私たちは、分子動力学シミュレーションで生成されたDNAの二重らせんの座標を共有するためのイジーŠponerに感謝しています。また、これらの構造をダウンロードの支援のために灘Spackovaを認める。 USPHS研究助成GM34809とGM096889を通してこの仕事のサポートは感謝して承諾されます。

<ol>
	<li>Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3DNA:+a+software+package+for+the+analysis,+rebuilding,+and+visualization+of+three-dimensional+nucleic+acid+structures.">3DNA: a software package for the analysis, rebuilding, and visualization of three-dimensional nucleic acid structures.</a> Nucleic Acids Res. 31, 5108-5121 (2003).</li><li>Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3DNA:+a+versatile,+integrated+software+system+for+the+analysis,+rebuilding,+and+visualization+of+three-dimensional+nucleic-acid+structures.">3DNA: a versatile, integrated software system for the analysis, rebuilding, and visualization of three-dimensional nucleic-acid structures.</a> Nature Protocols. 3, 1213-1227 (2008).</li><li>Zheng, G., Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web+3DNA-a+web+server+for+the+analysis,+reconstruction,+and+visualization+of+three-dimensional+nucleic-acid+structures.">Web 3DNA-a web server for the analysis, reconstruction, and visualization of three-dimensional nucleic-acid structures.</a> Nucleic Acids. Res. 37, W240-W246 (2009).</li><li>Xin, Y., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BPS:+a+database+of+RNA+base-pair+structures.">BPS: a database of RNA base-pair structures.</a> Nucleic Acids Res. 37, D83-D88 (2009).</li><li>Zheng, G., Colasanti, A. V., Lu, X. -. J., Olson, W. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-DART:+a+DNA+structure+modelling+server.">3D-DART: a DNA structure modelling server.</a> Nucleic Acids Res. 37, W235-W239 (2009).</li><li>Contreras-Moreira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-footprint:+a+database+for+the+structural+analysis+of+protein-DNA+complexes.">3D-footprint: a database for the structural analysis of protein-DNA complexes.</a> Nucleic Acids Res. 38, D91-D97 (2010).</li><li>Popenda, M., Szachniuk, M., Blazewicz, M., Wasik, S., Burke, E. K., Blazewicz, J., Adamiak, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+FRABASE+2.0:+an+advanced+web-accessible+database+with+the+capacity+to+search+the+three-dimensional+fragments+within+RNA+structures.">RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures.</a> BMC Bioinformatics. 11, 231 (2010).</li><li>&#268;ech, P., Svozil, D., Hoksza, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SETTER:+web+server+for+RNA+structure+comparison.">SETTER: web server for RNA structure comparison.</a> Nucleic Acids Res. , (2012).</li></ol>

利害の衝突は宣言されていない。

この記事で紹介した一連のプロトコルは、プログラムのみの3DNAスイートの能力に触れる。ツールは等、主鎖に沿っ塩基の重なりを測定するために、螺旋フラグメントの空間的な配置を定量化するために、そのようなペアリングが発生する二次構造のコンテキストを決定するために、非標準塩基対を識別するための構造をRNAに適用することができる再構築コマンドは、ユーザが図1を</stro...

DNA、RNA、およびタンパク質、薬物、及び他のリガンドとの複合体の三次元構造を理解し、それらの多様な生物学的機能を解読するための、および治療の合理的な設計を可能にするために重要である。解析（形状や相互作用のパターンを抽出する）、モデリング（エネルギー論と分子動力学を評価する）、および視覚化：そのような構造の探査には、3つの独立した、まだ密接に関連のコンポーネントを必要とする。構造解析とモデル構築は、本質的に表裏一体であり、可視化は、それらの両方を補完するものです。 コンピュータプログラムの3DNAスイートは、分析し構築し、三次元の核酸構造を可視化する機能を備えた人気が高まって、構造バイオインフォマティクスツールキットです。以前の出版物は、ソフトウェア1の機能を概説し選択したタスク2を実行するためのレシピを提供する、Webベースのインターフェイスを導入しましたソフトウェア3の一般的な機能には、構造的特徴の提示のデータベースが使用して収集 3DNA 4,5およびDNAとRNAの構造6の両方の解析にソフトウェアの有用性を示すように、7。 この記事の目的は、最先端の計算ツールとDNAとRNAの空間組織を調査するために利益および/またはニーズに実験室の科学者や他の人に3DNAソフトウェアキットをもたらすことです。ここで紹介するプロトコルは、構成塩基対のステップのレベルでDNA構造を分析し、修正するためのMac OS Xシステムでは、（II-III）、（上のソフトウェアをダウンロードしてインストールするためのステップバイステップの手順（i）を含むIV-v）を分析し、関連するDNA構造のセットを揃え、および（vi）ユーザーフレンドリーw3DNA Webインターフェイスを持つタンパク質装飾DNAチェーンのモデルを構築しています。ソフトウェアは、個々の構造だけでなく、大型X線結晶学的方法を用いて解く分析する能力を有する核磁気共鳴（NMR）法を用いて測定又はコンピュータシミュレーション技術によって生成された構造のアンサンブル。 構造は、ここで（i）はボレリアブルグドルフェリ 8（ヒト9,10にライム病を引き起こすダニ媒介性細菌）、（ii）の順次の二つの大きなセットからHBBタンパク質に結合したDNAの高分解能結晶構造を含む調べDの4,500スナップショット（GGCAAAATTTTGCC）2およびd（CCGTTTTAAAACGG）2計算中100ピコ秒単位で収集し、O3のDNAオペレーターのNMRベースの構造の（ⅲ）小アンサンブル-分子シミュレーション11で生産関連DNA分子大腸菌 lacリプレッサータンパク質12のヘッドピースにバインドされた。以下の手順では、これらの構造のそれぞれに関連付けられた原子座標のファイルにアクセスするだけでなく、使い方を3DNA（このファイルのコピーが発見された方法に関する情報を含むで3DNAフォーラム<a href="http://forum.x3dna.org/jove" target="_blank">http://forum.x3dna.org/jove</a> ）これらの構造を調べ、変更することができます。

analyzing and building nucleic acid structures with 3dna

3DNAソフトウェアパッケージは、解析構成し、三次元の核酸構造を可視化する機能を備えた人気があり、汎用性の高いバイオインフォマティクスツールである。この記事では、個々の構造と関連構造のアンサンブルの両方に適用3DNAで利用可能な新しい、人気の機能のサブセットのための詳細なプロトコルを提示します。プロトコル1は、ソフトウェアをダウンロードしてインストールするために必要な一連の命令を示しています。これは、塩基対の割り当てと、原子の再構成の記載により、プロトコル3で、構造を記述し、剛体パラメータの決定を含む核酸構造の分析により、プロトコール2において、続くその剛体パラメータから構造のモデル。 3DNAの最新バージョンは、2.1バージョンでは、このような核磁気共鳴（NMR）測定と分子動力学（MDから推定されるような構造のアンサンブルの分析と操作のための新しい機能を持っています）シミュレーション、これらの機能はプロトコル4と5に示されている。 3DNAスタンドアロンのソフトウェアパッケージに加えて、に位置w3DNA Webサーバ、 <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.eduは</a> 、ソフトウェアの選択された機能へのユーザーフレンドリーなインターフェースを提供します。プロトコル6は、ユーザーが指定した位置に結合したタンパク質で飾られた長いDNA分子のモデルを構築するためのサイトの新規な特徴を示しています。

3DNAソフトウェアツールを定期核酸構造を分析するために使用される。例えば、塩基対のアイデンティティとDNAとRNA構造の二重らせんの断片中の塩基の配列を特徴付ける剛体パラメータが自動的核酸データベース22の世界的なリポジトリに新しいエントリごとに計算して記憶する核酸構造情報。プロトコール2で決定剛体パラメータの値を容易に見つかり、そのような大きな正のロール...

Watch this Scientific Journal Video about Analyzing and Building Nucleic Acid Structures with 3DNA at JoVE.com

Analyzing and Building Nucleic Acid Structures with 3DNA

3DNAソフトウェアパッケージは、解析構成し、三次元の核酸構造を可視化する機能を備えた人気があり、汎用性の高いバイオインフォマティクスツールである。この記事では、個々の構造と関連構造のアンサンブルの両方に適用3DNAで利用可能な新しい、人気の機能のサブセットのための詳細なプロトコルを提示します。

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 ...

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JoVE Journal

Biology

20.4K ビュー.  Rutgers - The State University of New Jersey. 3DNAソフトウェアパッケージは、解析構成し、三次元の核酸構造を可視化する機能を備えた人気があり、汎用性の高いバイオインフォマティクスツールである。この記事では、個々の構造と関連構造のアンサンブルの両方に適用3DNAで利用可能な新しい、人気の機能のサブセットのための詳細なプロトコルを提示します。

ビデオ: Analyzing and Building Nucleic Acid Structures with 3DNA

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

マイクロ電極の構造によって生成されたAC動電現象

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

の蛍光標識ショウジョウバエハートの構造

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

二重共鳴コヒーレント反ストークスラマン散乱（CARS）と生物学的構造の差イメージング

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

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 ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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 ...

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 ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

細菌群集の特徴付けのための効率的な核酸抽出および16S rRNAの遺伝子配列決定

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.