Nous remercions les membres des Blaschke, Grunwald, et Wittwer laboratoires pour obtenir des conseils et une assistance technique. Ce travail est soutenu par la Fondation PCMC, NIH R01 MH092256 et DP2 MH100008, et le Mars de Dimes Foundation subvention de recherche n ° 1-AF13-425, à JLB.

Une. Préparation de l&#39;ADN <ol><li> Préparer le tampon de lyse de l&#39;ADN: 50 mM de KCl, 10 mM Tris-HCl pH 8,3, 0,3% de Tween 20, 0,3% de NP40. Ajouter fraîche protéinase K à une concentration finale de 1 mg / ml le jour de l&#39;utilisation 12. </li><li> collecte des tissus: <ol><li> Pour adultes pince poisson nageoire: <ol><li> Anesthésier les poissons: Placer le poisson dans 0,004% MS-222 (tricaine) solution. Attendez jusqu&#39;à ce que le mouvement des branchies ralentit. </li><li> Mettez le poisson sur une pile de 5-10 Kimwipes et couper un petit morceau de la nageoire caudale, environ 2-3 mm, avec une lame de rasoir stérile. </li><li> Placer rapidement le poisson dans un réservoir de l&#39;étiquette à l&#39;eau douce pour la récupération; étiqueter soigneusement la fois le réservoir et le tube correspondant qui contiendra la nageoire. Changez l&#39;eau et nourrir les poissons tous les jours. </li><li> Ramassez la nageoire avec une pointe de pipette stérile et le transférer dans un tube rempli de 100 tampon de lyse ADN ul. </li><li> Jeter la pointe de la pipette, et jeter la lame de rasoir dans un récipient pour objets tranchants. </li></ol></li><li> Pour embryon queue clip: <ol><li> Placez embryons dans 0,004% MS-222 (tricaine) solution. Attendez 2 min. </li><li> Coupez un morceau de queue avec une pince sous un microscope à dissection. </li><li> Mettez la queue dans un tube étiqueté rempli de 30 tampon de lyse d&#39;ADN ul. </li><li> Mettre rapidement à l&#39;embryon dans un tube contenant 100 ul de paraformaldehyde à 4%. </li><li> Identifier les tubes avec des embryons et leurs tubes de queue correspondantes. </li><li> Stockez les tubes avec des embryons à 4 ° C pendant la nuit pour la fixation. </li><li> Nettoyez les pinces avec de l&#39;eau Milli-Q et Kimwipes entre chaque embryon pour minimiser la contamination. </li></ol></li><li> Pour les embryons entiers: <ol><li> Placez embryons dans 0,004% MS-222 (tricaine) solution. Attendez 2 min. </li><li> Mettez 1-5 embryons dans un tube rempli de 100 tampon de lyse ADN ul. Dechorionation n&#39;est pas nécessaire. </li></ol></li></ol></li><li> Digestion de l&#39;ADN Incuber les tubes avec le tissu (nageoires ou de la queue des clips ou des embryons) à 55 ° Cpendant 4 heures jusqu&#39;à la nuit 12. </li><li> Inactiver la protéinase K tubes à chaleur à 95 ° C pendant 15 min 12. ADN doit être utilisée pour la PCR immédiatement, ou conservée à -20 ° C pendant jusqu&#39;à 3 mois. </li></ol> 2. PCR <ol><li> Concevoir des amorces soit manuellement ou par logiciel de conception d&#39;amorce (par exemple Lightscanner Primer logiciel de conception-Biofire). Les produits de PCR pour HRMA sont généralement 50-200 pb; produits de PCR pour la détection de SNP devraient être plus petits, généralement 50-80 pb. </li><li> PCR <ol><li> Effectuer réaction de PCR dans une plaque de 96 puits ou 384 puits. </li><li> Utilisez 10 pi volume total: 4 master mix de LightScanner pi (0,1 U chaud Commençons Taq-AB polymérase, tampon, 0,2 mM dNTPs, le chlorure de magnésium 2 mM, et 1x colorant fluorescent d&#39;ADN double brin contraignante); 5 pmol de chaque amorce, et 1 pl matrice d&#39;ADN génomique extrait de queue adulte aileron ou 3 pl de l&#39;ADN génomique de la queue embryonnaire 13. </li><li> échantillons de couverture avec 30 ul d&#39;huile minérale. </li&gt; <li> Couvrir la plaque avec un joint adhésif optiquement transparent. </li><li> Optimiser PCR vélo conditions-typiques conditions sont 95 ° C pendant 5 min et 30 cycles de 10 s à 95 ° C, 25 sec à 60 ° C, 30 secondes à 72 ° C, se terminant par 95 ° C pendant 30 sec et refroidi à 15 ° C. </li><li> Magasin plaques PCR à 4 ° C ou continuent directement à AGRH. </li><li> Confirmer notre produit de PCR par analyse de sa dimension en utilisant une électrophorèse sur gel d&#39;agarose pendant l&#39;optimisation initiale de la PCR, et, si indiqué, par séquençage du produit de PCR. </li></ol></li></ol> 3. HRMA <ol><li> chauffer les assiettes <ol><li> Placer la plaque dans un système d&#39;analyse à l&#39;état fondu. </li><li> Ouvrez le logiciel (Software LightScanner Call-IT 2.0). </li><li> Faites chauffer les assiettes de 60 à 95 ° C. </li><li> Créer un nouveau fichier de stockage de données. </li></ol></li><li> Analyser les données de HRMA (Figure 2). Plusieurs étapes d&#39;analyse sont possibles. Nous montrons l&#39;ensemble le plus couramment utilisé de données manipulations. <ol><li> réglage de sous-ensemble Cliquez sur l&#39;onglet Sous-ensemble. Sélectionnez les puits avec des échantillons. </li><li> Normalisation <ol><li> Sélectionnez l&#39;onglet Normaliser dans le panneau de gauche. Pour éliminer la fluorescence variance, positionner manuellement la double ligne parallèle en pré-(initiale) et les régions poste (finale)-fusion comme illustré (figure 2C, pointes de flèches) et normaliser préfusion et de fluorescence post-fusion des signaux de tous les échantillons de 1 et 0 , respectivement 14. </li><li> Ajuster le positionnement des lignes de sorte que la région de fusion est dans la région entre les pré-et post-fusion des lignes, mais pas sélectionné. En général, la Basse-Min et Max Basse (et supérieur Min et Max supérieur) lignes de température doivent être placés à environ 1 ° C dehors. </li><li> Choisissez positions de température de normalisation tels que tous les échantillons ont (100%) fluorescence maximale ou totale de la région de première fusion et un minimum ou pas (0%) fluorescence pour la région post-fusion (mieux que possible). La normalisation devrait conduire à une ligne presque horizontale à la fluorescence maximale de 1 qui s&#39;étend jusqu&#39;au point de fusion, puis une ligne presque horizontale à partir du point de la fluorescence minimum de 0 à l&#39;état fondu, il ne devrait pas y avoir des niveaux de fluorescence supérieures à 1 ou inférieures à 0 . Par exemple, sur la figure 2C, normaliser les courbes de préfusion en utilisant des plages de températures de 79-80 ° C. </li></ol></li><li> Groupement / Clustering Distinguer les génotypes en fonction de leur température de fusion (figure 2C, boîte verte). Sélectionnez Groupement. <ol><li> Sélectionnez Autogroup de la liste de sélection des normes dans la section Groupement. </li><li> Sélectionnez Normal ou Haut pour la fusion des profils avec des transitions simples ou multiples transitions respectivement dans la liste de sélection de sensibilité dans la section Groupement. </li><li> Sélectionnez ComputeGroupes sous la section Groupement. </li><li> Allez dans le menu Fichier et cliquez sur Enregistrer pour enregistrer les résultats. </li></ol></li></ol></li></ol>

<ol>
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G., Ahmed, J. N., Arkell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+melt+analysis+(HRMA);+a+viable+alternative+to+agarose+gel+electrophoresis+for+mouse+genotyping.">High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.</a> PloS one. 7 (9), (2012).</li><li>Vorkas, P. A., Poumpouridou, N., Agelaki, S., Kroupis, C., Georgoulias, V., Lianidou, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIK3CA+hotspot+mutation+scanning+by+a+novel+and+highly+sensitive+high-resolution+small+amplicon+melting+analysis+method.">PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method.</a> J. Mol. Diagn. 12 (5), 697-704 (2010).</li><li>Parant, J. M., George, S. A., Pryor, R., Wittwer, C. T., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+of+genotyping+zebrafish+mutants.">A rapid and efficient method of genotyping zebrafish mutants.</a> Dev. Dyn. 238 (12), 3168-3174 (2009).</li><li>Xing, L., Hoshijima, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+foxP2+zinc+finger+nuclease+mutant+has+normal+axon+pathfinding.">Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.</a> PloS one. 7 (8), (2012).</li><li>Raghavendra, A., Siji, A., Sridhar, T. S., Phadke, K., Vasudevan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+High+Resolution+Melting+analysis+as+an+alternate+tool+to+screen+for+risk+alleles+associated+with+small+kidneys+in+Indian+newborns.">Evaluation of High Resolution Melting analysis as an alternate tool to screen for risk alleles associated with small kidneys in Indian newborns.</a> BMC Nephrol. 12 (1), 60 (2011).</li><li>Herrmann, M. G., Durtschi, J. D., Wittwer, C. T., Voelkerding, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V+Expanded+instrument+comparison+of+amplicon+DNA+melting+analysis+for+mutation+scanning+and+genotyping.">V Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping.</a> Clin. Chem. 53 (8), 1544-1548 (2007).</li><li>Wittwer, C. 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Les auteurs n&#39;ont rien à révéler.

PCR combinée avec HRMA est une technologie puissante pour le génotypage du poisson zèbre. Les avantages de cette approche sont sa vitesse, la robustesse et la sensibilité à détecter même des mutations ponctuelles. L&#39;ensemble du protocole, à partir de la cassette d&#39;analyse ailette pour faire fondre la courbe, peut être effectuée en moins de huit heures par une seule personne. De plus, la technique est susceptible de criblage à haut débit, ne nécessite pas l&#39;utilisation de bromure d&#39;éthidium,...

Poisson zèbre (Danio rerio) est un système de modèle vertébré largement utilisé pour les études de développement et de modélisation de la maladie. Récemment, de nombreuses technologies transgéniques et de mutation ont été développés pour le poisson zèbre. Techniques de transgénèse rapides, généralement basés sur un système de transposon Tol2 1, ont été combinées avec une amélioration des options de clonage multiple pour l&#39;assemblage de fragments d&#39;ADN 2. Nucléases à doigts de zinc (ZFN) et transcription nucléases effectrices activateur comme (Talens) ont été utilisées pour cibler les lieux dans les cellules somatiques et germinales chez le poisson zèbre 3,4. Ces techniques peuvent générer efficacement des animaux génétiquement modifiés, avec la création de mutation à haute fréquence et la transmission de la lignée germinale 3,4. Malgré ces avancées, les techniques de génotypage traditionnels chez le poisson zèbre limiter la puissance des outils de mutagenèse et la transgenèse. PCR suivie d&#39;une électrophorèse sur gel, parfois combinée à RESTRICTIOn digestion avec des enzymes, est largement utilisée pour détecter la modification du génome, mais est longue et moins sensible pour identifier de petites insertions ou délétions. essais de sonde TaqMan ont des coûts initiaux élevés et nécessitent une optimisation soignée. Le séquençage des produits de PCR peut prendre plusieurs jours et n&#39;est pas pratique pour le dépistage à grande échelle. longueur des fragments de restriction (RFLP) analyse ne peut discriminer SNP affectant un nombre limité de sites de reconnaissance des enzymes de restriction. Analyse à haute résolution fusion (HRMA), un post-PCR méthode d&#39;analyse en tube fermé, est un procédé récemment mis au point qui est rapide, sensible, peu coûteux, et aptes à la sélection de grands nombres d&#39;échantillons. HRMA peut être utilisé pour détecter des SNP, les mutations et les transgènes 7.5. HRMA est basé sur la dénaturation thermique des ADN double brin, et chaque amplicon de PCR a une dissociation uniques (fondre) caractéristique 5. Les échantillons peuvent être victimes de discrimination en raison to leur composition différente de nucléotides, contenu GC, ou longueur, généralement en combinaison avec un colorant fluorescent qui se lie uniquement l&#39;ADN double brin 8. Ainsi, HRMA peut distinguer différents génotypes sur la base des caractéristiques différentes à l&#39;état fondu de courbe. Parce que HRMA utilise des réactifs à faible coût et est un processus post-PCR en une seule étape, il peut être utilisé pour des stratégies à haut débit. HRMA est non destructive, si suite à l&#39;analyse des amplicons PCR peuvent être utilisés pour d&#39;autres applications. HRMA a été appliqué dans de nombreux organismes, y compris les systèmes et les lignées cellulaires, les souris et les humains 9-11. Son utilisation a été récemment décrit dans le poisson zèbre pour détecter des mutations induites par les nucléases à doigt de zinc (ZFN) et Talens 6,12,13. Dans cet article, nous décrivons comment effectuer HRMA basée sur la PCR chez le poisson zèbre embryonnaire et adulte (figure 1). Ce protocole est adapté pour détecter des SNP, les transgènes et les mutations, notamment single changements de paires de bases, des insertions ou des suppressions.

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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>
</table>

rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

Le poisson zèbre est un système de modèle vertébré puissant pour étudier le développement, la modélisation de la maladie, et en effectuant le criblage de médicaments. Récemment, une variété d&#39;outils génétiques ont été mis en place, y compris des stratégies multiples pour induire des mutations et de générer des lignées transgéniques. Cependant, le dépistage à grande échelle est limitée par des méthodes de génotypage traditionnels, qui prennent beaucoup de temps et de main-d&#39;œuvre. Nous décrivons ici une technique pour analyser les génotypes de poisson zèbre par PCR combinées avec l&#39;analyse à haute résolution fusion (AGRH). Cette approche est rapide, sensible et peu coûteux, avec un risque plus faible d&#39;objets de contamination. Le génotypage par PCR avec HRMA peut être utilisé pour des embryons ou des poissons adultes, y compris dans des protocoles de criblage à haut débit.

Le protocole peut être effectué pendant une seule journée ou dans des étapes séparées sur plusieurs jours (diagramme de flux de travail est représentée sur la figure 1). extraction de l&#39;ADN est suivie par la fusion et l&#39;analyse des amplicons PCR. Les températures pour la fusion de l&#39;amplicon dépendent de la taille et de GC-contenu, mais en général, et de commencer à des températures de 50 ˚ C et 95 ˚ C terminales sont appropriées (figures 2A et 2B).<...

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 combinée à l&#39;analyse de fusion à haute résolution (AGRH) est démontré comme une méthode rapide et efficace de déterminer le génotype du poisson zèbre.

Le poisson zèbre rapide et efficace génotypage utilisant la PCR avec haute résolution Melt analyse

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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22.1K vues.  University of Utah School of Medicine.  PCR combinée à l'analyse de fusion à haute résolution (AGRH) est démontré comme une méthode rapide et efficace de déterminer le génotype du poisson zèbre. 

Vidéo: 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.

Génotypage rapide des tissus de souris en utilisant Sigma Extract-N-Amp PCR Kit Tissu

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 ...

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Biologie

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 entier Mont haute résolution double fluorescent In Situ Hybridation

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.

Rapide par PCR en utilisant thermocyclage Microscale convection thermique

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).

Une méthode de génotypage basé sur la PCR pour distinguer les variétés de type sauvage et les plantes ornementales 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.

Utilisation à haute résolution tomodensitométrie pour visualiser la structure tridimensionnelle et la fonction des vaisseaux sanguins des végétaux

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.

Isolation efficace et rapide des embryons à un stade précoce de<em&gt; Arabidopsis thaliana</em&gt; Graines

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.

Haute Résolution Mont entier<em&gt; In Situ</em&gt; Hybridation dans les embryons de poisson zèbre pour étudier l&#39;expression des gènes et la fonction

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.

Analyse qPCRTag - un débit élevé, PCR temps réel pour Sc2.0 Génotypage

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.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

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.

L&#39;imagerie à haute résolution et d&#39;analyse de l&#39;individu Astral microtubules Dynamique dans la levure bourgeonnante

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