1. Coleta de Amostras <ol><li> Coletar amostras de sangue de indivíduos com o consentimento informado adequado. Para cada pessoa, desenhar 4 a 5 ml de sangue para um tubo P100 (BD Diagnostics, Franklin Lake, Nova Iorque, EUA). Para fins de comparação, ao mesmo tempo recolher sangue num tubo contendo EDTA. </li><li> Os tubos P100 BD contêm 15,8 ml K2EDTA seca por pulverização e um vasto espectro de propriedade liofilizado coquetel de inibidores de protease para impedir a coagulação e estabilizar as proteínas do plasma. Elas contêm também um separador mecânico. Depois de recolher a amostra de sangue, deve manter os tubos à temperatura ambiente durante 60 min, durante a noite até que o processamento ocorre. </li><li> Centrifugar os tubos P100 BD a 2.500 g durante 15 minutos à temperatura ambiente. Transfira o plasma recolhido acima do separador mecânica em micro tubos de centrífuga. </li><li> Armazenar os P100 tubos contendo o sangue compactado abaixo do separador, a -80 ° C até que esteja pronto para começar o DNA extraction. </li></ol> 2.1 Do sangue de P100 tubo (P100_DNA) <ol><li> P100 Os tubos são armazenados a -80 ° C, após a extracção de plasma. Dentro de 3 a 4 meses, recuperar o tubo contendo o sangue P100 compactada do congelador e descongelamento a 37 ° C em banho-maria durante 5 min (Figura 1A). Remova qualquer plasma residual que fica acima do separador. Adicionar 2 ml de solução de sacarose a 17% (solução de gradiente testados em casa - dados não publicados) P100 nos tubos acima do separador (Figura 1B). Centrifugar os tubos à temperatura ambiente durante 20 minutos a 2.500 g, este processo envia o separador mecânico para o topo do tubo e produz três camadas de solução incluindo a crosta inflamatória (Figura 1C). Extrair manualmente o separador usando um steriled clipe de metal em forma de gancho (Figura 1D). </li><li> Depois de recuperar o separador mecânico de cada tubo (Figura 1D), retire e discard a camada superior sacarose. Transferir a camada do meio, representando a camada de revestimento buffy (BC), em um tubo cônico de 15 ml estéril. Adicionar Tampão Fosfato Salino (PBS), para aumentar o volume de creme leucocitário até 3 ml. Extrair o DNA do creme leucocitário utilizando reagentes da Qiagen Puregene Kit sangue conforme a recomendação do fabricante, exceto uma velocidade de centrifugação de 2500 g (em vez de 2.000 g). </li><li> Para lisar e remover os glóbulos vermelhos do sangue (RBC), adicionar 9 ml de lise de RBC a 3 ml de creme leucocitário. Inverter cada tubo várias vezes e incubar à temperatura ambiente durante 10 min, com inversão ocasional durante a incubação. Rodar cada uma das misturas para baixo a 2.500 g durante 5 minutos e descarta-se o sobrenadante. Tratar o sedimento restante em cada tubo com 3 ml de solução de lise celular e de vórtice durante 15 seg. Tratar o lisado com 1 ml de solução de precipitação de proteínas e vortex durante 15 seg. </li><li> Centrifugar a 2500 g durante 5 min, e transferir o sobrenadante para um tubo cónico de 15 mL fresco contendo3 ml de 100% de isopropanol. Inverta os novos tubos de 10 vezes e centrifugar a 2500 g por 3 min. Desprezar o sobrenadante e adicionar 1 ml de etanol 70%. Inverta os tubos cónicos algumas vezes para desalojar e lavar o sedimento de DNA. Centrifugar a 2.500 g por 1 min. Deixar o sedimento secar durante 3 min (o lugar do tubo de cabeça para baixo), à temperatura ambiente e depois suspender o ADN com 250 ml de solução de re-hidratação, a 37 ° C durante 10 minutos e transferir para um pré-rotulados microtubo de 1,7 mL. Armazenar os tubos a -80 ° C até utilização. </li></ol> 2.2 a partir de sangue de EDTA Ttube (EDTA_DNA) <ol><li> A amostra de sangue para testes de rotina imunoematologia é recolhido em tubos de plástico vaccutainer revestidas por pulverização com 10,8 mg de K 2 EDTA e armazenadas a -80 ° C. Como os tubos não contêm um separador mecânico, a extracção de ADN não inclui as etapas que envolvem a separação mecânica (2.1.1 e 2.1.2). </li><li> Dentro de 3 a 4 meses de armazenamento de sangue, o DNUm é extraído usando o KingFisher Flex (Thermo Fisher Scientific, Waltham, MA, EUA). Trata-se de um sistema de extracção de ADN com base na tecnologia de automação de partículas magnéticas e consiste de lise / ADN de ligação, vários passos de lavagem das células e a eluição do ADN. </li><li> O kit utilizado na extração de DNA é o KingFisher Flex 24 kit DNA Blood (Qiagen, Alemanha). </li></ol> 3. DNA Quantidade e Avaliação da Qualidade A quantidade, a qualidade e integridade do ADN genómico foram analisadas por uma combinação de método que inclui PicoGreen, espectroscopia e electroforese. <ol><li> A concentração do DNA genómico é determinado pela quantificação Nanodrop e PicoGreen. </li><li> A pureza é determinada a partir da medição rácio 260/280 em espectrofotômetro Nanodrop. </li><li> A amostra (500 ng) também é carregado em 1% de gel de agarose para avaliar a integridade (degradação) do DNA. </li></ol> 4. Genotyping Ensaios e Controle de Qualidade <ol><li> Cinco P100_DNA e suas amostras EDTA_DNA correspondentes são selecionados aleatoriamente para análise usando o Immunochip personalizado (Immuno DNA Análise BeadChip). Immunochip é um chip de genotipagem Infinium contendo 196.524 polimorfismos e é projetado para estudos de imunogenética 5. </li><li> Para cada amostra, a 200 ng de ADN é amplificado, fragmentada, precipitado e ressuspenso no tampão de hibridação apropriado. </li><li> As amostras desnaturadas são hibridizados em Immunochips a 48 ° C durante um mínimo de 16 horas. </li><li> Após a hibridação, as Immunochips são processados ​​para reacções de extensão de uma única base e coradas. </li><li> Os immunochips são então fotografada usando Ilumina Hiscan leitor matriz talão e os dados de intensidade do grânulo normalizados é carregada no software Ilumina GenomeStudio e convertido em genótipos. Genótipos são chamadas usando o algoritmo de autocalling GenomeStudio. O controle de qualidade emenvolve exclusão de polimorfismos de nucleotídeo único (SNPs) com uma taxa de chamada de &lt;95%. </li><li> A taxa de SNP é usado como uma medida da eficiência do ensaio immunochip. É calculada como a percentagem do número total de ensaios no chip (196,524 SNPs codificados em cada chip) que atingir a intensidade de sinal e qualidade suficientes para receber uma atribuição genótipo automatizado. ADN de alta qualidade (260/280 razão de 1,8 ou superior, e ausência de degradação) deverá atingir, pelo menos, a mesma taxa de chamada como o ADN de controlo HapMap incluído no ensaio immunochip. O controle de qualidade envolve a exclusão de SNPs com uma taxa de chamada de &lt;95% em cada conjunto de dados. </li></ol>

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Biotechnol. 46, 258-264 (2010).</li><li>Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+points+in+mapping+autoimmunity.">Fine points in mapping autoimmunity.</a> Nature. 43, 1173-1174 (2011).</li><li>Cooper, J. D., Simmonds, M. J., Walker, N. M., Burren, O., Brand, O. J., Guo, H., Wallace, C., Stevens, H., Coleman, G., Franklyn, J. A., Todd, J. A., Gough, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+newly+identified+loci+for+autoimmune+thyroid+disease.">Seven newly identified loci for autoimmune thyroid disease.</a> Human Molecular Genetics. , (2012).</li></ol>

Não há conflitos de interesse declarados.

Muitas doenças humanas têm causas genéticas e explorar a base genética da doença humana exige uma crescente DNA de alta qualidade. A fonte mais comum de ADN utilizado em estudos genéticos se sangue total anticoagulado. Porque maioria dos laboratórios clínicos realizar testes bioquímicos, sorológicos e viral como um primeiro passo na investigação da evolução fenotípica, o sangue é muitas vezes coletado em um tubo de plasma. Após a recolha do plasma, o sangue compactado é frequentemente descartados. Port...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>BD P100 tubes</td>
 <td>BD Diagnostics</td>
 <td>366448</td>
 </tr>
 <tr>
 <td>EDTA tubes</td>
 <td>BD Diagnostics</td>
 <td>367863</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 </tr>
 <tr>
 <td>Paper clip</td>
 <td>Office product</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Corning</td>
 <td>430052</td>
 </tr>
 <tr>
 <td>Red blood cells (RBC)</td>
 <td>Qiagen</td>
 <td>158389 (kit)</td>
 </tr>
 <tr>
 <td>Phosphate Buffer Saline (PBS)</td>
 <td>Thermo Scientific</td>
 <td>SH30256.01</td>
 </tr>
 <tr>
 <td>BioSprint 96 DNA Blood Kit (48) </td>
 <td>Qiagen</td>
 <td>940054</td>
 </tr>
 <tr>
 <td>1.7 ml microtubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 </tr>
 <tr>
 <td>Human Immuno DNA Analysis BeadChip Kit</td>
 <td>Illumina</td>
 <td>WG-352-1001</td>
 </tr>
 <tr>
 <td>Bead array reader</td>
 <td>Illumina</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>GenomeStudio Software</td>
 <td>Illumina</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation

Testes de laboratório pode ser feito nas porções celulares ou fluidos do sangue. O uso de diferentes tubos de colheita de sangue determina a porção do sangue que pode ser analisada (sangue completo, plasma ou soro). Os laboratórios que participam no estudo da base genética de doenças humanas dependem de sangue inteiro anti-coagulado recolhido em EDTA vacutainer contendo como fonte de ADN para a análise genética / genómico. Porque a maioria dos laboratórios clínicos realizar testes bioquímicos, sorológicos e virais, como um primeiro passo na investigação fenotípica resultado, o sangue anticoagulado é também recolhida no tubo contendo heparina (tubo de plasma). Portanto, quando o ADN e de plasma são necessários para a análise simultânea e em paralelo de dados, tanto ao genoma e proteoma, é comum recolher o sangue em EDTA e ambos tubos de heparina. Se o sangue pode ser recolhido num tubo e servir como uma fonte de plasma e de DNA, que o método poderia ser considerado como um avanço à metanfetamina existenteods. A utilização do sangue compactados, após extracção de plasma representa uma fonte alternativa de ADN genómico, minimizando assim a quantidade de amostras de sangue e processadas a redução do número de amostras necessário para cada paciente. Este acabaria por economizar tempo e recursos. O sistema de coleta de sangue P100 BD para a preservação proteínas plasmáticas foram criados como um método melhorado ao longo plasma anterior ou recolha de soro tubos 1, para estabilizar o teor de proteína do sangue, permitindo uma melhor descoberta de biomarcadores de proteínas e proteômica experimentação a partir de sangue humano. Os tubos P100 BD contêm 15,8 ml de K2EDTA seca por pulverização e um vasto espectro de propriedade liofilizado coquetel de inibidores de protease para impedir a coagulação e estabilizar as proteínas do plasma. Eles também incluem um separador mecânico, que proporciona uma barreira física entre o plasma e os sedimentos celulares após centrifugação. Alguns métodos foram desenvolvidos para extrair DNA de sangue coagulado SAmples coletadas em tubos de plasma de idade 2-4. Os desafios de estes métodos foram associados principalmente com o tipo de separador no interior dos tubos (separador de gel) e às dificuldades na recuperação do sangue coagulado, o inconveniente de fragmentação ou de dispersão do coágulo e obstrução do coágulo por extracção do gel de separação. Nós apresentamos o primeiro método que extrai e purifica o DNA genômico de sangue coletado nos novos tubos BD P100. Comparamos a qualidade da amostra de ADN a partir de P100 tubos para que a partir de tubos com EDTA. Nossa abordagem é simples e eficiente. Ela envolve quatro passos principais como se segue: 1) o uso de um BD P100 plasma (BD Diagnostics, Sparks, MD, USA) com separador mecânico de tubos para colheita de sangue, 2) a remoção do separador mecânico usando uma combinação de sacarose e um clipe estéril gancho metálico, 3) a separação da camada de cobertura amarelada que contém os glóbulos brancos e 4) o isolamento do ADN genómico dobuffy coat usando um kit de extração de DNA comercial regular ou um protocolo padrão similar.

Avaliação da qualidade do DNA e medição da concentração Os rendimentos de P100_DNAs foram significativamente inferiores aos dos EDTA_DNAs (Tabelas 1 e 2). A pureza do ADN representada pelo seu valor de rácio 260/280 é similar para ambos e conjunto de amostras P100_DNA EDTA_DNA (Tabelas 1 e 2). A qualidade do ADN é bastante uniforme dentro de cada conjunto de amostras, embora alguma degradação é visto nas amostras ED...

Watch this Scientific Journal Video about A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation at JoVE.com

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Estamos a descrever um novo método de isolamento de DNA genómico a partir de sangue total recolhido de plasma / sorologia. Após a coleta de plasma, o sangue compactado é geralmente descartado. O novo método represente uma melhoria significativa em relação aos métodos existentes e torna o ADN disponível e de plasma a partir de um único conjunto, sem solicitar sangue adicional.

Um método prático e inovador para extrair DNA genômico de Kits de coleta de sangue para Preservação Plasma Protein

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

a-practical-novel-method-to-extract-genomic-dna-from-blood-collection

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Biology

41.9K visualizações.  Emory University School of Medicine and Children's Health Care of Atlanta. Estamos a descrever um novo método de isolamento de DNA genómico a partir de sangue total recolhido de plasma / sorologia. Após a coleta de plasma, o sangue compactado é geralmente descartado. O novo método represente uma melhoria significativa em relação aos métodos existentes e torna o ADN disponível e de plasma a partir de um único conjunto, sem solicitar sangue adicional.

Vídeo: A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Isolation of Genomic DNA from Mouse Tails

Isolamento de DNA genômico de Tails rato

Biologia

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Coleta de sangue do caranguejo em ferradura americano, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extração de DNA genômico de alto peso molecular de solos e sedimentos

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Cromatografia de afinidade lipídica mediada por vesículas com separação de células Magnetic Ativado (LIMACS): um novo método para analisar proteínas lipídios Interação

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Genômica MRI - um recurso público para estudar os padrões de seqüência dentro de DNA genômico

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Captura selectivo de 5-hydroxymethylcytosine a partir de DNA genómico

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Um Guia Prático para Phylogenetics para não especialistas

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Colheita de sangue para testes repetidos de sangue em Zebrafish Adulto

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificação, sequenciamento de próxima geração, e Genomic DNA Mapeamento das retrovirais Integração Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...