Richard W. Beringer e David L. Ash, Aplicações Os cientistas, os produtos NanoDrop Thermo Scientific, de assistência técnica e filmagens.

1. Microvolume Quantificação de Ácido Nucleico Usando um NanoDrop 2000c Espectrofotômetro <ol><li> Para começar, limpar as superfícies superior e inferior óptica do sistema de retenção microvolume espectrofotômetro amostra pipetando 2-3 mL de água deionizada para limpar a superfície inferior óptica. </li><li> Fechar o braço de alavanca, garantindo que o pedestal superior entra em contato com a água deionizada. Levante o braço de alavanca e limpe ambas as superfícies ópticas com um pano limpo, sem fiapos, limpe a seco laboratório. </li><li> Abra o software NanoDrop e selecione o aplicativo de ácido nucléico. Use um volume pequeno, pipetador calibrado para realizar uma medição em branco, dispensa 1 mL de tampão para a superfície inferior óptica. Abaixe o braço de alavanca e selecione &quot;branco&quot; na aplicação de ácido nucléico. </li><li> Uma vez que a medição em branco é completa, limpa ambas as superfícies ópticas com um pano limpo, sem fiapos, limpe a seco laboratório. </li><li> Escolha a constante apropriada para o exemplo que está a ser medido. <table border="1"><tbody><tr><td> Tipo de amostra </td><td> Selecione Opção </td><td> Constante Utilizado para Calcular Concentração </td></tr><tr><td> dsDNA </td><td> DNA-50 </td><td> 50 </td></tr><tr><td> ssDNA </td><td> DNA-33 </td><td> 33 </td></tr><tr><td> RNA </td><td> RNA-40 </td><td> 40 </td></tr><tr><td> Oligo </td><td> Personalizado </td><td> 15-150 </td></tr></tbody></table> Tabela 1. Típicos varia de ácido nucléico de concentração para as medidas de absorbância direta A280 usando um NanoDrop microvolume espectrofotômetro, um espectrofotômetro cuvete tradicional baseado usado com uma microcélula TrayCell cuvete, e um fluorospectrometer NanoDrop microvolume em conjunto com o ensaio PicoGreen. </li><li> Dispense 1 mL de amostra de ácido nucléico para o pedestal inferior ópticas e fechar o braço de alavanca. Porque a medida é de volume independente, a amostra só precisa preencher a lacuna entre as duas superfícies ópticas para uma medição a ser feita. </li><li> Selecione &quot;Measure&quot; no software de aplicação. O software irá calcular automaticamente as taxas de ácido nucléico concentração e pureza. Após medição da amostra, analisar a saída espectral. </li><li> O software irá calcular automaticamente as taxas de ácido nucléico concentração e pureza. Após medição da amostra, rever a imagem espectral para avaliar a qualidade da amostra. </li><li> Uma amostra de ácido nucleico típico terá um perfil muito característico. <img src="/files/ftp_upload/2565/2565fig1.jpg" alt="Figura 1" /> Figura 1. Uma amostra de ácido nucleico típico terá um perfil muito característico. </li><li> As fontes comuns de contaminantes específicos associados com técnicas de isolamento de ácido nucléico incluem a extração de fenol / Trizol e coluna. No caso da extração de fenol / Trizol, a contaminação do reagente residual pode ser indicado por espectros anormal entre 220-240 nm, bem como por mudanças na região 260-280 nm. Por outro lado, guanidina residual da extração de coluna podem contribuir para um pico próximo 230 nm e uma mudança no cocho de 230 nm a cerca de 240 nm. <img src="/files/ftp_upload/2565/2565fig2.jpg" alt="Figura 2" /> Figura 2. As mudanças nos picos e depressões das amostras B e C, em comparação com amostra A ilustrar como contaminantes podem afetar os espectros de amostras de ácidos nucléicos. </li><li> Para avaliar com precisão a qualidade da amostra, 260/280 ou 260/230 índices deveriam ser analisados ​​em combinação com qualidade espectral global. Pure ácidos nucléicos normalmente produzem uma relação de 260 / 280 ~ 1,8 e uma relação de 260/280 de ~ 2.0 para DNA e RNA, respectivamente. Esta relação é dependente do pH e força iônica do buffer usado para fazer o branco e as medições da amostra. Soluções ácidas será sub-representação da relação por 0,2-0,3, enquanto uma solução básica sobre-representam a razão por 0,2-0,3. Relações pureza significativamente diferentes podem indicar a presença de proteínas, fenol ou outros contaminantes que absorvem fortemente em ou perto de 280 nm. O grau de pureza de 260/230 é uma segunda medida de pureza do DNA com os valores de um ácido nucléico &quot;puro&quot; em geral na faixa de 1,8-2,2. Relações pureza que são significativamente inferiores aos valores esperados podem indicar a técnica de isolamento utilizado pode exigir maior otimização. </li><li> Espectrofotômetros NanoDrop também podem ser usados ​​para quantificar proteínas usando absorbância direta ou por ensaios de proteína colorimétrico. Para garantir a formação adequada coluna e reprodutibilidade, 2 mL amostras são aconselhados para as amostras de proteína. Consulte o vídeo JOVE seguinte: Microvolume determinação da concentração de proteínas usando o espectrofotômetro NanoDrop <a href="http://www.jove.com/index/Details.stp?ID=1610">http://www.jove.com/index/Details.stp?ID=1610</a> </li></ol><ol><li> Para demonstrar a alta sensibilidade microvolume quantificação de ácidos nucléicos usando o 3300 NanoDrop, um kit PicoGreen fluorescência é usada. Para começar, equilibrar os padrões do kit e todas as amostras de ácido nucléico à temperatura ambiente. Amostras fluorescentes são sensíveis à luz e deve ser mantido em âmbar ou envolto tubos de alumínio. </li><li> Prepare o suficiente 1x tampão TE para todos os padrões e amostras a serem medidos, bem como para a solução PicoGreen de trabalho que serão necessários. Prepare tampão 1xTE diluindo fornecidas 20x tampão TE com água livre de nuclease por protocolo do fabricante. Volumes de reação pode variar de 200 mL para menos de 10 ul. Neste exemplo, 10 volumes de reação mL será preparado. </li><li> Prepare série padrão diluídas dsDNA em 2X a concentração final desejada em nuclease livre de frascos ou tubos. Transferir 5 mL de cada um dos padrões dsDNA diluído em um indivíduo rotulado nuclease livre de âmbar ou cobertos de folha de tubo. </li><li> ML alíquota 5 de cada amostra de interesse em dsDNA devidamente rotulados âmbar nuclease-free ou folhas cobertas de tubos. </li><li> Prepare PicoGreen solução de trabalho de acordo com o protocolo do fabricante. Transferência de um volume igual de solução PicoGreen trabalhando a cada tubo contendo um padrão ou amostra de interesse (5 mL, neste exemplo). </li><li> Combine volumes iguais de 1x tampão TE e solução PicoGreen de trabalho para preparar o controle negativo, que serve como uma solução de referência. </li><li> Misture o conteúdo de cada tubo de reacção completamente pipetando suave e incubar as reações à temperatura ambiente por 5 minutos. Todos os padrões e as amostras devem ser equilibrados à mesma temperatura antes da medição como sinal de fluorescência é afetado pela temperatura. </li><li> Para preparar o instrumento e efectuar medições, primeiro limpe as superfícies inferior e superior do sistema de retenção de amostra. Pipete 2-3 de água deionizada para a menor superfície óptica, feche em seguida, abra o braço de alavanca, e secar os pedestais superior e inferior com um pano limpo, sem fiapos, limpe laboratório. Assegurar as superfícies são livres de fiapos antes de prosseguir como fiapos podem apresentar fluorescência na presença de uma fonte de excitação e pode interferir com a medição. </li><li> Abra o software do aparelho, selecione a opção &quot;ácidos nucléicos&quot;, aplicação e selecione a opção &quot;PicoGreen-dsDNA&quot; opção. </li><li> Para o instrumento em branco, adicionar 2 mL de TE 1x ao menor pedestal, perto do braço, e selecione &quot;Blank&quot; no campo de software instrumento. Uma vez em branco é completa, limpe a solução em branco de ambas as superfícies do sistema de retenção de amostra. </li><li> Misturar a solução de referência por pipetagem suave. Usando dicas de retenção baixa, pipeta mL 2 sobre o pedestal inferior e fechar o braço. </li><li> Selecione &quot;referência&quot; no tipo de medição, e selecione as unidades desejadas. Clique em &quot;Measure&quot; para iniciar o ciclo de medição. </li><li> Quando o ciclo de medição estiver completo, abra o braço e completamente fora da amostra blot superfícies do sistema de retenção. Medir até cinco repetições de referência, utilizando uma nova porção para cada repetição. </li><li> Repita o processo para as normas complementares para construir uma curva padrão. Até sete padrões podem ser usados. O software é projetado para armazenar até cinco repetições para cada padrão. Replica das normas são em média para gerar uma curva padrão a partir do qual as concentrações da amostra são determinados automaticamente. </li><li> Uma vez que a curva padrão é completo, selecione a opção &quot;Samples&quot; guia, insira as informações ID respectivos amostra e medir cada amostra desconhecida. </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>

Philippe Desjardins e Debra Conklin são empregados por produtos Thermo Scientific NanoDrop, Inc, que produz os instrumentos utilizados neste artigo.

Os protocolos de quantificação aqui apresentados são baseados no mais aceita microvolume sistemas. Tais sistemas fornecem alternativas práticas para a metodologia de quantificação de ácidos nucleicos tradicionais, que contam com maiores volumes de amostra e do uso de aparelhos de contenção. NanoDrop microvolume tecnologia emprega um sistema de retenção de amostra que se baseia nas propriedades de tensão de superfície da amostra que está sendo medido para formar uma coluna de líquido. É essencial que a amostra entra em contato com a superfície superior e inferior de medição óptica para a formação de coluna adequada. Se em algum momento os resultados da medição não parece ser preciso ou não reprodutíveis, o mais provável é o resultado da heterogeneidade da amostra ou quebra de coluna líquida. Detergentes e álcool isopropílico não são recomendados agentes de limpeza, pois podem descondicionar as superfícies de medição pedestal. Um pedestal incondicionada ocorre quando as propriedades de superfície hidrofóbica do pedestal ter sido comprometida, resultando em um achatamento da gota de amostra. Pedestais incondicionado pode levar à quebra da coluna de líquido durante a medição. <img src="/files/ftp_upload/2565/2565fig3.jpg" alt="Figura 3" /> Figura 3. Incondicionado superfície é caracterizada por um achatamento de uma gota de água. Superfícies pedestal Uncondtioned podem ser recondicionados usando compostos Pedestal NanoDrop Recondicionamento (PR-1, os produtos Thermo Scientific NanoDrop). Uma fina camada de composto de recondicionamento é aplicada às superfícies pedestal superior e inferior, deixa-se secar para ~ 30 segundos, em seguida, esfregou fora de usar um pano limpo, sem fiapos, limpe a seco laboratório. Um estado com sucesso recondicionados é caracterizada por uma gota de água beading acima quando aplicados à superfície pedestal inferior. <img src="/files/ftp_upload/2565/2565fig4.jpg" alt="Figura 4" /> Figura 4. Recondicionados Uma superfície é caracterizada por uma beading de uma gota de água. Sistemas de quantificação Microvolume reduzir significativamente o consumo de amostra e aumentar drasticamente a faixa de concentração, quando comparado aos sistemas de quantificação mais tradicionais. Embora a quantificação microvolume tem freqüentemente se tornado uma tecnologia que permite em circunstâncias envolvendo massa celular limitados, tais como biópsias de agulhas e laser de captura de microdissecção, a eficiência ea facilidade de utilização desta metodologia tornou um alternativas amplamente aceitas para os métodos tradicionais de quantificação de ácidos nucleicos mesmo quando a amostra é abundante.

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<td>NanoDrop 2000c</td>
<td>&nbsp;</td>
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nanodrop microvolume quantitation of nucleic acids

Ensaios de biomolecular estão continuamente a ser desenvolvido, que utiliza quantidades progressivamente menores de material, muitas vezes impossibilitando o uso de cubeta convencional baseada em instrumentos para quantificação de ácidos nucleicos para aqueles que podem realizar quantificação microvolume. O NanoDrop microvolume sistema de retenção de amostra (Produtos NanoDrop Thermo Scientific) funções através da combinação de tecnologia de fibra óptica e propriedades tensão natural da superfície para captar e reter pequenas quantidades de amostra independente de aparelhos de contenção tradicionais, como tinas ou capilares. Além disso, o sistema utiliza comprimentos de caminho mais curto, que resultam em uma ampla gama de medições da concentração do ácido nucléico, essencialmente, eliminando a necessidade de realizar diluições. Reduzindo o volume de amostra necessário para análise espectroscópica também facilita a inclusão de etapas adicionais de controle de qualidade em todo muitos fluxos de trabalho molecular, aumentando a eficiência e, finalmente, levando a uma maior confiança nos resultados a jusante. A necessidade de análise de alta sensibilidade fluorescentes de massa limitada também surgiu com os recentes avanços experimentais. Usando a mesma tecnologia de retenção de amostra microvolume, medições fluorescentes pode ser realizada com 2 mL de material, permitindo fluorescentes requisitos ensaios volume a ser reduzido significativamente. Microreactions tais de 10 mL ou menos são agora possíveis através de um fluorospectrometer microvolume dedicados. Dois protocolos microvolume quantificação de ácidos nucleicos será demonstrado que o uso de sistemas integrados de retenção de amostra como alternativas práticas para cuvete tradicionais baseados em protocolos. Primeiro, um método de absorção direta A260 usando um espectrofotômetro microvolume é descrito. Isto é seguido por uma demonstração de um método baseado em fluorescência que permite redução de volume reações de fluorescência com uma fluorospectrometer microvolume. Estas novas técnicas permitem a avaliação das concentrações de ácido nucléico que variam de 1 pg / mL a 15.000 ng / mL com o consumo mínimo de amostra.

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

NanoDrop Microvolume Quantitation of Nucleic Acids

O uso de sistemas NanoDrop microvolume como alternativas práticas e eficientes para a metodologia de quantificação de ácidos nucleicos tradicional é descrito através da demonstração de dois protocolos microvolume quantificação de ácidos nucleicos.

NanoDrop quantificação Microvolume de Ácidos Nucléicos

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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Vídeo: 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.

Microvolume determinação da concentração de proteína utilizando o NanoDrop 2000c Espectrofotômetro

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

Biologia

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.

Quantificação de γH2AX Focos em amostras de tecido

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.

Determinação de concentração de ácidos nucléicos e proteínas Usando o Micro-volume Bio-spec Espectrofotômetro Nano

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.

Electricidade-Free, Ácido Nucleico seqüencial e isolamento de proteínas

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.

Análise e construção de estruturas de Ácido Nucleico com 3DNA

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.

A suplementação dietética de ácidos graxos poliinsaturados em<em&gt; 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.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

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.

Ligadura altamente eficiente de pequeno RNA moléculas microRNA para quantificação por sequenciamento de alto rendimento

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.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</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.

Simples massa de Leitura Digital de Ácido Nucleico Quantificação Assays

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