We thank members of the Blaschke, Grunwald, and Wittwer labs for advice and technical assistance. This work is supported by the PCMC Foundation, NIH R01 MH092256 and DP2 MH100008, and the March of Dimes Foundation research grant #1-FY13-425, to JLB.

1. DNA Preparation
<ol>
	<li>Prepare DNA lysis buffer: 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.3% Tween 20, 0.3% NP40. Add fresh Proteinase K to a final concentration of 1 mg/ml on the day of use12.</li>
	<li>Tissue collection:
	<ol>
		<li>For adult fish fin clip:
		<ol>
			<li>Anesthetize fish: Place the fish in 0.004% MS-222 (tricaine) solution. Wait until gill movement slows.</li>
			<li>Put fish on a stack of 5-10 Kimwipes and cut a small piece of the tail fin, about 2-3 mm, with a sterile razor blade.</li>
...

<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>

The authors have nothing to disclose.

PCR combined with HRMA is a powerful technology for zebrafish genotyping. The advantages of this approach are its speed, robustness, and sensitivity to detect even point mutations. The entire protocol, from fin-clip to melt-curve analysis, can be performed in less than eight hours by a single individual. In addition, the technique is amenable for high-throughput screening; does not require the use of ethidium bromide; and is sealed for all PCR and analysis steps which helps minimize contamination issues.
<p class="jo...

Zebrafish (Danio rerio) is a vertebrate model system widely used for studies of development and disease modeling. Recently, numerous transgenic and mutation technologies have been developed for zebrafish.&#160;Rapid transgenesis techniques, usually based on a Tol2 transposon system1, have been combined with improved cloning options for multiple DNA fragment assembly2. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used to target loci in both somatic and germline cells in zebrafish3,4. These techniques can efficiently generate genetically modified animals, with high-frequency mutation creation and germ-line transmission3,4.
Despite these advances, traditional genotyping techniques in zebrafish limit the full power of the mutagenesis and transgenesis tools.&#160;PCR followed by gel electrophoresis, sometimes combined with restriction enzyme digestion, is widely used to detect genome modification, but is time-consuming and less sensitive to identify small insertions or deletions. TaqMan probe assays have high initial costs and require careful optimization. Sequencing of PCR products can take several days and is not practical for large-scale screening.&#160;Restriction fragment length polymorphism (RFLP) analysis can only discriminate SNPs affecting a limited range of restriction enzyme recognition sites.

High-resolution melting analysis (HRMA), a closed-tube post-PCR analysis method, is a recently developed method that is rapid, sensitive, inexpensive, and amenable to screening large numbers of samples.&#160;HRMA can be used to detect SNPs, mutations, and transgenes5-7. HRMA is based on thermal denaturation of double-stranded DNAs, and each PCR amplicon has a unique dissociation (melt) characteristic5. Samples can be discriminated due to their different nucleotide composition, GC content, or length, typically in combination with a fluorescent dye that only binds double-stranded DNA8. Thus, HRMA can distinguish different genotypes based on the different melt-curve characteristics. Because HRMA uses low-cost reagents and is a single-step post-PCR process, it can be used for high-throughput strategies.&#160;HRMA is nondestructive, so following analysis the PCR amplicons can be used for other applications.&#160;HRMA has been applied in many organisms and systems, including cell lines, mice, and humans9-11. Its use has recently been described in zebrafish to detect mutations induced by zinc finger nucleases (ZFNs) and TALENs6,12,13.

In this paper, we describe how to perform PCR-based HRMA in embryonic and adult zebrafish (Figure 1). This protocol is suitable for detecting SNPs, transgenes, and mutations, including single base-pair changes, insertions, or deletions.

<table border="0" cellpadding="0" cellspacing="0" width="842">
	<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

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

The protocol can be performed during a single day or separated in steps over several days (flow diagram of work is shown in Figure 1). DNA extraction is followed by the melt and analysis of PCR amplicons.&#160;The temperatures for the melt of the amplicon depend on the size and GC-content, but generally start and end temperatures of 50 &#730;C and 95 &#730;C are appropriate (Figures 2A and 2B).&#160;Once the melt is performed, analysis of the fluorescence melt curves typ...

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 combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

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

22.0K Görüntüleme.  University of Utah School of Medicine.  Yüksek çözünürlüklü erime analizi (HRMA) ile bir araya PCR zebrafish genotip için hızlı ve etkili bir yöntem olarak gösterilmiştir. 