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.

<table><tbody><tr><td>100 Reaction LightScanner Master Mix</td><td>BioFire</td><td>HRLS-ASY-0002</td><td>www.biofiredx.com</td></tr><tr><td>Hard-Shell PCR 96-well BLK/WHT Plates</td><td>Bio-Rad Laboratories</td><td>HSP9665</td><td>www.bio-rad.com</td></tr><tr><td>Microseal &#039;B&#039; Adhesive Seals</td><td>Bio-Rad Laboratories</td><td>MSB1001</td><td>www.bio-rad.com</td></tr><tr><td>96-well LightScanner Instrument</td><td>BioFire</td><td>LSCN-ASY-0040</td><td>www.biofiredx.com</td></tr><tr><td>LightScanner Software with Call-IT 2.0</td><td>BioFire</td><td></td><td>www.biofiredx.com</td></tr><tr><td>High-resolution Melting Analysis 2.0</td><td>BioFire</td><td></td><td>www.biofiredx.com</td></tr><tr><td>LightScanner Primer Design Software</td><td>BioFire</td><td></td><td>www.biofiredx.com</td></tr><tr><td>Vector NTI Software</td><td>Invitrogen</td><td></td><td>www.invitrogen.com</td></tr><tr><td>Tricaine</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td></td><td></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 ...

rapid-efficient-zebrafish-genotyping-using-pcr-with-high-resolution

Research

JoVE Journal

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

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

Mit Hilfe eines Fluoreszenz-PCR-Kapillar-Gel-Elektrophorese-Technik CRISPR zum Genotyp / Cas9 vermittelte Knock-out Mutanten in einem Hochdurchsatz-Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Forschung

Genetik

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Effiziente Produktion und Kennzeichnung der CRISPR/Cas9 generiert gen Knockouts im Modellsystem Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA-Replikation Timing mit Zebrafisch als ein In Vivo Modell-System-Profiling

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Hochdurchsatz-DNA-Extraktion und Genotypisierung von 3dpf Zebrafischlarven durch Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Verschiebung der Zebrafisch letale Skelett mutierte Penetranz um Nachkommen testen

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Entwicklungsbiologie

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

Screening von Spermien zur schnellen Isolierung von Keimbahnveränderungen im Zebrafisch

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Genotypisierung und Quantifizierung der In-Situ-Hybridisierungsfärbung in Zebrafischen

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Methoden, um die Sensitivität der High Resolution Melting Single Nucleotide Polymorphism Genotypisierung in Malaria erhöhen

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Immunologie und Infektion

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Untersuchung der Muskelregeneration in Zebrafischmodellen von Muskelerkrankungen

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

High-Resolution Melting PCR to Determine Sequence Variations in Mutant DNA Sequences

Source: Kisserli, A., et al. <a href="https://app.jove.com/v/56012">High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#39;s Disease Gene Susceptibility Assessment.</a> J. Vis. Exp. (2017).
In this video, we describe a high-resolution melting PCR technique to determine the length polymorphism of DNA fragments of varying sequence lengths, based on melting temperature analysis.

Hochauflösende Schmelz-PCR zur Bestimmung von Sequenzvariationen in mutierten DNA-Sequenzen

Source: Kisserli, A., et al. High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#39;s ...