Agradecemos a membros das Blaschke, Grunwald, e Wittwer laboratórios para aconselhamento e assistência técnica. Este trabalho é apoiado pela Fundação PCMC, NIH R01 MH092256 e DP2 MH100008, ea Marcha de Dimes Foundation bolsa de investigação # 1-FY13-425, a JLB.

1. Preparação de DNA <ol><li> Preparar tampão de lise de ADN: 50 mM de KCl, 10 mM Tris-HCl pH 8,3, 0,3% de Tween 20, 0,3% de NP40. Adicionar fresco proteinase K para uma concentração final de 1 mg / mL, no dia da utilização 12. </li><li> Coleção de tecidos: <ol><li> Para o adulto clipe fin fish: <ol><li> Anestesiar peixe: Coloque o peixe em 0,004% solução (tricaina) MS-222. Espere até que o movimento de emalhar diminui. </li><li> Coloque os peixes em uma pilha de 5-10 Kimwipes e cortar um pequeno pedaço da barbatana caudal, cerca de 2-3 mm, com uma lâmina de barbear estéril. </li><li> Colocar rapidamente o peixe em um tanque rotulado com água fresca para a recuperação; rotular cuidadosamente tanto o tanque e correspondente tubo que contém o clipe de fin. Troque a água e alimentar os peixes todos os dias. </li><li> Pegue o clipe fin com uma ponta de pipeta estéril e transferi-lo para um tubo preenchido com 100 mL tampão de lise de DNA. </li><li> Descarte a ponta da pipeta, e descartar a lâmina de barbear em um recipiente apropriado. </li></ol></li><li> Por cauda embrião clipe: <ol><li> Coloque embriões em 0,004% solução (tricaina) MS-222. Espere 2 min. </li><li> Corte um pedaço da cauda com uma pinça sob um microscópio de dissecação. </li><li> Coloque a cauda para um tubo rotulado cheio com 30 uL de tampão de lise de ADN. </li><li> Rapidamente colocar o embrião para um tubo contendo 100 jil de 4% de paraformaldeído. </li><li> Tubos etiqueta com embriões e seus tubos de cauda correspondentes. </li><li> Armazenar os tubos com embriões a 4 ° C durante a noite para a fixação. </li><li> Limpar os fórceps usando água Milli-Q e Kimwipes entre cada embrião para minimizar a contaminação. </li></ol></li><li> Para embriões inteiros: <ol><li> Coloque embriões em 0,004% solução (tricaina) MS-222. Espere 2 min. </li><li> Colocar 1-5 embriões num tubo cheio com 100 ul de tampão de lise de ADN. Dechorionation não é necessário. </li></ol></li></ol></li><li> Digestão do DNA Incubar os tubos com o tecido (ou nadadeira da cauda clips ou embriões) a 55 ° Cpara 4 horas até durante a noite 12. </li><li> Desativar o Proteinase K Tubos de calor a 95 ° C durante 15 minutos 12. DNA deve ser utilizado para a PCR imediatamente ou armazenada a -20 ° C durante até 3 meses. </li></ol> 2. PCR <ol><li> Projeto Primers manualmente ou por software de desenho de primer (por exemplo Lightscanner Primer Projeto Software-Biofire). Os produtos de PCR para HRMA são geralmente de 50-200 pb, os produtos de PCR para a detecção de SNP pode ser menor, tipicamente 50-80 pb. </li><li> PCR <ol><li> Realizar a reacção de PCR em um de 96 poços ou de 384 poços da placa. </li><li> Utilizar 10 mL volume total: 4 ul de mistura principal LightScanner (0,1 L quente iniciar a Taq-polimerase AB, tampão, dNTP 0,2 mM, cloreto de magnésio 2 mM, 1x e corante de ligação a ADN de cadeia dupla fluorescente), 5 pmol de cada iniciador, e 1 uL de ADN genómico extraído de modelo de barbatana adulto ou 3 ul de DNA genómico a partir de cauda embrionária 13. </li><li> Amostras de cobertura com 30 mL de óleo mineral. </li&gt; <li> Cobrir a placa com um selo adesivo opticamente transparentes. </li><li> Optimizar PCR condições de ciclismo típicos, são condições de 95 ° C durante 5 min, e 30 ciclos de 10 seg a 95 ° C, 25 seg a 60 ° C, 30 seg a 72 ° C, terminando com 95 ° C durante 30 seg e arrefecida a 15 ° C. </li><li> Armazene PCR placas a 4 ° C ou continuar diretamente para HRMA. </li><li> Confirmar nosso produto de PCR por análise do seu tamanho utilizando electroforese em gel de agarose durante a optimização inicial de PCR e, se indicado, por sequenciação do produto de PCR. </li></ol></li></ol> 3. HRMA <ol><li> Placas de calor <ol><li> Coloque a placa em um sistema de análise de fusão. </li><li> Abra o software (Software LightScanner Chame-IT 2.0). </li><li> Placas de calor 60-95 ° C. </li><li> Crie um novo arquivo de armazenamento de dados. </li></ol></li><li> Analisar dados HRMA (Figura 2). Várias etapas de análise são possíveis. Mostramos o conjunto mais comumente usado de dados manipulations. <ol><li> Definição de subconjunto Clique na aba subconjunto. Selecione os poços com amostras. </li><li> Normalização <ol><li> Selecione a guia Normalize no painel à esquerda. Para eliminar variação de fluorescência, posicionar manualmente a dupla linha paralela na pré-(inicial) e pós regiões (final) derreter como ilustrado (Figura 2C, pontas de seta) e normalizar os sinais de fluorescência premelt e pós-fusão de todas as amostras a 1 e 0 , respectivamente 14. </li><li> Ajustar o posicionamento das linhas de modo que a região de fusão é, na região entre as linhas de pré-e pós-melt, mas não seleccionado. Em geral, quanto menor Min e Baixa Max (e Superior Min e Max superior) linhas de temperatura deve ser colocado cerca de 1 ° C à parte. </li><li> Escolha posições de temperatura para a normalização de tal forma que todas as amostras têm (100%) de fluorescência máxima ou total para a região premelt e mínima ou nenhuma (0%) de fluorescência para a região pós-fusão (da melhor forma posvel). A normalização deve resultar em uma linha quase horizontal na parte de fluorescência máxima de 1 que se estende até ao ponto de fusão e, em seguida, uma linha quase horizontal a partir do ponto de fusão na fluorescência mínima de 0, não deve haver níveis de fluorescência acima ou abaixo de 1 0 . Por exemplo, na figura 2C, normalizar as curvas premelting usando gamas de temperatura de 79-80 ° C. </li></ol></li><li> Agrupamento / Clustering Distinguir os genótipos com base na sua temperatura de fusão (Figura 2C, caixa verde). Selecione Agrupamento. <ol><li> Selecione autogroup na lista de seleção Padrões na seção Agrupamento. </li><li> Selecione Normal ou Alta para a fusão de perfis com transições individuais ou várias transições, respectivamente, na lista de seleção de sensibilidade na seção Agrupamento. </li><li> Selecione ComputeGrupos na seção Agrupamento. </li><li> Vá para o menu Arquivo e clique em Salvar para salvar os resultados. </li></ol></li></ol></li></ol>

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PCR combinada com HRMA é uma tecnologia poderosa para a genotipagem do peixe-zebra. As vantagens desta abordagem são a sua velocidade, robustez, e a sensibilidade para detectar mesmo mutações pontuais. O protocolo de todo, a partir de aleta-grampo análise para derreter-curva, pode ser realizada em menos de oito horas, por um único indivíduo. Além disso, a técnica é tratável através de rastreio de alto rendimento; não requer a utilização de brometo de etídio, e é selada por todas as etapas de PCR e de an...

Peixe-zebra (Danio rerio) é um sistema de modelo animal vertebrado amplamente utilizado para estudos de desenvolvimento e modelagem de doenças. Recentemente, várias tecnologias transgênicas e mutação foram desenvolvidos para zebrafish. Técnicas de transgenia rápidos, geralmente com base em um sistema de transposon Tol2 1, foram combinados com melhores opções de clonagem para uma montagem múltipla fragmento de DNA 2. Nucleases de dedos de zinco (ZFNs) e de transcrição nucleases efectoras activadores do tipo (TALENS) têm sido utilizadas para direccionar loci em ambas as células somáticas e germinais em peixes-zebra 3,4. Estas técnicas podem gerar de forma eficiente animais geneticamente modificados, com alta freqüência de criação e transmissão de mutação germinal 3,4. Apesar destes avanços, técnicas de genotipagem tradicionais em zebrafish limitar o poder das ferramentas de mutagênese e transgenia. PCR, seguida por electroforese em gel, por vezes combinados com DISSEMINAÇdigestão enzimática n, é amplamente utilizado para detectar a modificação do genoma, mas é demorada e menos sensível à identificação de pequenas inserções ou deleções. Ensaios de sonda TaqMan têm altos custos iniciais e requerem otimização cuidadosa. A sequenciação de produtos de PCR pode levar vários dias e não é prática para a triagem em grande escala. Análise do polimorfismo de fragmentos de restrição (RFLP) só pode discriminar SNPs que afetam um número limitado de sítios de reconhecimento de enzimas de restrição. A análise de alta resolução de fusão (HRMA), um tubo fechado, o método de análise pós-PCR, é um método recentemente desenvolvido que é rápido, sensível, pouco dispendioso, e passíveis de rastreio de grandes números de amostras. HRMA pode ser utilizado para detectar SNPs, mutações e transgenes 5-7. HRMA é baseada em desnaturação térmica de ADN de cadeia dupla, e cada fragmento amplificado de PCR tem um único dissociação (derreter) característica 5. As amostras podem ser discriminados devido to a sua composição de nucleótidos diferente, teor de GC, ou comprimento, tipicamente em combinação com um corante fluorescente que se liga somente o ADN de cadeia dupla 8. Assim, HRMA pode distinguir diferentes genótipos com base nas diferentes características de fusão-curva. Porque HRMA utiliza reagentes de baixo custo e é um processo de pós-PCR de etapa única, que pode ser usado para as estratégias de alto rendimento. HRMA é não destrutiva, de modo que após a análise dos produtos de amplificação de PCR podem ser usados ​​para outras aplicações. HRMA foi aplicado em muitos organismos e sistemas, incluindo linhas celulares, ratos e seres humanos 9-11. Seu uso foi recentemente descrito no peixe-zebra para detectar mutações induzidas por nucleases dedo de zinco (ZFNs) e Talens 6,12,13. Neste artigo, descrevemos como executar HRMA baseado em PCR em zebrafish embrionárias e adultas (Figura 1). Este protocolo é adequado para a detecção de SNPs, os transgenes e mutações, incluindo simudanças ngle de pares de bases, inserções ou exclusões.

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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>
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		<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>
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		<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

Zebrafish é um poderoso sistema modelo para o estudo de vertebrados desenvolvimento, modelagem de doenças, e realizar a triagem de drogas. Recentemente, uma variedade de ferramentas genéticas foram introduzidas, incluindo várias estratégias para induzir mutações e gerar linhagens transgênicas. No entanto, o rastreio em larga escala é limitada pelos métodos de genotipagem tradicionais, que são consumidoras de tempo e de trabalho intensivo. Aqui nós descrevemos uma técnica para analisar genótipos zebrafish por PCR combinada com análise de alta resolução de fusão (HRMA). Esta abordagem é rápida, sensível e de baixo custo, com menor risco de artefatos de contaminação. A genotipagem por PCR com HRMA pode ser usado para embriões e peixes adultos, incluindo protocolos de triagem de alto rendimento.

O protocolo pode ser realizada durante um único dia ou separada em passos ao longo de vários dias (diagrama de fluxo de trabalho é mostrado na Figura 1). Extracção de ADN é seguida pela análise de fusão e de produtos de amplificação de PCR. As temperaturas para a fusão do fragmento amplificado dependerá do tamanho e conteúdo GC, mas geralmente iniciar e as temperaturas finais de 50 ˚ C e 95 ˚ C são adequados (Figuras 2A e 2B). Uma vez que a fusão é rea...

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 com a análise de fusão de alta resolução (HRMA) é demonstrado como um método rápido e eficiente para a genotipagem de peixe-zebra.

Rápida e eficiente Zebrafish Genotipagem Usando PCR com alta resolução Análise Melt

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 visualizações.  University of Utah School of Medicine.  PCR combinada com a análise de fusão de alta resolução (HRMA) é demonstrado como um método rápido e eficiente para a genotipagem de peixe-zebra. 

Vídeo: 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.

A genotipagem rápida de tecido Mouse Usando Extract-N-Amp Tissue 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 ...

Biologia

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.

Zebrafish Whole Mount Alta Resolução Duplo Fluorescente In Situ Hibridização

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.

Rápida ciclagem térmica PCR usando convecção térmica em micro

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).

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 ...

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.

Usando tomografia de tórax para visualizar a estrutura tridimensional e Função da Usina 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.

Isolamento eficiente e rápida de embriões em início de carreira a partir de<em&gt; Arabidopsis thaliana</em&gt; Sementes

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 resolução Monte Whole<em&gt; In Situ</em&gt; Hibridização dentro embriões Zebrafish para estudar a expressão gênica e função

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.

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 ...

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.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

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.

Imagens de alta resolução e Análise de individuais Astral microtúbulos Dynamics em brotamento Yeast

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