Damos las gracias a Alan Tasker (USDA-APHIS, Riverdale, MD), Stephen Compton (Clemson), Sherry Aultman (Clemson), Craig Ramsey (USDA-APHIS, Fort Collins, CO), y Betty Marose (UMD) para la asistencia en la obtención de muestras . Damos las gracias a los estudiantes Andrew Adrian (UA-Huntsville) y Derek Thacker (UA-Huntsville) por su ayuda en las pruebas de este protocolo, y Herdy José por su trabajo en la filmación del video. Este trabajo fue financiado por la National Fish and 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. J., 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=Molecular+diagnostic+technologies+for+invasive+plants.">Molecular diagnostic technologies for invasive plants.</a> CPHST Fort Collins Laboratory 2009 Annual Report. , 27-28 (2009).</li><li>Dozier, H., Gaffney, J. F., McDonald, S. -. K., Johnson, E. R. R. L., Shilling, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cogongrass+in+the+United+States:+History,+ecology,+impacts,+and+management.">Cogongrass in the United States: History, ecology, impacts, and management.</a> Weed Technology. 12, 737-743 (1998).</li><li>Chikoye, D., Ekeleme, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weed+flora+and+soil+seedbanks+in+fields+dominated+by+Imperata+cylindrica+in+the+moist+savannah+of+West+Africa.">Weed flora and soil seedbanks in fields dominated by Imperata cylindrica in the moist savannah of West Africa.</a> Weed Research. 41, 475-490 (2001).</li><li>Weber, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Invasive Plant Species of the World: A Reference Guide to Environmental Weeds. , (2003).</li><li>Patterson, D. 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>

No tenemos nada que revelar.

<html> El vivero de EE.UU. y las industrias del paisaje prosperan en el cultivo y venta de especies de plantas exóticas y la novela. Esto, junto con la creciente globalización del comercio, aumenta las posibilidades de que las especies de plantas invasoras entran, establecer y difundir en los EE.UU. La posibilidad de regular por el gobierno federal tales plantas se basa en la información que a menudo no es disponible, incluyendo el potencial de convertirse en invasoras , la taxonomía correcta, y la distinción genética de taxones nativos y naturalizados. Debido a que nuestro conocimiento de las plantas invasoras es a menudo limitada, las plantas importadas con ocultos características invasoras se han introducido voluntariamente sólo para descubrir después que invaden nuestros recursos agrícolas y naturales. Este protocolo tiene como objetivo hacer frente a este tipo de problemas asociados con la I. cylindrica variedades, proporcionando el primer método simplificado molecular que puede distinguir exactamente entre la naturaleza y tipo de plantas de cogongrass tengan y la forma de revertir de su contraparte JBG ornamentales. jove_content &quot;&gt; Para el desarrollo de este protocolo, de tipo salvaje plantas de cogongrass tengan se recogió a partir de poblaciones naturalizadas de la Unidad de Pond Creek Forestal en el Condado de Santa Rosa cerca de Jay, Florida, en junio de 2008. JBG fue adquirido en un vivero comercial (Bluebird Nursery, Inc. ..) en junio de 2008, así como de la colección de propietario de una casa en Columbia, MO JBG vuelve se obtuvieron desde el patio del Museo Campbell Geológica de la Universidad de Clemson, Carolina del Sur en junio de 2008, de la Universidad Park en Riverdale, MA en junio de 2009, y en el patio delantero de un dueño de una casa en Columbia, MO en 2009 (identificados por Leland Cseke). Todas las plantas se mantuvieron en un invernadero ubicado en la Universidad de Alabama en Huntsville (Huntsville, AL). La secuenciación genética de ADN recogidas en esas plantas incluidas en la comparación de la profundidad de 9 regiones de ADN independientes, comúnmente utilizados para 2 plantas de código de barras. En todos los casos, las secuencias de JBG eran un partido 100% a las de la JBG revertir, ayudando así averificar que en efecto, la JBG revertir a una forma verde, invasivo. Sólo el ITS nuclear y las regiones del cloroplasto F trnL-tienen diferencias que pueden ser utilizados para distinguir entre plantas de cogongrass tengan genéticamente y JBG. La región ITS cuenta con un total de 3 SNPs (polimorfismos de nucleótido único) entre plantas de cogongrass tengan y la JBG, mientras que la región trnL-F tiene dos SNPs y indeles 2 (inserciones y deleciones). Estas diferencias genéticas específicas permite gran variedad-PCR a desarrollar que puede distinguir entre la naturaleza y tipo de plantas de cogongrass tengan y vuelve JBG. Los resultados más fiables provienen de primers derivados de la plastidio trnL-F región. Por lo tanto, este protocolo se basa en la secuencia de las diferencias entre las regiones trnL-F del genoma del cloroplasto de plantas de cogongrass tengan, JBG y revertidas JBG (Figura 4). Para ayudar a generar cebadores que son más específicos para las variedades en cuestión y para ayudar a evitar los falsos positivos de las especies estrechamente relacionadas, all conocidos trnL-F secuencias de I. variedades cylindrica se compararon con trnL-F secuencias de especies de gramíneas (43 relacionados con las secuencias independientes de 29 especies, por ejemplo, Cymbopogon citratus, Sorghastrum incompletum y Coix lacryma-jobi, Miscanthus sinensis, Saccharum officinarum, Sorghum halepense). Aunque, hemos examinado específicos variedad-secuencias de los cebadores a través de 29 especies de hierba, la especificidad de los cebadores específicos variedad-no ha sido ampliamente examinado por su capacidad para amplificar el ADN de la mayoría de otras especies gramíneas. En consecuencia, el tejido utilizado para la extracción de ADN deben ser cuidadosamente identificados como I. cylindrica antes de iniciar este protocolo. Si la hierba no se puede identificar, ya sea como plantas de cogongrass tengan o JBG, entonces le sugerimos la secuenciación del producto de PCR para asegurarse de que las secuencias son una coincidencia exacta con plantas de cogongrass tengan o JBG. Actualmente, el método más exacto para verificar la identidad de un espécimen hierba dado es llevar a cabo la PCR en tanto el trnl-F y de sus regiones, seguida de verificación de la secuencia de los productos de la PCR y la comparación de las secuencias a secuencias conocidas de los taxones identificados con precisión. ADN puede ser amplificado utilizando cebadores de control que se detallan en este protocolo (para la región trnL-F) o cebadores otros disponibles en otras publicaciones 13-15. La secuencia es la mano de obra mucho más y costosa que el uso de nuestro procedimiento simplificado. La calidad de los cebadores utilizados para la PCR es crítico para el éxito del procedimiento. Hemos hecho los iniciadores de este procedimiento disponible en oblicuo Bio, Inc. ( <a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> , Huntsville, AL). La ventaja de ordenar los cebadores de Bio oblicua es que almacenar un gran número de partes alícuotas de cada cebador de la serie de muestras de producción idénticos como los cebadores que se utilizan para la optimización en nuestro laboratorio. En consecuencia, no sólo cebadores tienen la misma secuencia, pero they provienen del lote de producción exactamente el mismo que se utilizó en este protocolo. El uso de primers de un mismo lote, puede ayudar a evitar las variables extrañas en el procedimiento que pueda resultar de las diferencias en la calidad de los cebadores de PCR. Asimismo, mientras que otras polimerasas Taq debería funcionar bien para la PCR, la calidad de la Taq polimerasa utilizada tendrá un impacto sobre la calidad de los resultados de la PCR. Para permitir una mejor consistencia en los reactivos de PCR, hemos optimizado el protocolo de uso de los reactivos de Clontech. La ventaja de dos polimerasa (Clontech, Mountain View, CA, Cat # 639201 o 639202) es una mezcla de un sistema robusto, de arranque en caliente Taq polimerasa y una enzima prueba de lectura que ayuda a proporcionar una alta especificidad y ampliaciones más precisos. Debido a que este protocolo se basa en el ADN del cloroplasto, que es heredado de la madre en los pastos, los sucesos de hibridación entre plantas de cogongrass tengan JBG y los genotipos no pueden ser capturados con nuestro procedimiento de identificación molecular. En los casos en que es hypridization suspeDirección Ejecutiva, le recomendamos que utilice las regiones nucleares que se heredan de ambos padres. Los más comúnmente utilizados no plastídico región variable a considerar en la determinación del genotipo de plantas es la región ITS ribosomal nuclear 13-15,17. En la actualidad, estamos avanzando hacia la multiplexación de la amplificación de los cloroplastos trnL-F región con la de su región de la central nuclear en el mismo tubo de PCR. Multiplexación plastidio con las regiones de ADN nuclear potencialmente eludir las limitaciones de utilizar ya sea solo, sin embargo, estos métodos requieren la optimización y evaluación adicional para determinar la viabilidad de una base de caso por caso. El uso de indicadores cuantitativos PCR en tiempo real (qPCR) y las nuevas tecnologías, tales como marcadores moleculares (sondas fluorescentes de imprimación), también están siendo evaluados como métodos de genotipado a prueba de fallos y exacta de la planta. El protocolo que aquí se presenta ofrece un método rápido y fiable para distinguir la JBG volver a la de tipo salvaje plantas de cogongrass tengan. Le recomendamos utilizarrs de este protocolo para comunicarse con nosotros para informar sobre los resultados derivados de la utilización de este protocolo. Dicha información compartida ayudará a proporcionar información sobre la distribución de la JBG se revierte. Esto también ayudará a los reguladores en el Departamento de Agricultura a tomar decisiones informadas sobre las acciones que pueden ser necesarias para evitar la propagación y la hibridación potencial de las variedades Cogongrass altamente invasivos.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DNeasy Plant Mini Kit </td><td> Qiagen, Valencia, CA </td><td> 69104 o 69106 </td><td> Cualquier planta de renombre genómico kit de ADN que se obtiene ADN de buena calidad debe trabajar muy bien para estos procedimientos. </td></tr><tr><td> TRNF (GAA)-F </td><td> Oblicua Bio, Inc. </td><td> 3-0578 </td><td> De control de avance positivo imprimación </td></tr><tr><td> trnL (5 &#39;del exón)-C </td><td> Oblicua Bio, Inc. </td><td> 3-0579 </td><td> Invertir cebador control positivo </td></tr><tr><td> trnLF-C-F1 </td><td> Oblicua Bio, Inc. </td><td> 3-0864 </td><td> Avance de tipo salvaje plantas de cogongrass tengan imprimación </td></tr><tr><td> trnLF-C-R1 </td><td> Oblicua Bio, Inc. </td><td> 3-0865 </td><td> Marcha atrásde tipo salvaje plantas de cogongrass tengan imprimación </td></tr><tr><td> trnLF-R-F2 </td><td> Oblicua Bio, Inc. </td><td> 3-0866 </td><td> Adelante y la JBG JBG volver imprimación </td></tr><tr><td> trnLF-R-R2 </td><td> Oblicua Bio, Inc. </td><td> 3-0867 </td><td> Invertir JBG JBG y volver imprimación </td></tr><tr><td> Ventaja UltraPure PCR dNTP Mix </td><td> Clontech, Mountain View, CA </td><td> 639125 </td><td></td></tr><tr><td> Ventaja 2 de la polimerasa </td><td> Clontech, Mountain View, CA </td><td> 639201 o 639202 </td><td> Una buena prueba de lectura, de arranque en caliente de Taq polimerasa </td></tr></tbody></table>

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

Wild-tipo I. cylindrica (plantas de cogongrass tengan) es uno de los diez peores plantas invasoras en el mundo, afectando negativamente a los recursos agrícolas y naturales en 73 países diferentes de África, Asia, Europa, Nueva Zelanda, Oceanía y las Américas, 1-2. Cogongrass forma rápida propagación-, stands monodominantes que desplazan a una gran variedad de especies de plantas nativas y, a su vez amenazan a los animales nativos que dependen de las especies de plantas nativas desplazadas para el forraje y refugio. Para agravar el problema, una variedad ornamental [I. cylindrica var. koenigii (Retzius)] es ampliamente comercializado bajo el nombre de &#39;Rubra&#39; Imperata cylindrica, Barón Rojo, y la hierba la sangre japonesa (JBG). Esta variedad es supuestamente estéril y no invasiva y es considerado como una planta ornamental deseable para sus hojas de color rojo. Sin embargo, en las condiciones correctas, la JBG se puede producir semillas viables (Carol Holko, 2009 comunicación personal) y puede volver a una zona verde invasive forma que es a menudo indistinguibles de plantas de cogongrass tengan, ya que toma las características distintivas de la variedad invasivo de tipo salvaje 4 (Figura 1). Esto hace que la identificación con la morfología de una tarea difícil, incluso para los taxonomistas de plantas bien entrenados. Reversión de la JBG a un fenotipo agresivo verde no es también una rara ocurrencia. Uso de comparaciones de secuencias de codificación y las regiones variables del ADN tanto nuclear y del cloroplasto, que han confirmado que la JBG ha vuelto a la verde invasiva dentro de los estados de Maryland, Carolina del Sur y Missouri. JBG ha sido vendido y se plantaron en el estado de casi todos en los EE.UU. continentales donde no hay una infestación de plantas de cogongrass tengan activa. La magnitud del problema en volver no se entiende bien porque las plantas revertidas son indocumentados y destruyó a menudo. La aplicación de este protocolo molecular proporciona un método para identificar JBG vuelve y puede ayudar a mantener estas variedades de co-produciendo unND posiblemente hibridar. Cogongrass es un outcrosser obligado y, al cruzarse con un genotipo diferente, puede producir el viento dispersa las semillas viables que se propagan plantas de cogongrass tengan en distancias 5-7 de ancho. JBG tiene un genotipo ligeramente diferente que plantas de cogongrass tengan y puede ser capaz de formar híbridos viables con plantas de cogongrass tengan. Para agravar el problema, JBG es más frío y tolerante a la sombra de plantas de cogongrass tengan 10.08, y el flujo genético entre estas dos variedades es probable que generen híbridos que son más agresivos, tolerante a la sombra, y resistente al frío que de tipo salvaje plantas de cogongrass tengan. Si bien de tipo salvaje plantas de cogongrass tengan actualmente infesta más de 490 millones de hectáreas en todo el mundo, en los EE.UU. el sudeste que infesta más de 500.000 hectáreas y es capaz de ocupar la mayor parte de los EE.UU., ya que rápidamente se extiende hacia el norte, debido a su nicho de amplia exposición geográfica y el potencial 3,7,11. El potencial de un cruce genético es un motivo de grave preocupación para el Programa de USDA-APHIS Federal Semana nocivas. En la actualidad, el USDA-APHIS prohíbe la JBG en los estados quantes que hay grandes infestaciones Cogongrass (por ejemplo, la Florida, Alabama, Mississippi). Sin embargo, la prevención de las dos variedades de la combinación puede resultar más difícil, ya que plantas de cogongrass tengan JBG y ampliar su distribución. Además, la distribución de la JBG revertir es actualmente desconocido y sin la capacidad de identificar estas variedades a través de la morfología, algunos infestaciones Cogongrass puede ser el resultado de JBG revierte. Desafortunadamente, los métodos actuales de identificación molecular normalmente se basan en AFLP (Amplified polimorfismos de longitud de fragmentos) y secuenciación de DNA, los cuales son el tiempo y es costosa. A continuación, presentamos el primero de costo-efectiva y fiable basado en la PCR método de genotipificación molecular para distinguir con precisión entre las plantas de cogongrass tengan JBG y volver.

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

Ofrecemos un protocolo de genotipificación molecular de costo-efectiva y rápida que emplea a gran específicos-PCR que se dirigen a diferencias en las secuencias de ADN dentro de la región del cloroplasto espaciador trnL-F para diferenciar entre las variedades de<em&gt; Imperata cylindrica</em&gt; (Plantas de cogongrass tengan) que no se pueden distinguir por morfología solo. Estas variedades incluyen la maleza nociva en la lista federal, plantas de cogongrass tengan y estrechamente relacionada, extendida variedad ornamental, que<em&gt;. cylindrica</em&gt; Var.<em&gt; Koenigii</em&gt; (Hierba sangre japonesa).

Una basada en PCR genotipificación método para distinguir entre las variedades de tipo silvestre y 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 ...

a-pcr-based-genotyping-method-to-distinguish-between-wild-type

Research

JoVE Journal

Biology

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

31.4K vistas.  The University of Alabama, Huntsville. Ofrecemos un protocolo de genotipificación molecular de costo-efectiva y rápida que emplea a gran específicos-PCR que se dirigen a diferencias en las secuencias de ADN dentro de la región del cloroplasto espaciador trnL-F para diferenciar entre las variedades de Imperata cylindrica (Plantas de cogongrass tengan) que no se pueden distinguir por morfología solo. Estas variedades incluyen la maleza nociva en la lista federal, plantas de cogongrass tengan ...

Video: 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: Un método simple para determinar el genotipo exacto

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

Investigación

Biología

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.

La identificación de los efectos de las mutaciones BRCA1 en recombinación homóloga utilizando células que expresan endógeno de tipo salvaje 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 ...

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.

De proteínas de membrana de superposición de ensayo: Un protocolo de prueba de interacción entre las proteínas soluble e insoluble 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.

PCR optimizado basado en la detección 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.

Capa de hielo: Un método para el cultivo Arabidopsis Y plantas de tomate en las placas de 96 pocillos de alto rendimiento para genotipado

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.

El genotipado de muestras de plantas y animales, sin previa purificación del ADN

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.

Ensayo Infinium para aplicaciones a gran escala de genotipado de SNP

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

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rápido y Eficiente de pez cebra Genotipado Utilizando PCR con análisis del derretimiento de alta resolución

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

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.

Determinación de ácidos siálicos en hígado y muestras de leche de tipo salvaje y<em&gt; CMAH</em&gt; Ratones knock-out.

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