;ハイブリダイゼーション

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

ゼブラフィッシュ胚を用いた高スループットへのアプローチマニュアル小分子スクリーン

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFEアッセイを用いた高スループットにおけるmiRNA標的の検出

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

ハイスループットスクリーニングを使用したキナーゼ基質ペアの同定


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...