Agradecemos Alan Tasker (USDA-APHIS, Riverdale, MD), Stephen Compton (Clemson), Sherry Aultman (Clemson), Craig Ramsey (USDA-APHIS, Fort Collins, CO), e Betty Marose (UMD) para a assistência na obtenção de amostras . Agradecemos aos alunos Andrew Adrian (UA-Huntsville) e Derek Thacker (UA-Huntsville) por sua assistência em testar este protocolo, e José Herdy por seu trabalho nas filmagens do vídeo. Este trabalho foi financiado pelo National Peixe e Wildlife Foundation.

<ol>
	<li>Holm, L. G., Pancho, J. V., Herberger, J. P., Plucknett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Geographical Atlas of World Weeds. , (1979).</li><li>CABI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crop+Protection+Compendium.">Crop Protection Compendium.</a> Commonwealth Agricultural Bureau International (CABI). , (2007).</li><li>MacDonald, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cogongrass+(Imperata+cylindrica)+-+Biology,+ecology,+and+management.">Cogongrass (Imperata cylindrica) - Biology, ecology, and management.</a> Critical Reviews in Plant Sciences. 23, 367-380 (2004).</li><li>Talley, S. M., Cseke, L. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shading+effects+on+growth+and+partitioning+of+plant+biomass+in+Cogongrass+(Imperata+cylindrica)+from+shaded+and+exposed+habitats.">Shading effects on growth and partitioning of plant biomass in Cogongrass (Imperata cylindrica) from shaded and exposed habitats.</a> Weed Science. 28, 735-740 (1980).</li><li>Cole, J. T., Cole, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ornamental+grass+growth+response+to+three+shade+intensities.">Ornamental grass growth response to three shade intensities.</a> Journal of Environmental Horticulture. 18, 18-22 (2000).</li><li>Bryson, C. T., Koger, C. H., Byrd, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+exposure+period+to+heat+on+cogongrass+(Imperata+cylindrica)+viability.">Effects of temperature and exposure period to heat on cogongrass (Imperata cylindrica) viability.</a> Weed Technology. 21, 141-144 (2007).</li><li>Capo-chichi, L. J. A., Faircloth, W. H., Williamson, A. G., Patterson, M. G., Miller, J. H., van Santen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+Dynamics+and+Genotypic+Diversity+of+Cogongrass+(Imperata+cylindrica)+at+the+Point+of+Introduction+in+the+Southeastern+United+States.">Invasion Dynamics and Genotypic Diversity of Cogongrass (Imperata cylindrica) at the Point of Introduction in the Southeastern United States.</a> Invasive Plant Science and Management. 1 (2), 133-141 (2008).</li><li>Talley, S. M., Ramsey, C. L., Zink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimentally+assessing+the+invasive+potential+of+plants.">Experimentally assessing the invasive potential of plants.</a> CPHST Laboratory 2009 Annual Report. , 29-30 (2009).</li><li>Hodkinson, T. R., Chase, M. W., Lled&#243;, M. D., Salamin, N., Renvoize, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetics+of+Miscanthus,+Saccharum+and+related+genera+(Saccharinae,+Andropogoneae,+Poaceae)+based+on+DNA+sequences+from+ITS+nuclear+ribosomal+DNA+and+plastid+trnLintron+and+trnL-F+intergenic+spacers.">Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers.</a> J. Plant Res. 115, 381-392 (2002).</li><li>Kress, W. J., Wurdack, K. J., Zimmer, E. A., Weigt, L. A., Janzen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+DNA+barcodes+to+identify+flowering+plants.">Use of DNA barcodes to identify flowering plants.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (23), 8369-8374 (2005).</li><li>Roodt-Wilding, R., Spies, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+relationships+in+southern+African+chloridoid+grasses+(Poaceae)+based+on+nuclear+and+chloroplast+sequence+data.">Phylogenetic relationships in southern African chloridoid grasses (Poaceae) based on nuclear and chloroplast sequence data.</a> Systematics and Biodiversity. 4, 401-415 (2006).</li><li>Brody, J. R., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+boric+acid:+a+Tris-free,+cooler+conductive+medium+for+DNA+electrophoresis.">Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.</a> BioTechniques. 36, 214-216 (2004).</li><li>Chou, C. -. H., Tsai, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+intergenic+spacer+of+ribosomal+DNA+of+Imperata+cylindrica+(L.)+Beauv.+var.+major+(Cogongrass)+populations+in+Taiwan.">Genetic variation in the intergenic spacer of ribosomal DNA of Imperata cylindrica (L.) Beauv. var. major (Cogongrass) populations in Taiwan.</a> Botanical Bulletin of Academia Sinica (Taipei). 40, 319-332 (1999).</li></ol>

Não temos nada a divulgar.

<html> O viveiro EUA e indústrias paisagem prosperar no cultivo e venda de espécies de plantas exóticas e romance. Isso, juntamente com a crescente globalização do comércio, aumenta as chances de que uma espécie de planta invasora entrarão, estabelecer e difundir os EUA em A capacidade de governo federal regular tais plantas depende de informações que muitas vezes não é disponível, incluindo o potencial para se tornar invasiva , taxonomia correta e distinção genética da taxa nativos e naturalizados. Porque o nosso conhecimento de plantas invasoras é muitas vezes limitado, plantas importadas, com características ocultas invasivas têm sido introduzidos voluntariamente para descobrir depois que eles invadem nossos recursos agrícolas e naturais. Este protocolo visa resolver esses problemas associados I. variedades cylindrica, proporcionando o primeiro método simplificado molecular que pode distinguir com precisão entre tipo selvagem cogongrass e reverteu a forma da sua contraparte JBG ornamentais. &gt; jove_content &quot;Para o desenvolvimento deste protocolo, tipo selvagem cogongrass foram coletados a partir de populações naturalizadas na Unidade Florestal Pond Creek, em Santa Rosa County perto de Jay, FL, em junho de 2008. JBG foi obtido a partir de um viveiro comercial (Bluebird Nursery, Inc ..) em junho de 2008, bem como da coleção de um proprietário em Columbia, MO JBG reverte foram obtidos a partir do pátio do Museu Campbell Geológica na Universidade de Clemson, SC, em junho de 2008; da Universidade Park, em Riverdale, MA, em junho de 2009; a partir da frente estaleiro de um proprietário em Columbia, MO em 2009 (identificados por Leland Cseke). Todas as plantas foram mantidas em uma estufa localizado na Universidade do Alabama em Huntsville (Huntsville, AL). A sequência genética de DNA recolhidos a partir destas plantas incluiu o em comparações profundidade de 9 regiões de DNA independentes comumente usados ​​para plantas de código de barras 2. Em todos os casos, as sequências de JBG eram um fósforo de 100% para os do JBG reverter, ajudando assim averificar que JBG, de fato, reverter para um formulário, verde invasivo. Apenas o ITS nuclear e os cloroplastos trnL F-regiões têm diferenças que podem ser utilizados para distinguir entre geneticamente cogongrass e JBG. A região ITS tem um total de 3 SNPs (polimorfismos de nucleotídeo único) entre cogongrass e JBG, enquanto a região trnL F-tem 2 SNPs e 2 indels (inserções e deleções). Estas diferenças genéticas permitiu variedade primers específicos de PCR para ser desenvolvido que pode distinguir entre tipo selvagem cogongrass e JBG reverte. Os resultados mais fiáveis ​​ter vindo de iniciadores derivados da região trnL F-plastidial. Assim, este protocolo é baseada nas diferenças de sequência entre as regiões F-trnL do genoma do cloroplasto de cogongrass, JBG e reverteu JBG (Figura 4). Para ajudar a gerar primers que são mais específicos para as variedades em questão e para evitar falsos positivos de espécies estreitamente relacionadas, all conhecidos trnL F-seqüências de I. variedades cylindrica foram comparados com trnL-F seqüências de espécies de gramíneas relacionados (43 seqüências independentes de 29 espécies, por exemplo, Cymbopogon citratus, Sorghastrum incompletum e Coix lacryma-jobi sinensis Miscanthus, Saccharum officinarum, Sorghum halepense). Embora, examinámos sequências variedade de iniciadores específicos através 29 espécies de relva, a especificidade dos iniciadores específicos variedade não tenha sido exaustivamente examinados para a sua capacidade para amplificar DNA da maioria das outras espécies de gramíneas. Consequentemente, o tecido usado para extração de DNA devem ser cuidadosamente identificados como I. cylindrica antes de iniciar este protocolo. Se a grama não pode ser identificada como a cogongrass ou JBG, então nós sugerimos o seqüenciamento do produto de PCR para se certificar de que as seqüências são uma correspondência exata para cogongrass ou JBG. Actualmente, o método mais preciso para verificar a identidade de um espécime grama dada é a realização de PCR em ambos os trnl-F e das suas regiões, seguido por verificação sequência de produtos de PCR e comparação das sequências para sequências conhecidas de taxa identificado com precisão. O ADN pode ser amplificado utilizando iniciadores de controlo descritos no presente protocolo (para a região trnL-F) ou iniciadores outros disponíveis em outras publicações 13-15. A sequenciação é muito mais trabalho e dispendiosa do que usar o nosso procedimento simplificado. A qualidade dos iniciadores utilizados para a PCR é crítica para o sucesso do procedimento. Fizemos os iniciadores para esse procedimento disponível a partir de Bio oblíqua, Inc. ( <a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> , Huntsville, AL). A vantagem de ordenação dos iniciadores a partir de Bio oblíqua é que eles armazenar grandes números de alíquotas de cada iniciador a partir da série idêntica de amostra de produção como os iniciadores foram utilizados para a optimização no nosso laboratório. Consequentemente, não só primers têm a mesma sequência, mas they vêm do lote de produção exatamente o mesmo que foi usado neste protocolo. Usando iniciadores a partir do mesmo lote, pode ajudar a evitar variáveis ​​estranhas no procedimento que pode resultar de diferenças na qualidade dos iniciadores de PCR. Da mesma forma, enquanto outras polimerases Taq deve funcionar bem para PCR, a qualidade da polimerase Taq usado terá um impacto sobre a qualidade dos resultados de PCR. Para permitir uma melhor consistência na PCR reagentes, temos otimizado o protocolo utilizando reagentes de Clontech. A vantagem 2 Polimerase (Clontech, Mountain View, CA, Cat. # 639201 ou 639202) é uma mistura de um sólido, a polimerase Taq de arranque a quente e uma enzima prova de leitura de que ajuda a proporcionar alta especificidade e ampliações mais precisos. Como este protocolo baseia-DNA do cloroplasto, que é herdada da mãe em gramíneas, eventos de hibridação entre genótipos JBG cogongrass e não pode ser capturado com o nosso processo de identificação molecular. Nos casos em que é hypridization suspeCTED, recomendamos o uso de regiões nucleares que são herdadas de ambos os pais. O mais comumente utilizado região não plastid variável a considerar na genotipagem das plantas é a região ITS nuclear ribossomal 13-15,17. Actualmente, estão a avançar em direcção a multiplexação a amplificação do cloroplasto região trnL-F com a da região ITS nuclear no tubo de PCR mesmo. Multiplexação plastid com regiões do DNA nuclear seria potencialmente contornar as limitações do uso sozinho, no entanto, estes métodos requerem otimização e avaliação adicional para determinar a viabilidade caso a caso. O uso de métodos quantitativos PCR em tempo real (qPCR) e novas tecnologias, tais como faróis molecular (sondas fluorescentes primers), também estão sendo avaliados como métodos de genotipagem não-seguras e precisas de plantas. O protocolo apresentado aqui proporciona uma abordagem rápida e fiável para distinguir o JBG reverter da de tipo selvagem cogongrass. Encorajamos a utilizaçãors deste protocolo em contactar-nos para relatar os resultados derivados do uso deste protocolo. Tal informação compartilhada ajudará a fornecer informações sobre a distribuição de JBG reverte. Isso também irá ajudar os reguladores no USDA tomar decisões informadas sobre as ações que podem ser necessárias para contornar a propagação e hibridação potencial das variedades altamente invasivos cogongrass.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DNeasy Usina Mini Kit </td><td> Qiagen, Valencia, CA </td><td> 69104 ou 69106 </td><td> Qualquer respeitável genômica kit DNA da planta que produz DNA de boa qualidade deve funcionar bem para estes procedimentos. </td></tr><tr><td> trnF (GAA)-F </td><td> Oblique Bio, Inc. </td><td> 3-0578 </td><td> Cartilha controle positivo para a frente </td></tr><tr><td> trnL (5 &#39;do exão)-C </td><td> Oblique Bio, Inc. </td><td> 3-0579 </td><td> Reverter cartilha controle positivo </td></tr><tr><td> trnLF-C-F1 </td><td> Oblique Bio, Inc. </td><td> 3-0864 </td><td> Atacante do tipo selvagem cartilha cogongrass </td></tr><tr><td> trnLF-C-R1 </td><td> Oblique Bio, Inc. </td><td> 3-0865 </td><td> Revertertipo selvagem cartilha cogongrass </td></tr><tr><td> trnLF-R-F2 </td><td> Oblique Bio, Inc. </td><td> 3-0866 </td><td> Encaminhar JBG e JBG reverter cartilha </td></tr><tr><td> trnLF-R-R2 </td><td> Oblique Bio, Inc. </td><td> 3-0867 </td><td> Reverter JBG e JBG reverter cartilha </td></tr><tr><td> Vantagem UltraPure PCR dNTP Mix </td><td> Clontech, Mountain View, CA </td><td> 639125 </td><td></td></tr><tr><td> Polimerase vantagem de 2 </td><td> Clontech, Mountain View, CA </td><td> 639201 ou 639202 </td><td> Uma boa leitura de prova, hot-start Taq polimerase </td></tr></tbody></table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

De tipo selvagem I. cylindrica (cogongrass) é um dos melhores piores dez plantas invasoras do mundo, impactando negativamente os recursos agrícolas e naturais em 73 países diferentes na África, Ásia, Europa, Nova Zelândia, da Oceania e das Américas 1-2. Cogongrass forma rápida propagação, estandes monodominantes que deslocam uma grande variedade de espécies de plantas nativas e, por sua vez ameaçam os animais nativos que dependem dos deslocados espécies de plantas nativas para forragem e abrigo. Para aumentar o problema, uma variedade ornamental [I. cylindrica var. koenigii (Retzius)] é amplamente comercializado sob os nomes de &#39;Rubra&#39; Imperata cylindrica, Barão Vermelho, e grama sangue japonês (JBG). Esta variedade é supostamente estéril e não-invasivo e é considerado um ornamental desejável para suas folhas de cor vermelha. No entanto, nas condições corretas, JBG pode produzir sementes viáveis ​​(Carol Holko, 2009 comunicação pessoal) e pode reverter para um verde iforma nvasive que muitas vezes é indistinguível da cogongrass à medida que assume as características distintivas da variedade de tipo selvagem invasiva 4 (Figura 1). Isto torna a identificação usando morfologia uma tarefa difícil mesmo para taxonomistas bem treinados plantas. Reversão de JBG a um fenótipo agressivo verde também não é uma ocorrência rara. Usando a comparação de seqüências de codificação e regiões variáveis ​​do DNA nuclear e do cloroplasto tanto, temos confirmado que JBG foi revertido para o invasivo verde dentro dos estados de Maryland, Carolina do Sul e Missouri. JBG foi vendido e plantada no estado em quase todos os os EUA continental, onde não há uma infestação cogongrass ativo. A extensão do problema em reverter não é bem compreendido porque as plantas são revertidos em situação irregular e, muitas vezes destruída. A aplicação deste protocolo molecular fornece um método para identificar JBG reverte e pode ajudar a manter estas variedades de co-ocorrência de umnd possivelmente hibridação. Cogongrass é um outcrosser obrigatório e, quando cruzou com um genótipo diferente, pode produzir vento dispersa sementes viáveis ​​que se espalham sobre grandes distâncias cogongrass 5-7. JBG tem um genótipo ligeiramente diferente do que cogongrass e pode ser capaz de formar híbridos viáveis ​​com cogongrass. Para aumentar o problema, JBG é mais frio e mais tolerantes do que cogongrass 8-10, eo fluxo gênico entre estas duas variedades é susceptível de gerar híbridos que são mais agressivas, tolerantes, e résistente frio do que do tipo selvagem cogongrass. Enquanto tipo selvagem cogongrass atualmente infesta mais de 490 milhões de hectares no mundo todo, os EUA Sudeste que infesta mais de 500.000 hectares e é capaz de ocupar mais de os EUA como se espalha rapidamente para o norte devido ao seu nicho amplo e geográfica 3,7,11 potencial. O potencial de um cruzamento genético é uma preocupação séria para o USDA-APHIS Programa Semana Federal nocivas. Atualmente, o USDA-APHIS proíbe JBG em estados where há infestações maiores cogongrass (por exemplo, Flórida, Alabama, Mississippi). No entanto, impedindo que as duas variedades da combinação pode ser mais difícil como cogongrass e JBG expandir suas distribuições. Além disso, a distribuição do JBG reverter é actualmente conhecida e sem a capacidade de identificar esses variedades por morfologia, alguns infestações cogongrass pode ser o resultado de JBG reverte. Infelizmente, os actuais métodos de identificação molecular de dependem normalmente AFLP (Polimorfismos comprimento de fragmentos amplificados) e sequenciação de ADN, ambos os quais são demorados e dispendiosos. Aqui, nós apresentamos o primeiro rentável e confiável baseada em PCR método de genotipagem molecular para distinguir com precisão entre cogongrass e JBG reverter.

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Nós fornecer um protocolo de genotipagem custo-eficácia e rápida molecular que emprega iniciadores variedade-PCR específicos que as diferenças de sequência alvo de ADN dentro da região espaçadora cloroplasto trnL-F para diferenciar entre as variedades de<em&gt; Imperata cylindrica</em&gt; (Cogongrass) que não podem ser distinguidos pela morfologia sozinho. Estas variedades incluem a erva federal listado nocivo, cogongrass e estreitamente relacionada, a variedade difundida ornamental, eu<em&gt;. cylindrica</em&gt; Var.<em&gt; Koenigii</em&gt; (Grama sangue japonês).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

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Research

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Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

31.5K visualizações.  The University of Alabama, Huntsville. Nós fornecer um protocolo de genotipagem custo-eficácia e rápida molecular que emprega iniciadores variedade-PCR específicos que as diferenças de sequência alvo de ADN dentro da região espaçadora cloroplasto trnL-F para diferenciar entre as variedades de Imperata cylindrica (Cogongrass) que não podem ser distinguidos pela morfologia sozinho. Estas variedades incluem a erva federal listado nocivo, cogongrass e estreitamente relacionada, a variedade ...

Vídeo: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Um método simples para genotipagem Accurate

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biologia

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identificar os efeitos das mutações BRCA1 em Recombinação Homóloga usando células que expressam BRCA1 tipo selvagem endógena

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Protein Assay Overlay membrana: um protocolo para testar Interação entre proteínas solúveis e insolúveis In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Detecção otimizada baseados em PCR de Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice Cap: Um Método para Crescer Arabidopsis Plantas e tomate em placas de 96 poços para High-Throughput Genotipagem

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

Genotipagem de amostras de plantas e animais, sem purificação DNA Antes

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium Ensaio para a grande escala Applications genotipagem

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análise qPCRTag - A High Throughput, PCR em Tempo Real Ensaio para Sc2.0 Genotipagem

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinação de ácidos sialic em amostras de fígado e leite de tipo selvagem e<em&gt; CMAH</em&gt; Ratinhos de eliminação.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Novo método de entrega de DNA do plasmídeo para Mouse bexiga Urotélio por eletroporação

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica

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