Name: Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis (Video) | JoVE Journal
Uploaded: 2014-02-05T17:30:00.000+00:00
Duration: 6 min 30 s
Description: Learn rapid zebrafish genotyping using PCR with high-resolution melt analysis (HRMA). This sensitive method detects single nucleotide changes and small insertions or deletions efficiently.

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5 minutes

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>
			<li>Quickly place the fish in a labeled tank with fresh water for recovery; carefully label both the tank and corresponding tube that will contain the fin clip. Change the water and feed the fish every other day.</li>
			<li>Pick up the fin clip with a sterile pipette tip and transfer it into a tube filled with 100 &#956;l DNA lysis buffer.</li>
			<li>Discard the pipette tip, and discard the razor blade into a sharps container.</li>
		</ol>
		</li>
		<li>For embryo tail clip:
		<ol>
			<li>Place embryos into 0.004% MS-222 (tricaine) solution. Wait 2 min.</li>
			<li>Cut a piece of tail with forceps under a dissecting microscope.</li>
			<li>Put the tail into a labeled tube filled with 30 &#956;l DNA lysis buffer.</li>
			<li>Quickly put the embryo into a tube containing 100 &#956;l 4% paraformaldehyde.</li>
			<li>Label tubes with embryos and their corresponding tail tubes.</li>
			<li>Store the tubes with embryos at 4 &#176;C overnight for fixation.</li>
			<li>Clean the forceps using Milli-Q water and Kimwipes between each embryo to minimize contamination.</li>
		</ol>
		</li>
		<li>For whole embryos:
		<ol>
			<li>Place embryos into 0.004% MS-222 (tricaine) solution. Wait 2 min.</li>
			<li>Put 1-5 embryos in a tube filled with 100 &#956;l DNA lysis buffer. Dechorionation is not necessary.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>DNA digestion 
	Incubate tubes with tissue (fin or tail clips or embryos) at 55 &#176;C for 4 hr up to overnight12.</li>
	<li>Inactivate the Proteinase K 
	Heat tubes to 95 &#176;C for 15 min12. DNA should be used for PCR immediately, or stored at -20 &#176;C for up to 3 months.</li>
</ol>
2. PCR
<ol>
	<li>Design Primers either manually or by primer design software (e.g. Lightscanner Primer Design Software- Biofire). PCR products for HRMA are usually 50-200 bp; PCR products for SNP detection should be smaller, typically 50-80 bp.</li>
	<li>PCR
	<ol>
		<li>Perform PCR reaction in a 96-well or 384-well plate.</li>
		<li>Use 10 &#956;l total volume: 4 &#956;l LightScanner master mix (0.1 U hot-start Taq-AB polymerase, buffer, 0.2 mM dNTPs, 2 mM magnesium chloride, and 1x fluorescent double-stranded DNA-binding dye); 5 pmol each primer, and 1 &#956;l genomic DNA template extracted from adult tail fin or 3 &#956;l genomic DNA from embryonic tail13.</li>
		<li>Cover samples with 30 &#956;l mineral oil.</li>
		<li>Cover the plate with an optically-transparent adhesive seal.</li>
		<li>Optimize PCR cycling conditions- typical conditions are 95 &#176;C for 5 min and 30 cycles of 10 sec at 95 &#176;C, 25 sec at 60 &#176;C, 30 sec at 72 &#176;C, ending with 95 &#176;C for 30 sec and cooled to 15 &#176;C.</li>
		<li>Store PCR plates at 4 &#176;C or continue directly to HRMA.</li>
		<li>Confirm our PCR product by analysis of its size using agarose gel electrophoresis during initial optimization of PCR; and if indicated, by sequencing of the PCR product.</li>
	</ol>
	</li>
</ol>
3. HRMA
<ol>
	<li>Heat plates
	<ol>
		<li>Place plate in a melt analysis system.</li>
		<li>Open the software (LightScanner Software Call-IT 2.0).</li>
		<li>Heat plates from 60-95 &#176;C.</li>
		<li>Create a new data storage file.</li>
	</ol>
	</li>
	<li>Analyze HRMA data (Figure 2). 
	Multiple steps of analysis are possible. We show the most commonly used set of data manipulations.
	<ol>
		<li>Subset setting 
		Click Subset tab. Select the wells with samples.</li>
		<li>Normalization
		<ol>
			<li>Select the Normalize tab in the left panel. To eliminate fluorescence variance, manually position the parallel double-line in pre- (initial) and post (final) -melt regions as illustrated (Figure 2C, arrowheads) and normalize premelt and post-melt fluorescence signals of all samples to 1 and 0, respectively14.</li>
			<li>Adjust the positioning of the lines so that the melt region is in the region between the pre- and post-melt lines, but not selected. In general, the Lower Min and Lower Max (and Upper Min and Upper Max) temperature lines should be placed approximately 1 &#176;C apart.</li>
			<li>Choose temperature positions for normalization such that all samples have maximum or full (100%) fluorescence&#160;for the premelt region and minimum or no (0%) fluorescence for the post-melt region (as best as possible).&#160;Normalization should result in a near-horizontal line at the maximum fluorescence of 1 that extends until the melt point and then a near-horizontal line from the melt point at the minimum fluorescence of 0; there should not be fluorescence levels above 1 or below 0.&#160;For example, in Figure 2C, normalize the premelting curves using temperature ranges of 79-80 &#176;C.</li>
		</ol>
		</li>
		<li>Grouping/Clustering 
		Distinguish genotypes based on their melting temperature (Figure 2C, green box). Select Grouping.
		<ol>
			<li>Select Autogroup from the Standards selection list under the Grouping section.</li>
			<li>Select Normal or High for melting profiles with single transitions or multiple transitions respectively from the Sensitivity selection list under the Grouping section.</li>
			<li>Select Compute Groups under the Grouping section.</li>
			<li>Go to the File menu and click Save to save the results.</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/pubmed?term=((A+transposon-mediated+gene+trap+approach+identifies+developmentally+regulated+genes+in+zebrafish%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">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/pubmed?term=((The+Tol2kit%3A+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">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/pubmed?term=((Targeted+gene+disruption+in+somatic+zebrafish+cells+using+engineered+TALENs%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Heritable+gene+targeting+in+zebrafish+using+customized+TALENs%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((High-resolution+melting+analysis+%28HRMA%29%3A+more+than+just+sequence+variant+screening%5BTitle%5D)+AND+%22Hum.+Mutation%22%5BJournal%5D)">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/pubmed?term=((Simple+methods+for+generating+and+detecting+locus-specific+mutations+induced+with+TALENs+in+the+zebrafish+genome%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">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/pubmed?term=((Genotyping+of+single-nucleotide+polymorphisms+by+high-resolution+melting+of+small+amplicons%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">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/pubmed?term=((V+Amplicon+DNA+melting+analysis+for+mutation+scanning+and+genotyping%3A+cross-platform+comparison+of+instruments+and+dyes%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">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., Martínez, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+resolution+melting+analysis+for+the+identification+of+novel+mutations+in+DKC1+and+TERT+genes+in+patients+with+dyskeratosis+congenita%5BTitle%5D)+AND+%22Blood+Cell.+Mol.+Dis.%22%5BJournal%5D)">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/pubmed?term=((High+resolution+melt+analysis+%28HRMA%29%3B+a+viable+alternative+to+agarose+gel+electrophoresis+for+mouse+genotyping%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((PIK3CA+hotspot+mutation+scanning+by+a+novel+and+highly+sensitive+high-resolution+small+amplicon+melting+analysis+method%5BTitle%5D)+AND+%22J.+Mol.+Diagn%22%5BJournal%5D)">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/pubmed?term=((A+rapid+and+efficient+method+of+genotyping+zebrafish+mutants%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">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/pubmed?term=((Zebrafish+foxP2+zinc+finger+nuclease+mutant+has+normal+axon+pathfinding%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+High+Resolution+Melting+analysis+as+an+alternate+tool+to+screen+for+risk+alleles+associated+with+small+kidneys+in+Indian+newborns%5BTitle%5D)+AND+%22BMC+Nephrol%22%5BJournal%5D)">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/pubmed?term=((V+Expanded+instrument+comparison+of+amplicon+DNA+melting+analysis+for+mutation+scanning+and+genotyping%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">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/pubmed?term=((High-Resolution+Genotyping+by+Amplicon+Melting+Analysis+Using+LCGreen%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">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/pubmed?term=((A+closed-tube+methylation-sensitive+high+resolution+melting+assay+%28MS-HRMA%29+for+the+semi-quantitative+determination+of+CST6+promoter+methylation+in+clinical+samples%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">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/pubmed?term=((Comparative+analysis+of+two+broad-range+PCR+assays+for+pathogen+detection+in+positive-blood-culture+bottles%3A+PCR-high-resolution+melting+analysis+versus+PCR-mass+spectrometry%5BTitle%5D)+AND+%22J.+Clin.+Microbiol%22%5BJournal%5D)">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><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

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. The temperatures for the melt of the amplicon depend on the size and GC-content, but generally start and end temperatures of 50 ˚C and 95 ˚C are appropriate (Figures 2A and 2B). Once the melt is performed, analysis of the fluorescence melt curves typically requires norm...

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 ...

The overall goal of this procedure is to rapidly screen for different genotypes, mutations or transgenes in zebrafish. This is accomplished by first extracting DNA from thin clips and then amplifying the DNA by PCR. The next step is to denature the PCR Amplicons while recording the melt curves, which are analyzed by software.Ultimately, the results show distinguishable melt curves for different genotypes. The main advantage of this technique over existing method like gel electrophoresis volume PCR, is that is sensitive to single nucleotide changes, small insertion, all deletions, and this technique is fast and reliable. Though this technique may be used to rapidly, effectively genotype zebra fish, it can also be applied to other systems and model organisms, including cell lines, mice, and other animals.On the day of the DNA preparation, add fresh proteinase K to the lysis buffer at a concentration of one milligram per milliliter. Tissue can be collected from an adult fish using a thin clip or from an embryonic fish. First anesthetize the fish in Trica solution.Wait until the gill movements slow. Then put the fish on a stack of tissues and with a sterile razor blade, cut off a small piece of the tail fin about two to three millimeters long. Quickly place the fish in a labeled tank with fresh water for recovery.Pick up the fin clip with a sterile pipette tip and transfer it into a tube filled with 100 microliters of DNA lysis buffer. Be sure to label both the animal's tank and the tube, incubate all the collected tissues for at least four hours and up to overnight at 55 degrees Celsius after the incubation. Inactivate the protein ACE K by heating the tubes at 95 degrees Celsius for 15 minutes.These samples should be used for PCR immediately, but can also be stored at minus 20 degrees Celsius for up to three months. Perform the PCR in a 96 or 384 well plate for each reaction. Combine no more than one microliter of the adult DNA with four microliters of light scanner.Master mix containing the fluorescent probe and primers at a final concentration of five pico molar each. If the DNA was from an embryo, use up to three microliters. Cover the reactions with 30 microliters of mineral oil and then cap them shut.Next, cover the loaded PCR plate with an optically transparent adhesive seal. Now optimize the PCR cycling conditions. A typical condition set begins with five minutes of melt time.This is followed by 30 PCR cycles with ten second melts, 25 seconds of a kneeling time and 30 seconds of elongation. The reaction should end with an added 32nd melt and then cool to 15 degrees Celsius. Analyze the PCR plate by placing it into a melt analysis system in the software.Set up the temperature and create a new data storage file. Set up a subset. Select the wells with samples on the lower left side of the screen.Select the normalized tab to eliminate fluorescent variants. Manually position the parallel double line in prem, melt and post melt regions and normalize premel and post melt fluorescent signals of all samples to one and zero respectively. Next, distinguish the genotypes based on their melting temperature by first selecting grouping.Then select auto group from the standard selection list. Under the grouping section, select normal or high for melting profiles with single transitions or multiple transitions respectively. They're in the sensitivity selection list under the grouping section.Now select compute groups under the grouping section and then go to the file menu and click save to save the results. The protocol can be performed during a single day or separated in steps over several days. When performing PCR, the temperatures for the melt of the amplicon depends on the size and GC content, but generally start and end temperatures of 60 degrees Celsius and 95 degrees Celsius are appropriate once the melt is performed.Analysis of the fluorescent melt curves typically requires normalization of the variation of the different sample curves using pre and post melt regions as standards. This improves comparison of results from different samples in which the variation of fluorescence is related to minor experimental variations. Each pair of temperature lines for normalization should be placed approximately one degree Celsius apart.The data can be represented in two ways by a graph that shows the melt curve profiles files, or by a graph that shows a subtractive difference plot in comparison to a reference sample following HRMA analysis mutations or transgenes can be detected. Two different mutations in the EIF two B five gene are noted by the red and blue curves. These mutant samples are identified by their significant difference in their melt curve, shapes, or change in fluorescence when compared to the standard wild type curves.This example of four different genotypes in a single collection of embryos illustrates the power and sensitivity of the method. Once master, this technique can be done in less than eight hours. After watching this video, you should have a good understanding of how to perform HRMA for efficient large scale screening of zebrafish genotypes.

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

Research

JoVE Journal

Biology

This article describes DNA extraction from zebrafish tail clip. The protocol demonstrates a rapid and efficient method to analyze zebrafish genotypes.

DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

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.

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

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 Sperm for the Rapid Isolation of Germline Edits in Zebrafish

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

Learn rapid zebrafish genotyping using PCR with high-resolution melt analysis (HRMA). This sensitive method detects single nucleotide changes and small insertions or deletions efficiently.

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

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