Damos las gracias a los miembros de las Blaschke, Grunwald, y Wittwer laboratorios para el asesoramiento y la asistencia técnica. Este trabajo es apoyado por la Fundación PCMC, NIH R01 MH092256 y DP2 MH100008, y la Fundación March of Dimes beca de investigación # 1-FY13-425, a JLB.

1. Preparación de ADN <ol><li> Preparar ADN tampón de lisis: 50 mM de KCl, 10 mM de Tris-HCl, pH 8,3, 0,3% de Tween 20, 0,3% de NP40. Añadir fresco proteinasa K a una concentración final de 1 mg / ml en el día de uso 12. </li><li> Colección de tejidos: <ol><li> Para el adulto clip de aleta de los pescados: <ol><li> Anestesie peces: Coloque el pescado en un 0,004% de MS-222 solución (tricaína). Espere hasta que el movimiento de enmalle se desacelera. </li><li> Coloque el pescado en una pila de 5-10 Kimwipes y cortar un pequeño trozo de la aleta de la cola, de unos 2-3 mm, con una hoja de afeitar estéril. </li><li> Coloque rápidamente a los peces en un tanque de la etiqueta con agua dulce para la recuperación; etiquetar cuidadosamente tanto el depósito y el tubo correspondiente que contendrá el clip de la aleta. Cambie el agua y alimentar a los peces todos los días. </li><li> Levante el clip de la aleta con una punta de pipeta estéril y la transfiere en un tubo relleno con 100 l de tampón de lisis de ADN. </li><li> Desechar la punta de la pipeta, y deseche la hoja de afeitar en un recipiente para objetos filosos. </li></ol></li><li> Para clip de la cola del embrión: <ol><li> Coloque embriones en 0,004% MS-222 solución (tricaína). Espere 2 min. </li><li> Corte un pedazo de cola con pinzas bajo un microscopio de disección. </li><li> Ponga la cola en un tubo etiquetado lleno de 30 l de tampón de lisis de ADN. </li><li> Poner rápidamente el embrión en un tubo que contiene 100 l 4% de paraformaldehído. </li><li> Marque los tubos con los embriones y sus tubos de cola correspondientes. </li><li> Guarde los tubos con los embriones a 4 ° C durante la noche para la fijación. </li><li> Limpie la pinza usando agua Milli-Q y Kimwipes entre cada embrión para minimizar la contaminación. </li></ol></li><li> Para los embriones enteros: <ol><li> Coloque embriones en 0,004% MS-222 solución (tricaína). Espere 2 min. </li><li> Ponga 1-5 embriones en un tubo relleno con 100 l de tampón de lisis de ADN. Dechorionation no es necesario. </li></ol></li></ol></li><li> La digestión de ADN Incubar los tubos con tejido (o de aleta de cola clips o embriones) a 55 ° Cdurante 4 horas o hasta el día 12. </li><li> Inactivar la proteinasa K Tubos de calor a 95 ° C durante 15 min 12. ADN se debe utilizar para PCR inmediatamente o se almacenó a -20 ° C durante un máximo de 3 meses. </li></ol> 2. PCR <ol><li> Primers diseño ya sea manualmente o mediante imprimación de software de diseño (por ejemplo Lightscanner Primer Software de Diseño-Biofire). Productos de la PCR para HRMA son generalmente 50-200 pb; productos de PCR para la detección de SNP deben ser más pequeñas, típicamente 50-80 pb. </li><li> PCR <ol><li> Realizar reacción de PCR en una placa de 96 pocillos o 384 pocillos. </li><li> Utilice 10 l volumen total: 4 l mezcla maestra LightScanner (0,1 U hot-start Taq polimerasa-AB, tampón, dNTPs 0,2 mM, cloruro de magnesio 2 mM, y 1x colorante fluorescente de unión a ADN de doble cadena), 5 pmol de cada cebador, y 1 l de plantilla de ADN genómico extraído de aleta de la cola adulto o 3 l de ADN genómico de la cola embrionaria 13. </li><li> Muestras de cubierta con 30 l de aceite mineral. </li&gt; <li> Cubra la placa con un sello adhesivo ópticamente transparente. </li><li> Optimizar PCR ciclismo condiciones típicas de condiciones son de 95 ° C durante 5 min y 30 ciclos de 10 segundos a 95 ° C, 25 seg a 60 ° C, 30 seg a 72 ° C, terminando con 95 ° C durante 30 segundos y se enfrió a 15 ° C. </li><li> Tienda placas PCR a 4 ° C o siguen directamente a HRMA. </li><li> Confirmar nuestro producto de PCR mediante el análisis de su tamaño utilizando electroforesis en gel de agarosa durante la optimización inicial de la PCR, y si está indicado, por secuenciación del producto de PCR. </li></ol></li></ol> 3. HRMA <ol><li> Placas de calor <ol><li> Coloque la placa en un sistema de análisis de fusión. </li><li> Abra el software (Software LightScanner Call-IT 2.0). </li><li> Placas de calor de 60-95 º C. </li><li> Crear un nuevo archivo de almacenamiento de datos. </li></ol></li><li> Analizar los datos HRMA (Figura 2). Son posibles varias etapas de análisis. Mostramos el conjunto de datos m más utilizadoanipulations. <ol><li> Ajuste de subconjuntos Haga clic en la pestaña de subconjuntos. Seleccione los pocillos con muestras. </li><li> Normalización <ol><li> Seleccione la pestaña Normalizar en el panel izquierdo. Para eliminar la varianza de fluorescencia, coloque manualmente la doble línea paralela en pre-(inicial) y las regiones posteriores (final) de fusión, como se ilustra (Figura 2 C, puntas de flecha) y normalizar Premelt y fluorescencia después de la fusión en las señales de todas las muestras a 1 y 0 , respectivamente 14. </li><li> Ajuste la posición de las líneas de modo que la región de fusión es en la región entre los pre-y post-fusión en las líneas, pero no se selecciona. En general, el Min Baja y el Bajo Max (y Superior y Superior Min Max) líneas de temperatura se deben colocar aproximadamente 1 ° C de diferencia. </li><li> Elija posiciones de temperatura para la normalización de tal manera que todas las muestras tienen (100%) de fluorescencia máxima o total de la región Premelt y mínima o ninguna (0%) de fluorescencia para la región de post-fusión (como mejor como POSble). La normalización debería dar lugar a una línea casi horizontal en la fluorescencia máxima de 1 que se extiende hasta el punto de fusión y luego una línea casi horizontal desde el punto de fusión a la fluorescencia mínima de 0, no debería haber niveles de fluorescencia por encima o por debajo de 1 0 . Por ejemplo, en la Figura 2C, normalizar las curvas de fusión previa usando los rangos de temperatura de 79-80 ° C. </li></ol></li><li> Agrupación / Clustering Distinguir los genotipos en función de su temperatura de fusión (Figura 2 C, caja de color verde). Seleccione Agrupación. <ol><li> Seleccione Autogroup de la lista de selección de Normas en la sección Agrupación. </li><li> Seleccione Normal o Alta para la fusión de perfiles con transiciones individuales o múltiples transiciones, respectivamente, desde la lista de selección de sensibilidad en la sección Agrupación. </li><li> Seleccionar ComputeGrupos menores de la sección Agrupación. </li><li> Vaya al menú Archivo y haga clic en Guardar para guardar los resultados. </li></ol></li></ol></li></ol>

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PCR combinada con HRMA es una tecnología de gran alcance para el genotipado de pez cebra. Las ventajas de este enfoque son su velocidad, robustez, y la sensibilidad para detectar incluso mutaciones puntuales. El protocolo de todo, desde el análisis hasta derretir curva fin-clip, se puede realizar en menos de ocho horas por un solo individuo. Además, la técnica es susceptible de cribado de alto rendimiento; no requiere el uso de bromuro de etidio, y se sella para todos PCR y pasos de análisis que ayuda a minimizar l...

El pez cebra (Danio rerio) es un sistema modelo de vertebrados ampliamente utilizada para estudios de desarrollo y modelado de la enfermedad. Recientemente, numerosas tecnologías transgénicas y de mutación se han desarrollado para el pez cebra. Técnicas de transgénesis rápida, generalmente basados ​​en un sistema transposón Tol2 1, se han combinado con mejores opciones de clonación para el montaje fragmento de ADN múltiple 2. Nucleasas de dedos de zinc (ZFNs) y de transcripción nucleasas efectoras del tipo activador (Talens) se han utilizado para dirigir los loci tanto en las células somáticas y de línea germinal en el pez cebra 3,4. Estas técnicas pueden generar eficientemente animales modificados genéticamente, con la creación de mutación de alta frecuencia y la transmisión de la línea germinal 3,4. A pesar de estos avances, las técnicas de genotipado tradicionales en el pez cebra limitando la plena potencia de las herramientas de mutagénesis y transgénesis. PCR seguida de electroforesis en gel, a veces combinada con restriccioneN enzima de digestión, se utiliza ampliamente para detectar la modificación del genoma, pero consume tiempo y menos sensible para identificar pequeñas inserciones o deleciones. Ensayos con sondas TaqMan tienen altos costos iniciales y requieren una cuidadosa optimización. La secuenciación de los productos de la PCR puede tomar varios días y no es práctico para el cribado a gran escala. Polimorfismo de longitud de fragmentos de restricción (RFLP) sólo puede discriminar SNPs que afectan a una gama limitada de sitios de reconocimiento de enzimas de restricción. Análisis de alta resolución de fusión (HRMA), un método de análisis post-PCR en tubo cerrado, es un método recientemente desarrollado que es rápido, sensible, de bajo costo, y es susceptible de análisis de gran número de muestras. HRMA se puede utilizar para detectar los SNPs, mutaciones, y los transgenes 5-7. HRMA está basado en la desnaturalización térmica de los ADN de doble cadena, y cada uno de amplificación de PCR tiene un único disociación (fundir) característica 5. Las muestras pueden ser discriminados por to su composición de nucleótidos diferente, contenido de GC, o longitud, típicamente en combinación con un colorante fluorescente que sólo se une de doble cadena de ADN 8. Por lo tanto, HRMA puede distinguir diferentes genotipos en base a las diferentes características en estado fundido de la curva. Debido a HRMA utiliza reactivos de bajo costo y es un proceso de post-PCR de un solo paso, que puede ser utilizado para las estrategias de alto rendimiento. HRMA es no destructivo, por lo siguiente análisis de los amplicones de PCR se pueden utilizar para otras aplicaciones. HRMA se ha aplicado en muchos organismos y sistemas, incluyendo líneas celulares, ratones y seres humanos 9-11. Su uso ha sido recientemente descrito en el pez cebra para detectar mutaciones inducidas por las nucleasas de dedos de zinc (ZFNs) y talens 6,12,13. En este trabajo se describe la forma de realizar HRMA PCR basada en el pez cebra embrionarias y adultas (Figura 1). Este protocolo es adecuado para la detección de SNPs, los transgenes, y mutaciones, incluyendo SINGLE cambios de pares de bases, inserciones o deleciones.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">100 Reaction LightScanner Master Mix</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">HRLS-ASY-0002</td>
			<td style="width:157px;">www.biofiredx.com</td>
			<td style="width:167px;">Store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Hard-Shell PCR 96-well BLK/WHT Plates</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">HSP9665</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">MSB1001</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">96-Well LightScanner Instrument</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">LSCN-ASY-0040</td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Software with Call-IT 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">High-Resolution Melting Analysis 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Primer Design Software</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vector NTI Software</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:141px;"></td>
			<td>www.invitrogen.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Paraformaldehyde</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
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rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

El pez cebra es un modelo de sistema de vertebrados de gran alcance para el estudio del desarrollo, modelado de la enfermedad, y la realización de la detección de drogas. Recientemente se ha introducido una variedad de herramientas genéticas, incluyendo las múltiples estrategias para inducir mutaciones y la generación de líneas transgénicas. Sin embargo, el cribado a gran escala está limitada por los métodos de genotipificación tradicionales, que consumen mucho tiempo y mano de obra intensiva. Aquí se describe una técnica para analizar los genotipos de pez cebra mediante PCR en combinación con el análisis de alta resolución de fusión (HRMA). Este enfoque es rápido, sensible y de bajo costo, con menor riesgo de artefactos de contaminación. La genotipificación por PCR con HRMA se puede utilizar para los embriones o los peces adultos, incluidos en los protocolos de selección de alto rendimiento.

El protocolo se puede realizar durante un solo día o separado en pasos lo largo de varios días (diagrama de flujo de trabajo se muestra en la Figura 1). La extracción de ADN es seguido por la masa fundida y el análisis de los amplicones de PCR. Las temperaturas de la masa fundida del amplicón dependen del tamaño y contenido de GC, pero generalmente comienzan y las temperaturas extremas de 50 ˚ C y 95 ˚ C son apropiadas (Figuras 2A y 2B). Una vez que se realiza l...

Watch this Scientific Journal Video about Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis at JoVE.com

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

PCR combinada con el análisis de fusión de alta resolución (HRMA) se demuestra como un método rápido y eficiente para determinar el genotipo de pez cebra.

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 ...

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Biology

22.1K vistas.  University of Utah School of Medicine.  PCR combinada con el análisis de fusión de alta resolución (HRMA) se demuestra como un método rápido y eficiente para determinar el genotipo de pez cebra. 

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

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genotipificación rápida de tejido de ratón utilizando extracto-N-Amp tejido Sigma PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Investigación

Biología

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Pez cebra todo el montaje de alta resolución doble fluorescente In Situ La hibridación

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

PCR rápido ciclo térmico con convección térmica microescala

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

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

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

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

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

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 ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

El uso de alta resolución de tomografía computarizada para visualizar la estructura tridimensional y la función de la Planta Vasculatura

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Aislamiento eficaz y rápida de los embriones en etapa temprana de<em&gt; Arabidopsis thaliana</em&gt; Semillas

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta resolución todo el montaje de<em&gt; In Situ</em&gt; La hibridación en embriones de pez cebra para estudiar la expresión génica y la función

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Imágenes de alta resolución y análisis de la dinámica de microtúbulos astrales individuales de levadura en ciernes

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...