Este estudo foi financiado em parte pelo Instituto Nacional de Saúde (GM071440 a CH e NS051630/MH076090/MH078972 para PJ).

1. A fragmentação de ADN genómico Fragmento de ADN genómico utilizando sonicação de uma gama de tamanhos desejada adequada para a plataforma de sequenciamento do genoma. (Nós normalmente sonicar a ~ 300 pb.) Verificar a distribuição do tamanho do ADN genómico fragmentado em 1% de gel de agarose (Figura 1). 2. Preparação de ADN Determinar as quantidades de ADN a partir baseadas na abundância de 5 HMC em ADN genómico. Desde 5-HMC níveis variam significativamente em diferentes tipos de tecidos, quantidades de DNA a partir dependem dos níveis de 5-HMC das amostras. Por favor, consulte a Tabela 1 para exemplos. 3. β-GT reacção catalisada (reacção de transferência de Glicose) <ol><li> Misturar por pipetagem da mistura, como detalhado na Tabela 2 e incuba-se em um banho de água a 37 ° C durante 1 h. </li><li> Após a incubação, limpar-se a mistura reaccional com remoção Kit QIAquick Nucleotide, utilizando 10 jig de DNA porcoluna. Eluir com 30 ul de água por coluna e combinar. </li></ol> 4. Reação Biotinilação (Clique Química) <ol><li> Adicionar DBCO-SS-PEG3-biotina conjugado de solução de trabalho (1 mM) na solução de ADN eluído (do passo 3) para uma concentração final de 150 uM (ou seja, 5 uL de solução de trabalho por 30 ul de solução de ADN). </li><li> Misturar por pipetagem, e incubar em banho de água a 37 ° C durante 2 horas. </li><li> Limpe a reação com QIAquick Removal Kit Nucleotide. O volume de eluição total ideal é de 100 uL. </li><li> Quantificar a quantidade de DNA recuperado usando microlitro espectrofotômetro escala (por exemplo., NanoDrop). </li></ol> 5. Captura de 5 HMC contendo DNA <ol><li> Lave 50 ul de Dynabeads MyOne Estreptavidina C1 3 vezes com 1 ml de 1X tampão B &amp; W de acordo com as instruções do fabricante. Separa-se as pérolas com um suporte magnético. </li><li> Adicionar um volume igual de 2X tampão B &amp; W para o D recuperado biotiniladoNA (100 ul) às pérolas lavadas. </li><li> Incubar durante 15 min à temperatura ambiente com rotação suave num agitador rotativo. </li><li> Separa-se as pérolas com um suporte magnético e lavar as pérolas três vezes com 1 ml de 1X tampão B &amp; W. </li><li> Eluir o DNA através da incubação as pérolas em 100 ul de DTT mM preparada de fresco 50 durante 2 h à temperatura ambiente, com rotação suave num agitador rotativo. </li><li> Separa-se as pérolas com um suporte magnético. Aspirar o eluente e uma carga para Micro Bio-Spin 6 Coluna de acordo com a instrução de fabrico para remover o DTT. O ADN alvo é a solução agora. </li><li> Purifica-se o DNA eluído a partir do passo anterior, Qiagen Purification Kit MinElute PCR DNA e elui-se em 10 ul de tampão EB. Quantificar DNA usando Fluorometer Qubit ou NanoDrop se a concentração é superior a 20 ng / ul. O DNA está pronto para jusante preparação biblioteca genômica sequenciação. </li></ol> 6. Resultados representativos Se a qualidade de of DNA genómico é elevada, o rendimento de recuperação típicos após a β-GT e reacções de biotinilação são ~ 60-70%. No entanto, a eficiência da captação variar significativamente com os tipos de tecidos diferentes, dependendo dos níveis de 5-HMC das amostras. Normalmente, a eficiência da captação de DNA genómico cérebro é ~% 4-9, e, em alguns casos extremos, a eficiência pode atingir até 12%. No caso de células ES, a eficiência da captação média é de aproximadamente 2-4%, em contraste com a ~ 0,5% para as células estaminais neurais. A menor eficiência visto até agora foi para o DNA genômico de células cancerosas. Todos DNA enriquecido está pronto para o padrão da próxima geração de protocolos de preparação de bibliotecas. Além disso, o ADN capturado pode também ser usado como molde para a PCR em tempo real para detectar o enriquecimento de alguns fragmentos em comparação com o ADN de entrada, se os iniciadores relacionados estão disponíveis. <img alt="Figura 1" src="/files/ftp_upload/4441/4441fig1.jpg" /> Figura 1. Sonicados fragmentos genómicos humanos de DNA em1% de gel de agarose. De 10 ug de DNA genómico isolado a partir de células humanas do iPS em 120 ul de tampão TE 1X foi sonicada usando um dispositivo de ultra-sons (Covaris). Após sonicação, 2 ul do DNA sonicado foi carregada num gel de agarose a 1% utilizando 100 pb do marcador de DNA para comparar os tamanhos dos fragmentos de DNA sonicados. <table border="1"><tbody><tr><td> Componente </td><td> Volume </td><td> Concentração Final </td></tr><tr><td> Água </td><td> _ Ul </td><td></td></tr><tr><td> 10 X tampão de reacção β-GT </td><td> 2 ul </td><td> 1 X </td></tr><tr><td> Até 10 ug de ADN genómico </td><td> _ Ul </td><td> Até 500 ng / uL </td></tr><tr><td> UDP-N-6, 3-Glc (3 mM) </td><td> 0,67 ul </td><td> 100 uM </td></tr><tr><td> β-GT (40 uM) </td><td> 1 ul </td><td> 2 uM </td></tr><tr><td> Volume total </td><td> 20 ul </td&gt; <td></td></tr></tbody></table> i) Para o DNA genômico de tecidos (conteúdo 5 HMC alta&gt; 0,1%) <table border="1"><tbody><tr><td> Componente </td><td> Volume </td><td> Concentração Final </td></tr><tr><td> Água </td><td> _ Ul </td><td></td></tr><tr><td> 10 X tampão de reacção β-GT </td><td> 10 ul </td><td> 1 X </td></tr><tr><td> Até 20 ug de ADN genómico </td><td> _ Ul </td><td> Até 500 ng / uL </td></tr><tr><td> UDP-6-N3-Glc (3 mM) </td><td> Ul 1,33 </td><td> 100 uM </td></tr><tr><td> β-GT (40 uM) </td><td> 2 ul </td><td> 2 uM </td></tr><tr><td> Volume total </td><td> 40 ul </td><td></td></tr></tbody></table> ii) Para o ADN genómico de células estaminais (mediana 5 HMC conteúdo ~ 0,05%) <table border="1"><tbody><tr><td> Componente </td><td> Volume </td><td> Concentração Final </td></tr><tr><td> Água </td><td> _ Ul </td><td></td></tr><tr><td> 10 X tampão de reacção β-GT </td><td> 10 ul </td><td> 1 X </td></tr><tr><td> Até 50 ug de ADN genómico </td><td> _ Ul </td><td> Até 500 ng / uL </td></tr><tr><td> UDP-6-N3-Glc (3 mM) </td><td> 3,33 ul </td><td> 100 uM </td></tr><tr><td> β-GT (40 uM) </td><td> 5 ul </td><td> 2 uM </td></tr><tr><td> Volume total </td><td> 100 ul </td><td></td></tr></tbody></table> iii) Para o câncer de DNA genômico de células (conteúdo 5 HMC baixo ~ 0,01%) Tabela 1. Exemplos de quantidades de ADN de entrada e de reacções de etiquetagem utilizando as amostras com diferentes níveis de HMC-5 através do método selectivo química rotulagem. <table border="1"><tbody><tr><td> Amostra </td><td> 5 HMC nível </strong&gt; </td><td> ADN de partida (mg) </td><td> A recuperação após a etiquetagem (entrada de esferas) (ig) </td><td> Rendimento de recuperação </td><td> Pull-down DNA (ng) </td><td> Puxe para baixo rendimento </td></tr><tr><td> Cerebelo do rato adulto </td><td> 0,4% </td><td> 10 </td><td> 7,5 </td><td> 75% </td><td> 236 </td><td> 3,1% </td></tr><tr><td> Pós-natal do rato 7 dias cerebelo </td><td> 0,1% </td><td> 11 </td><td> 9 </td><td> 82% </td><td> 140 </td><td> 1,6% </td></tr><tr><td> Rato de células ES E14 </td><td> 0,05% </td><td> 60 </td><td> 42 </td><td> 70% </td><td> 350 </td><td> 0,8% </td></tr></tbody></table> Tabela 2. Resultados representativos a partir de tecidos e células do cérebro de rato ES.

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5-hydroxymethylcytosine (5 HMC) é uma modificação do presente recentemente identificado epigenética em quantidades substanciais em certos tipos de células de mamíferos. O método aqui apresentado é para determinar a distribuição de todo o genoma de 5 HMC. Usamos bacteriófago T4 β-glicosiltransferase para transferir uma porção glucose engenharia contendo um grupo azida para o grupo hidroxilo de 5 HMC. O grupo azida pode ser quimicamente modificados com biotina para a detecção, o enriquecimento por afinidade, e sequenciamento de fragmentos de 5 HMC contendo ADN em genomas de mamíferos. Este processo tem vantagens sobre 5 HMC DNA imunoprecipitação com anticorpos hidroximetilada (hMeDIP-Seq) 13-15, embora o hMeDIP é simples, que tem uma forte tendência para a alta densidade de 5-HMC regiões e geralmente dá resultados inconsistentes de puxar independente -downs. Nosso método não introduzir viés. Há, contudo, dois problemas possíveis em termos de rotulagem e a capturar meCTO. A primeira preocupação seria a especificidade da marcação e de captura. Uma vez que um estudo recente indicou que o 5 hydroxymethyluracil-(5-HMU) também pode servir como um substrato de β-GT, sinais falsos positivos podem ser introduzidos, levando a fragmentos enriquecidos, que são não-específica para 5 HMC-relação 16. Esta preocupação pode ser infundadas, embora, desde que altamente activos de 5-hydroxymethyluracil ADN-glicosilase são constantemente removendo este dano no DNA, resultando em essencialmente indetectável 5-HMU no genoma dos mamíferos 17,18. A segunda preocupação é a eficiência da rotulagem e da captura. Uma vez que os níveis de 5-HMC variar significativamente em diferentes tecidos e células, variando entre 0,5% em tecidos de cérebro de 0,05% em células de MES e de 0,01% para as células cancerosas, é essencial que os nossos métodos são aplicáveis ​​a todos estes tecidos em termos de a jusante aplicações do ADN marcado e capturado genómico. A nossa experiência mostra que os métodos descritos aqui são, de facto sensível e específicosuficiente para analisar todos estes tecidos para o propósito de comparação, quer a quantificação baseado de 5-HMC níveis entre os tecidos ou para aplicações a jusante, tais como a sequenciação de profundidade para mapear a distribuição de 5 HMC no genoma 19-21. A chave para o nosso método é a utilização de uma quantidade apropriada de partida de ADN genómico. Se a concentração de ADN genómico é muito baixa, a eficiência e especificidade de rotulagem diminuirá na mesma proporção. Com base na nossa experiência, apesar da abundância de 5 HMC é suficientemente elevada no DNA a partir de tecidos do cérebro, se a concentração é inferior a 25 ng / ul em 20 ul de reacção, a etiquetagem e a eficiência de captura, bem como a especificidade, será significativamente reduzido. Para as amostras de baixa abundância de ADN, para além do requisito de concentração, a quantidade total de ADN é essencial para obter elevada e eficiência de captura específico. Assim, embora se necessita apenas de 25 ng de fragmentos de 5 HMC enriquecidas de DNA para o st jusanteprotocolos de biblioteca andard geração empregadas pelas plataformas da próxima geração de sequenciação, a quantidade inicial de ADN genómico a partir de tecidos cerebrais não deve ser inferior a 2 ug (e a quantidade ideal de partida é ug 5-10). Através de tentativa e erro, temos aperfeiçoado o montante inicial de DNA genômico a partir de tecidos diferentes, com variáveis ​​5-HMC abundâncias para obter eficiência de marcação e especificidade, como detalhado na Tabela 2. Em resumo, nosso método é qualificado para rotular precisamente genômica 5 HMC e especificamente capturar os fragmentos 5 HMC contendo, tornando-se um protocolo bem sucedido em termos de sensibilidade e especificidade para a jusante ensaios de seqüenciamento da próxima geração.

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Catalogo </td><td> Comentário </td></tr><tr><td></td><td></td><td></td><td> Reagentes </td></tr><tr><td> O cloreto de sódio 5 M (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> PH 8,0 0,5 M de ácido etilenodiaminotetracético (EDTA) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma base (Tris) pH 7,5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1M, pH 7,4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> Cloreto de magnésio (MgCl2) 1M </td><td> Ambion </td><td> AM9530g </td><td></td></tr><tr><td> Dimetil-sulfóxido (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Tween 20 </td><td> Fisher biorreagentes </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3-biotina conjugado </td><td> Clique em Ferramentas Química </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-Ditiotreitol, ultrapura (TDT) suprapuro </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick Removal Kit Nucleotide </td><td> Qiagen </td><td> 28304 </td><td></td></tr><tr><td> Micro Bio-Spin 6 Coluna </td><td> Bio-Rad </td><td> 732-6222 </td><td></td></tr><tr><td> Dynabeads MyOne </td><td> Invitrogen </td><td> 650-01 </td><td></td></tr><tr><td> Estreptavidina C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen MinElute PCR Purification Kit </td><td> Qiagen </td><td> 28004 </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-glucose </td><td> Motivo ativa </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Enzima </td></tr><tr><td> β-glicosiltransferase (β-GT) </td><td> New England Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> Dispositivo sonicação </td><td> Covaris </td><td></td><td></td></tr><tr><td> Centrífuga de mesa </td><td></td><td></td><td></td></tr><tr><td> Banho de água </td><td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Aparelho de gel de corrida </td><td> Bio-Rad </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Labquake Tubo Shaker </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tubo Shaker </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> Suporte de Separação Magnética </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> Qubit 2,0 Fluorometer </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> Configuração de reagente 10 X Reacção β-GT tampão (500 mM de HEPES pH 7,9, 250 mM de MgCl 2) 2 X de ligação e de lavagem (B &amp; W) de tampão (10 mM Tris pH 7,5, EDTA 1 mM, NaCl 2 M, Tween a 0,02% 20) . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-metilcitosina (5-mC) constitui ~ 2-8% das citosinas totais no DNA genómico humano e um impacto de uma ampla gama de funções biológicas, incluindo a expressão de genes, a manutenção da integridade do genoma, a impressão parental, inactivação do cromossoma X, a regulação dos desenvolvimento, envelhecimento, cancro e 1. Recentemente, na presença de um oxidado 5-mC, 5-hydroxymethylcytosine (5 HMC), foi descoberto em células de mamíferos, em particular no estaminais embrionárias (ES) e células neuronais 2-4. 5 HMC é gerado por oxidação de 5-mC catalisada por TET família de ferro (II) / α-cetoglutarato-dependente dioxigenases 2, 3. 5 HMC é proposto para ser envolvido na manutenção de células estaminais embrionárias (MES), a hematopoiese normal e doenças malignas, e o desenvolvimento do zigoto 2, 5-10. Para melhor compreender a função da 5-HMC, um sistema de sequenciação fiável e simples é essencial. Seqüenciamento bissulfito tradicional não pode distinguir 5 HMC de 5-MC 11 </sup&gt;. Para desvendar a biologia de 5 HMC, temos desenvolvido um método químico altamente eficaz e selectivo para etiquetar e capturar 5 HMC, aproveitando-se de uma enzima de bacteriófago que adiciona uma porção de glucose a 5 HMC especificamente 12. Aqui descrevemos um procedimento em duas etapas simples para rotulagem química seletiva de 5-HMC. No passo de rotulagem em primeiro lugar, 5 HMC em ADN genómico é marcado com um catalisada 6-azida-glucose por β-GT, uma glicosiltransferase de bacteriófago T4, de uma maneira que transmite a 6-azida-glucose a 5 HMC do cofactor modificado, UDP-6-N3-Glc (6-N3UDPG). No segundo passo, biotinilação, de um ligante de biotina de dissulfureto é ligado ao grupo azida por química do clique. Ambos os passos são altamente específicos e eficiente, levando à completa marcação, independentemente da abundância de 5 HMC em regiões genómicas e dando de fundo extremamente baixos. Seguindo a biotinilação de 5 HMC, os fragmentos de 5 HMC contendo ADN são então selectivamente capturadautilizando contas de estreptavidina de um modo independente de densidade. Os fragmentos resultantes de 5 HMC enriquecidas de DNA pode ser utilizado para as análises a jusante, incluindo a última geração de sequenciação. Nossa marcação selectiva e protocolo de captura confere alta sensibilidade, aplicável a qualquer fonte de DNA genômico com variáveis ​​/ diverso 5 HMC-abundâncias. Embora o objetivo principal deste protocolo é a sua aplicação a jusante (ou seja., Sequenciamento de última geração para mapear a distribuição de 5 HMC no genoma), é compatível com molécula única, em tempo real SMRT (DNA) de seqüenciamento, que é capaz de fornecer uma única base de sequenciação de resolução de 5 HMC.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Descrita é um processo de rotulagem em duas etapas usando β-glicosiltransferase (β-GT) para transferir uma azida de glucose para 5 HMC, seguido por química do clique para transferir de um ligante de biotina para o enriquecimento fácil e densidade independente. Este método de marcação eficiente e específico permite o enriquecimento de 5 HMC com fundo extremamente baixo e de alto rendimento de mapeamento epigenômico via sequenciamento de última geração.

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 ...

selective-capture-of-5-hydroxymethylcytosine-from-genomic-dna

Nanyang Technological University

Research

JoVE Journal

Biology

11.9K visualizações.  Emory University School of Medicine. Descrita é um processo de rotulagem em duas etapas usando β-glicosiltransferase (β-GT) para transferir uma azida de glucose para 5 HMC, seguido por química do clique para transferir de um ligante de biotina para o enriquecimento fácil e densidade independente. Este método de marcação eficiente e específico permite o enriquecimento de 5 HMC com fundo extremamente baixo e de alto rendimento de mapeamento epigenômico via sequenciamento de última geração.

Vídeo: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Isolation of Genomic DNA from Mouse Tails

Isolamento de DNA genômico de Tails rato

Biologia

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Grandes Inserir Produção Ambiental Biblioteca Genômica

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

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 ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

Captura Primer Extensão: Recuperação Sequence alvejado a partir de Fontes de DNA altamente degradadas

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Geração de RNA / DNA híbrido em DNA genômico por transformação por meio de RNA contendo Oligonucleotídeos

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

Purificação simplificada do DNA plasmidial das culturas Procarióticas

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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 ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitiva Screens Genomic de Bibliotecas Yeast Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Extração de DNA de alto peso molecular de Esteiras Microbianas

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

Detecção de variantes raras genômico de Seqüenciamento combinados usando SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...