Wir danken Mitglieder der Blaschke, Grunwald, Wittwer und Labors für die Beratung und technische Unterstützung. Diese Arbeit wird von der Stiftung PCMC, NIH R01 MH092256 und DP2 MH100008 und der March of Dimes Foundation Forschungsstipendium # 1-FY13-425, bis JLB unterstützt.

1. DNA-Präparation <ol><li> Vorbereitung DNA Lysepuffer: 50 mM KCl, 10 mM Tris-HCl pH 8,3, 0,3% Tween 20, 0,3% NP40. Frisches Proteinase K bis zu einer Endkonzentration von 1 mg / ml am Tag der Verwendung 12. </li><li> Tissue Sammlung: <ol><li> Für erwachsene Fischflosse Clip: <ol><li> Anesthetize Fisch: Den Fisch in 0,004% MS-222 (Tricaine)-Lösung. Warten Sie, bis Kiemenbewegung verlangsamt. </li><li> Setzen Sie Fisch auf einem Stapel von 5-10 Kimwipes und schneiden Sie ein kleines Stück der Schwanzflosse, ca. 2-3 mm, mit einer sterilen Rasierklinge. </li><li> Schnell stellen die Fische in einem markierten Tank mit Frischwasser für die Wiederherstellung, sorgfältig beschriftet sowohl den Tank und Schlauch, der den entsprechenden fin Clip enthalten wird. Wechseln Sie das Wasser und die Fische füttern jeden zweiten Tag. </li><li> Nehmen Sie den Fin-Clip mit einer sterilen Pipettenspitze und übertragen sie in ein Rohr mit 100 ul DNA-Lyse-Puffer gefüllt. </li><li> Entsorgen Sie die Pipettenspitze, und entsorgen Sie die Rasierklinge in einen Behälter für scharfe Gegenstände. </li></ol></li><li> Für Embryo Schwanz-Clip: <ol><li> Zeigen Embryonen in 0,004% MS-222 (Tricaine)-Lösung. Warten Sie 2 min. </li><li> Schneiden Sie ein Stück Schwanz mit einer Pinzette unter einem Binokular. </li><li> Setzen Sie den Schwanz in ein beschriftetes Röhrchen mit 30 ul DNA-Lyse-Puffer gefüllt. </li><li> Setzen schnell den Embryo in ein Röhrchen mit 100 ul 4% Paraformaldehyd. </li><li> Beschriften Sie die Röhrchen mit Embryonen und ihre entsprechenden Endrohre. </li><li> Die Röhrchen mit Embryonen bei 4 ° C über Nacht zur Fixierung. </li><li> Reinigen Sie die Zange mit Milli-Q-Wasser und Kimwipes zwischen jedem Embryo auf Kontamination zu minimieren. </li></ol></li><li> Für ganze Embryonen: <ol><li> Zeigen Embryonen in 0,004% MS-222 (Tricaine)-Lösung. Warten Sie 2 min. </li><li> Setz 1-5 Embryonen in einem Rohr mit 100 ul DNA-Lysepuffer gefüllt. Dechorionation ist nicht erforderlich. </li></ol></li></ol></li><li> DNA-Verdau Rohre mit Gewebe (fin oder Schwanz Clips oder Embryonen), bei 55 ° C inkubieren4 Stunden bis zu 12 über Nacht. </li><li> Inaktivierung der Proteinase K Wärmerohren auf 95 ° C für 15 min 12. DNA für die PCR sollte sofort verwendet werden oder bei -20 ° C für 3 Monate gelagert. </li></ol> 2. PCR <ol><li> Design-Primer entweder manuell oder durch Primer-Design-Software (zB Lightscanner Primer-Design-Software-Biofire). PCR-Produkte sind in der Regel für HRMA 50-200 bp, PCR-Produkte für die SNP-Nachweis sollte kleiner sein, in der Regel 50-80 bp. </li><li> PCR <ol><li> Führen Sie die PCR-Reaktion in einem 96-Well oder 384-Well-Platte. </li><li> Verwenden Sie 10 ul Volumen: 4 ul LightScanner Master-Mix (0,1 U Hot-Start Taq-AB-Polymerase, Puffer, 0,2 mM dNTPs, 2 mM Magnesiumchlorid und 1x Leuchtstoffdoppelstrang-DNA-bindenden Farbstoff), 5 pmol von jedem Primer, und 1 ul genomische DNA-Vorlage aus adulten Schwanzflosse oder 3 ul genomischer DNA aus embryonalen Schwanz 13 extrahiert. </li><li> Titel Proben mit 30 ul Mineralöl. </li&gt; <li> Die Platte mit einem optisch transparenten Klebesiegel. </li><li> Optimierung der PCR-Zyklusbedingungen sind typische Bedingungen 95 ° C für 5 min und 30 Zyklen von 10 s bei 95 ° C, 25 sec bei 60 ° C, 30 sec bei 72 ° C, endend mit 95 ° C für 30 sec und gekühlt 15 ° C. </li><li> Speicher PCR-Platten bei 4 ° C oder direkt weiter HRMA. </li><li> Bestätigen unsere PCR-Produkt durch Analyse seiner Größe unter Verwendung von Agarose-Gel-Elektrophorese bei der ersten Optimierung der PCR, und, wenn angegeben, durch Sequenzierung des PCR-Produkts. </li></ol></li></ol> 3. HRMA <ol><li> Wärmeplatten <ol><li> Zeigen Platte in einem Schmelzanalyse-System. </li><li> Öffnen Sie die Software (Software LightScanner Call-IT 2.0). </li><li> Wärmeplatten von 60 bis 95 ° C </li><li> Erstellen Sie einen neuen Datenspeicher-Datei. </li></ol></li><li> Analysieren HRMA Daten (Abbildung 2). Mehrere Analyseschritte möglich sind. Wir zeigen die am häufigsten verwendeten Satz von Daten manipulations. <ol><li> Subset-Einstellung Klicken Sie auf Subset-Registerkarte. Wählen Sie die Vertiefungen mit Proben. </li><li> Normalisierung <ol><li> Wählen Sie die Registerkarte Normalisieren in der linken Panel. Um Fluoreszenz Varianz zu beseitigen, manuell positionieren Sie den parallelen Doppellinie in der Vor-(initial) und Post (final) Schmelz Regionen dargestellt (2C, Pfeilspitzen) und normalisieren Vorschmelze und Post-Schmelze Fluoreszenzsignale aller Proben 1 und 0 bzw. 14. </li><li> Einstellen der Positionierung der Leitungen, so daß der Schmelzbereich in dem Bereich zwischen den Pre-und Post-Schmelzeleitungen, aber nicht ausgewählt. Im Allgemeinen ist die Nieder Min-und Max Nieder (und Ober Min-und Max Ober) Temperaturleitungen sollten getrennt aufgestellt werden, etwa 1 ° C. </li><li> Wählen Temperatur Positionen für die Normalisierung, so dass alle Proben Maximum oder voll (100%) für die Fluoreszenz Vorschmelze Region und minimale oder gar keine (0%) Fluoreszenz für die Post-Schmelzbereich (am besten wie möglichlich). Die Normalisierung sollte in einer nahezu horizontalen Linie bei der maximalen Fluoreszenz von 1, die bis zum Schmelzpunkt und dann eine nahezu horizontale Linie aus der Schmelze Punkt bei der minimalen Fluoreszenz von 0 erstreckt sich führen, es sollte nicht Fluoreszenzwerte über 1 oder unter 0 sein . Beispielsweise in Fig. 2C, normalisieren die Vorschmelztemperatur Kurven mit Temperaturbereichen von 79-80 ° C. </li></ol></li><li> Gruppierung / Clustering Unterscheiden Genotypen basierend auf ihren Schmelztemperatur (2C, grün Kasten). Wählen Sie Gruppierung. <ol><li> Wählen Sie aus dem Autogroup Standards Auswahlliste unter dem Abschnitt Gruppierung. </li><li> Wählen Sie Normal oder Hoch zum Schmelzen Profile mit einzelnen Übergänge oder mehrere Übergänge jeweils von der Empfindlichkeit Auswahlliste unter dem Abschnitt Gruppierung. </li><li> Wählen ComputeGruppen unter der Gruppierung Abschnitt. </li><li> Gehen Sie auf das Menü Datei und klicken Sie auf Speichern, um die Ergebnisse zu speichern. </li></ol></li></ol></li></ol>

<ol>
	<li>Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N., Mishina, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon-mediated+gene+trap+approach+identifies+developmentally+regulated+genes+in+zebrafish.">A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.</a> Dev. Cell. 7 (1), 133-144 (2004).</li><li>Kwan, K. M., Fujimoto, E., 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236 (11), 3088-3099 (2007).</li><li>Sander, J. D., Cade, L., 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=Targeted+gene+disruption+in+somatic+zebrafish+cells+using+engineered+TALENs.">Targeted gene disruption in somatic zebrafish cells using engineered TALENs.</a> Nat. Biotechnol. 29 (8), 697-698 (2011).</li><li>Huang, P., Xiao, A., Zhou, M., Zhu, Z., Lin, S., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+gene+targeting+in+zebrafish+using+customized+TALENs.">Heritable gene targeting in zebrafish using customized TALENs.</a> Nat. Biotechnol. 29 (8), 699-700 (2011).</li><li>Vossen, R. H. A. M., Aten, E., Roos, A., Den Dunnen, J. T. <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+melting+analysis+(HRMA):+more+than+just+sequence+variant+screening.">High-resolution melting analysis (HRMA): more than just sequence variant screening.</a> Hum. Mutation. 30 (6), 860-866 (2009).</li><li>Dahlem, T. J., 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=Simple+methods+for+generating+and+detecting+locus-specific+mutations+induced+with+TALENs+in+the+zebrafish+genome.">Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.</a> PLoS Genet. 8 (8), (2012).</li><li>Liew, M., Pryor, R., 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=Genotyping+of+single-nucleotide+polymorphisms+by+high-resolution+melting+of+small+amplicons.">Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.</a> Clin. Chem. 50 (7), 1156-1164 (2004).</li><li>Herrmann, M. G., Durtschi, J. D., Bromley, L. K., 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+Amplicon+DNA+melting+analysis+for+mutation+scanning+and+genotyping:+cross-platform+comparison+of+instruments+and+dyes.">V Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes.</a> Clin. Chem. 52 (3), 494-503 (2006).</li><li>Carrillo, J., Mart&#237;nez, P., 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=High+resolution+melting+analysis+for+the+identification+of+novel+mutations+in+DKC1+and+TERT+genes+in+patients+with+dyskeratosis+congenita.">High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.</a> Blood Cell. Mol. Dis.. 49 (3-4), 140-146 (2012).</li><li>Thomsen, N., Ali, R. 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. T. <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+Genotyping+by+Amplicon+Melting+Analysis+Using+LCGreen.">High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen.</a> Clin. Chem. 49 (6), 853-860 (2003).</li><li>Dimitrakopoulos, L., Vorkas, P. A., 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=A+closed-tube+methylation-sensitive+high+resolution+melting+assay+(MS-HRMA)+for+the+semi-quantitative+determination+of+CST6+promoter+methylation+in+clinical+samples.">A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.</a> BMC Cancer. 12, 486-48 (2012).</li><li>Jeng, K., Gaydos, C. A., 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=Comparative+analysis+of+two+broad-range+PCR+assays+for+pathogen+detection+in+positive-blood-culture+bottles:+PCR-high-resolution+melting+analysis+versus+PCR-mass+spectrometry.">Comparative analysis of two broad-range PCR assays for pathogen detection in positive-blood-culture bottles: PCR-high-resolution melting analysis versus PCR-mass spectrometry.</a> J. Clin. Microbiol. 50 (10), 3287-3292 (2012).</li></ol>

Die Autoren haben nichts zu offenbaren.

PCR in Kombination mit HRMA ist eine leistungsstarke Technologie für die Zebrafisch-Genotypisierung. Die Vorteile dieses Ansatzes sind seine Geschwindigkeit, Robustheit und Empfindlichkeit, auch Punktmutationen. Das gesamte Protokoll, aus fin-Clip zum Schmelzkurvenanalyse kann in weniger als acht Stunden von einer einzelnen Person durchgeführt werden. Darüber hinaus ist die Technik zugänglich für Hochdurchsatz-Screening, die Verwendung von Ethidiumbromid nicht erforderlich, und wird für alle PCR und Analyseschritt...

Zebrafisch (Danio rerio), ein Wirbeltier-Modellsystem für die Untersuchung der weithin Entwicklung und Krankheit Modellierung verwendet. Kürzlich wurden zahlreiche transgenen und Mutationstechniken für Zebrafisch entwickelt. Rapid-Techniken Transgenese, in der Regel auf einem Tol2 Transposonsystem 1 basiert, haben mit verbesserten Klonen Optionen für mehrere DNA-Fragment Anordnung 2 zusammengefasst. Zink-Finger-Nukleasen (ZFNs) und Transkriptionsaktivator-Effektor wie Nukleasen (Talens) verwendet worden, um Loci in beiden somatischen und Keimbahnzellen im Zebrafisch 3,4 Ziel. Diese Techniken können effizient genetisch veränderte Tiere zu erzeugen, mit Hochfrequenz-Mutation Erstellung und Keimbahn-Übertragung 3,4. Trotz dieser Fortschritte, traditionelle Techniken, Genotypisierung im Zebrafisch begrenzen die volle Leistung der Mutagenese und Transgenese-Tools. PCR, gefolgt von Gelelektrophorese, manchmal kombiniert mit EINSCHRÄNKn Enzymverdauung, wird häufig zur Modifikation Genom nachzuweisen, ist jedoch zeitaufwendig und weniger empfindlich gegenüber kleinen Insertionen oder Deletionen zu identifizieren. TaqMan-Sondentests haben eine hohe Anfangskosten und erfordern eine sorgfältige Optimierung. Die Sequenzierung von PCR-Produkten kann mehrere Tage dauern und ist nicht praktisch für große Screening. Restriktionsfragmentlängen-Polymorphismus (RFLP)-Analyse kann nur unterscheiden SNPs mit Auswirkungen auf eine begrenzte Anzahl von Restriktionsenzym-Erkennungsstellen. Hochauflösende Schmelzanalyse (HRMA), eine geschlossene Röhre Post-PCR-Analyseverfahren, ist eine kürzlich entwickelte Methode, die schnell, empfindlich, kostengünstig und zugänglich Screening einer großen Anzahl von Proben. HRMA kann verwendet werden, um SNPs, Mutationen und Transgene 5-7 zu erkennen. HRMA auf die thermische Denaturierung von doppelsträngigen DNAs basiert, und jedes PCR-Amplicon eine eindeutige Dissoziation (Schmelze) Kennlinie 5. Die Proben unterschieden werden durch to ihrer unterschiedlichen Nukleotid-Zusammensetzung, GC-Gehalt, oder Länge, in der Regel in Kombination mit einem Fluoreszenz-Farbstoff bindet, die nur doppelsträngige DNA-8. Somit kann HRMA verschiedenen Genotypen basierend auf den unterschiedlichen Schmelzkurvencharakteristika unterscheiden. Da HRMA verwendet Low-Cost-Reagenzien und ist eine Einzelschritt-Post-PCR-Verfahren, kann es für Hochdurchsatz-Strategien verwendet werden. HRMA ist nicht destruktiv, so dass folgende Analyse die PCR-Produkte auch für andere Anwendungen verwendet werden. HRMA in vielen Organismen und Systeme, einschließlich Zelllinien, Mäusen und Menschen 9-11 angewandt. Sein Einsatz hat kürzlich im Zebrafisch beschrieben worden, um Mutationen, die durch Zink-Finger-Nukleasen (ZFNs) und Talens 6,12,13 induziert wird. In diesem Papier beschreiben wir, wie man auf PCR-Basis HRMA in embryonalen und adulten Zebrafisch (Abbildung 1) durchzuführen. Dieses Protokoll ist für die Detektion von SNPs, Transgene, und Mutationen, einschließlich single Basenpaar-Veränderungen, Insertionen oder Deletionen.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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

Der Zebrafisch ist ein leistungsfähiges Wirbeltier-Modellsystem für die Untersuchung der Entwicklung, Modellierung Krankheit und Arzneimittel-Screening durchführen. Kürzlich wurde eine Vielzahl von genetischen Werkzeugen eingeführt, darunter mehrere Strategien zur Induktion von Mutationen und zur Erzeugung transgener Linien. Jedoch wird großen Screening durch Genotypisierung traditionellen Methoden, die zeitaufwändig und arbeitsintensiv sind begrenzt. Hier beschreiben wir eine Technik, um Zebrafisch-Genotypen durch PCR in Kombination mit hochauflösenden Schmelzanalyse (HRMA) zu analysieren. Dieser Ansatz ist eine schnelle, empfindliche und preiswert, mit einem geringeren Risiko der Kontamination Artefakte. Genotypisierung mittels PCR mit HRMA für Embryonen oder ausgewachsene Fische verwendet werden, auch im Hochdurchsatz-Screening-Protokolle.

Das Protokoll kann an einem einzigen Tag über mehrere Tage durchgeführt wird oder in Stufen getrennt (Flußdiagramm der Arbeit ist in Fig. 1 gezeigt). Die DNA-Extraktion wird durch die Schmelze und Analyse von PCR-Amplikons, gefolgt. Die Temperaturen für die Schmelze des Amplikons sind abhängig von der Größe und der GC-Gehalt, aber in der Regel Anfang und Ende Temperaturen von 50 ˚ C und 95 ˚ C falls (2A und 2B) sind. Sobald die Schmelze durchgeführt wird, die ...

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 in Kombination mit hochauflösenden Schmelzanalyse (HRMA) als schnelle und effiziente Methode, um Zebrafisch-Genotyp nachgewiesen.

Schnelle und effiziente Verwendung von Zebrafisch-Genotypisierung PCR mit Hochauflösende Schmelzanalyse

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 Aufrufe.  University of Utah School of Medicine.  PCR in Kombination mit hochauflösenden Schmelzanalyse (HRMA) als schnelle und effiziente Methode, um Zebrafisch-Genotyp nachgewiesen. 

Serie: 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.

Schnelle Genotypisierung von Maus Tissue Mit Sigma 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 ...

Forschung

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.

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

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.

Rapid PCR Thermocycling mit Microscale thermische Konvektion

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).

Ein PCR-basierte Genotypisierung Methode, zwischen Wildtyp-und Ziersorten Unterscheiden von 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.

Mit hochauflösenden Computertomographie, die dreidimensionale Struktur und Funktion pflanzlicher Gefäßsystem Visualisieren

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.

Effiziente und schnelle Isolierung von Frühphasen-Embryonen aus<em&gt; Arabidopsis thaliana</em&gt; Seeds

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.

Hohe Auflösung Ganze Berge<em&gt; In Situ</em&gt; Hybridisierung innerhalb Zebrafischembryonen Gene Expression und Funktion zu untersuchen

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.

qPCRTag Analyse - ein hoher Durchsatz, Echtzeit-PCR-Assay für 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 ...

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.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

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.

Hochauflösende Bildgebung und Analyse einzelner Astral Dynamik der Mikrotubuli in Bäckerhefe

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