All figures were created with Biorender.com under a license to Texas A&#38;M University. This work was supported by funds from the National Institute of Allergy and Infectious Disease (AI137112 and AI115138 to Z.N.A.), Texas A&#38;M AgriLife Research under the Insect Vectored Disease Grant Program, and the USDA National Institute of Food and Agriculture, Hatch project 1018401.

1. Scanning for single nucleotide polymorphisms (SNPs), HRMA primer design, and primer validation
<ol>
	<li>SNP identification in wild-type laboratory colony mosquitoes
	<ol>
		<li>Select the target exon to disrupt proper polypeptide translation. 
		NOTE: The target should be close to the start codon or amongst key residues required for protein function. The shorter the exon (e.g., &#8804;200 bases), the more difficult it is to target and analyze. Avoid editing close to the boundaries of an exon, as this forces on...

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+Guidelines+for+diagnosis,+treatment,+prevention+and+control.">Dengue Guidelines for diagnosis, treatment, prevention and control.</a> WHO. , 160 (2009).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+epidemiology+update.">Zika epidemiology update.</a> WHO. , 1-13 (2019).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+prevention+and+control+of+Chikungunya+fever.">Guidelines for prevention and control of Chikungunya fever.</a> WHO. , (2009).</li><li>World malaria report 2020: 20 years of global progress and challenges. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/i/item/9789240015791">https://www.who.int/publications/i/item/9789240015791</a> (2020)</li><li>Qiu, 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=Mutation+detection+using+Surveyor+nuclease.">Mutation detection using Surveyor nuclease.</a> Biotechniques. 36 (4), 702-707 (2004).</li><li>Babon, J. J., McKenzie, M., Cotton, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+resolvases+T4+endonuclease+VII+and+T7+endonuclease+I+in+mutation+detection.">The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection.</a> Molecular Biotechnology. 23 (1), 73-81 (2003).</li><li>Erali, M., Voelkerding, K. V., 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+melting+applications+for+clinical+laboratory+medicine.">High resolution melting applications for clinical laboratory medicine.</a> Experimental and Molecular Pathology. 85 (1), 50-58 (2008).</li><li>Reed, G. H., 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=Sensitivity+and+specificity+of+single-nucleotide+polymorphism+scanning+by+high-resolution+melting+analysis.">Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis.</a> Clinical Chemistry. 50 (10), 1748-1754 (2004).</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> Developmental Dynamics. 238 (12), 3168-3174 (2009).</li><li>Tong, S. Y., Giffard, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+applications+of+high-resolution+melting+analysis.">Microbiological applications of high-resolution melting analysis.</a> Journal of Clinical Microbiology. 50 (11), 3418-3421 (2012).</li><li>Simko, I. <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+DNA+melting+analysis+in+plant+research.">High-resolution DNA melting analysis in plant research.</a> Trends in Plant Science. 21 (6), 528-537 (2016).</li><li>Basu, S., 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=Silencing+of+end-joining+repair+for+efficient+site-specific+gene+insertion+after+TALEN/CRISPR+mutagenesis+in+Aedes+aegypti.">Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (13), 4038-4043 (2015).</li><li>O'Leary, S., Adelman, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+knockout+of+female-biased+genes+AeAct-4+or+myo-fem+in+Ae.+aegypti+results+in+a+flightless+phenotype+in+female,+but+not+male+mosquitoes.">CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes.</a> PLoS Neglected Tropical Diseases. 14 (12), 0008971 (2020).</li><li>Kojin, B. B., 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=Aedes+aegypti+SGS1+is+critical+for+Plasmodium+gallinaceum+infection+of+both+the+mosquito+midgut+and+salivary+glands.">Aedes aegypti SGS1 is critical for Plasmodium gallinaceum infection of both the mosquito midgut and salivary glands.</a> Malaria Journal. 20 (1), 11 (2021).</li><li>Ye, J., 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=Primer-BLAST:+a+tool+to+design+target-specific+primers+for+polymerase+chain+reaction.">Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.</a> BMC Bioinformatics. 13, 134 (2012).</li><li>Larkin, M. 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=Clustal+W+and+Clustal+X+version+2.0.">Clustal W and Clustal X version 2.0.</a> Bioinformatics. 23 (21), 2947-2948 (2007).</li><li>Notredame, C., Higgins, D. G., Heringa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Coffee:+A+novel+method+for+fast+and+accurate+multiple+sequence+alignment.">T-Coffee: A novel method for fast and accurate multiple sequence alignment.</a> Journal of Molecular Biology. 302 (1), 205-217 (2000).</li><li>Bassett, A. R., Tibbit, C., Ponting, C. P., Liu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+targeted+mutagenesis+of+Drosophila+with+the+CRISPR/Cas9+system.">Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.</a> Cell Reports. 6 (6), 1178-1179 (2014).</li><li>Zuker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mfold+web+server+for+nucleic+acid+folding+and+hybridization+prediction.">Mfold web server for nucleic acid folding and hybridization prediction.</a> Nucleic Acids Research. 31 (13), 3406-3415 (2003).</li><li>Hill, J. T., 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=Poly+peak+parser:+Method+and+software+for+identification+of+unknown+indels+using+sanger+sequencing+of+polymerase+chain+reaction+products.">Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.</a> Developmental Dynamics. 243 (12), 1632-1636 (2014).</li><li>Tsujimoto, H., Anderson, M. A. E., Myles, K. M., Adelman, Z. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+candidate+iron+transporters+from+the+ZIP/ZnT+gene+families+in+the+mosquito+Aedes+aegypti.">Identification of candidate iron transporters from the ZIP/ZnT gene families in the mosquito Aedes aegypti.</a> Frontiers in Physiology. 9, 380 (2018).</li><li>Slomka, M., Sobalska-Kwapis, M., Wachulec, M., Bartosz, G., Strapagiel, D. <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+(HRM)+for+high-throughput+genotyping-limitations+and+caveats+in+practical+case+studies.">High resolution melting (HRM) for high-throughput genotyping-limitations and caveats in practical case studies.</a> International Journal of Molecular Sciences. 18 (11), 2316 (2017).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5 (3), 407-415 (2015).</li></ol>

High-resolution melt analysis offers a simple and fast solution for the identification of indels generated by CRISPR/Cas9 technology in the vector mosquito Ae. aegypti. It provides flexibility, enabling the genotyping of mosquitoes mutated for a wide range of genes from flight muscle to iron metabolism and more13,14. HRMA can be performed in just a few hours from sample collection to the final analyses. Additional time is required for primer design and w...

As vectors of pathogens such as dengue1, Zika2, and chikungunya3 viruses, as well as malarial parasites4, mosquitoes represent a significant public health threat to humans. For all these diseases, there is a substantial focus of transmission intervention on the control of mosquito vectors. Study of the genes important in, for example, permissiveness to pathogens, mosquito fitness, survivorship, reproduction, and resistance to insecticides is key for developing novel mosquito control strategies. For such purposes, genome editing in mosquitoes is becoming a common practice, especially with the development of technologies such as HEs, ZFNs, TALENs, and most recently, CRISPR with Cas9. The establishment of gene-edited strains typically involves backcrossing individuals carrying the desired mutations for a few generations to minimize off-target and founder (bottleneck) effects, followed by crossing heterozygous individuals to generate homozygous or trans-heterozygous lines. In the absence of a dominant marker, molecular genotyping is necessary in this process because, in many cases, no clear phenotypic traits can be detected for heterozygous mutants.
Although sequencing is the gold standard for genotypic characterization, performing this across hundreds, or possibly thousands of individuals, poses significant costs, labor, and time required to obtain results, which is especially critical for organisms with short lifespans such as mosquitoes. Commonly used alternatives are Surveyor nuclease assay5 (SNA), T7E1 assay6, and high resolution melt analysis (HRMA, reviewed in7). Both SNA and T7E1 use endonucleases that cleave only mismatched bases. When a mutated region of the heterozygous mutant genome is amplified, DNA fragments from mutant and wild-type alleles are annealed to make mismatched double-stranded DNA (dsDNA). SNA detects the presence of mismatches via digestion with a mismatch-specific endonuclease and simple agarose gel electrophoresis. Alternatively, HRMA uses the thermodynamic properties of dsDNA detected by dsDNA-binding fluorescent dyes, with the disassociation temperature of the dye varying based on the presence and type of mutation. HRMA has been used for the detection of single-nucleotide polymorphisms (SNPs)8, mutant genotyping of zebra fish9, microbiological applications10, and plant genetic research11, among others.
This paper describes HRMA, a simple method of molecular genotyping for mutant mosquitoes generated by CRISPR/Cas9 technology. The advantages of HRMA over alternative techniques include 1) flexibility, as it has been proven useful for various genes, a wide range of indel sizes, as well as the distinction between different indel sizes and heterozygous, homozygous, and trans-heterozygous differentiation12,13,14, 2) cost, as it is based on commonly used PCR reagents, and 3) time-saving, as it can be performed in just a few hours. In addition, the protocol uses a small body part (a leg) as a source of DNA, allowing the mosquito to survive the genotyping process, permitting the establishment and maintenance of mutant lines.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>70% ethanol solution in water</td>
		</tr>
		<tr>
			<td>96-well PCR and Real-time PCR plates</td>
			<td>VWR</td>
			<td>82006-636</td>
			<td>For obtaining genomic DNA (from the mosquito leg)</td>
		</tr>
		<tr>
			<td>96-well plate templates</td>
			<td></td>
			<td></td>
			<td>House-made printed, for genotype recording</td>
		</tr>
		<tr>
			<td>Bio Rad CFX96</td>
			<td>Bio Rad</td>
			<td></td>
			<td>PCR machine with gradient and HRMA capabilities</td>
		</tr>
		<tr>
			<td>Diversified Biotech reagent reservoirs</td>
			<td>VWR</td>
			<td>490006-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Exo-CIP Rapid PCR Cleanup Kit</td>
			<td>New England Biolabs</td>
			<td>E1050S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri Dish</td>
			<td>VWR</td>
			<td>89001-246</td>
			<td>150 mm x 20 mm</td>
		</tr>
		<tr>
			<td>Hard-shell thin-wall 96-well skirted PCR plates</td>
			<td>Bio-rad</td>
			<td>HSP9665</td>
			<td>For HRMA</td>
		</tr>
		<tr>
			<td>Multi-channel pipettor (P10)</td>
			<td>Integra Biosciences</td>
			<td>4721</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor (P300)</td>
			<td>Integra Biosciences</td>
			<td>4723</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Polyolefin Acrylate Sealing tape, Thermo Scientific</td>
			<td>VWR</td>
			<td>37000-548</td>
			<td>To use with the 96-well&#160; PCR plates for obtaining genomic DNA</td>
		</tr>
		<tr>
			<td>Optical sealing tape</td>
			<td>Bio-rad</td>
			<td>2239444</td>
			<td>To use with the 96-well skirted PCR plates for HRMA</td>
		</tr>
		<tr>
			<td>Phire Animal tissue direct PCR Kit (without sampling tools)</td>
			<td>Thermo Fisher</td>
			<td>F140WH</td>
			<td>For obtaining genomic DNA and performing PCR</td>
		</tr>
		<tr>
			<td>Plastic Fly Vial Dividers</td>
			<td>Genesee</td>
			<td>59-128W</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Melt Analysis Software</td>
			<td>Bio Rad</td>
			<td>1845025</td>
			<td>Used for genotyping the mosquito DNA samples and analyzing the thermal denaturation properties of double-stranded DNA (see protocol step 3.3)</td>
		</tr>
		<tr>
			<td>SeqMan Pro</td>
			<td>DNAstar Lasergene software</td>
			<td></td>
			<td>For multiple sequence alignment</td>
		</tr>
		<tr>
			<td>Single-channel pipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers Dumont #5 11 cm</td>
			<td>WPI</td>
			<td>14098</td>
			<td></td>
		</tr>
		<tr>
			<td>White foam plugs</td>
			<td>VWR</td>
			<td>60882-189</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Drosophila Vials, Polystyrene</td>
			<td>Genesee</td>
			<td>32-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Fly Vial Tray, Blue</td>
			<td>Genesee</td>
			<td>59-164B</td>
			<td></td>
		</tr>
	</tbody>
</table>

indel detection following crispr/cas9 mutagenesis using high-resolution melt analysis in the mosquito aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Mosquitoes containing mutations in the genes AaeZIP11 (putative iron transporter21) and myo-fem (a female-biased myosin gene related to flight muscles13) were obtained using CRISPR/Cas9 technology, genotyped using HRMA, and sequence-verified (Figure 5). Figure 5A and Figure 5C show the normalized fluorescence intensity from the HRM curves from AaeZIP11 and <e...

Watch this Scientific Journal Video about Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti at JoVE.com

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

This protocol is a very simple and efficient way to detect mutants generated by CRISPR/Cas9 technology from numerous individuals. The two main advantages of this technique are that it can be done in a few hours and that it is applicable to a multitude of organisms. Primer design to can be challenging for certain genes and multiple rounds of primer design and verification may be required to make HRMA efficient.To perform a gradient PCR, start by preparing a master mix. Then remove 19 microliters of the master mix for the non-template control or NTC in a 96-well plate. Then, add the template to the remaining master mix.Aliquot 20 microliters of the sample mix into the 96-well plate. Perform PCR following the cycling parameters and then generate the thermal melt profiles using the parameters mentioned in the manuscript. After gathering all the required material, prepare a master mix with 0.5 microliters of the DNA release reagent and 20 microliters of dilution buffer per individual to be genotyped.Using a multichannel pipet, aliquot 20 microliters of the mix into a PCR plate and leave the PCR plate on ice. Next, place the anesthetized G1 mosquitoes in a glass Petri dish to keep them sedated and place eight wild-type mosquitoes in the second Petri dish. Wipe a pair of tweezers with 70%ethanol and use the disinfected tweezers to remove one of the mosquitoes hind legs.Then, submerge the leg in the DNA release reagent solution. Place the mosquito in the corresponding vial and close the vial with a sponge. Once done, wipe the tweezers with 70%ethanol before proceeding with removing the leg from the next mosquito until the 96-well plate is completed.Later, seal the plate with an optical PCR plate seal and incubate the PCR plate containing the legs at room temperature for two to five minutes, followed by incubation at 98 degrees Celsius for two minutes. Allow the plate to cool down to room temperature. For PCR, prepare a master mix containing the preferred components and then use a multichannel pipette to transfer 19 microliters of the master mix into each well of the 96-well plate.After transferring one microliter of the DNA release solution containing previously prepared mosquito DNA to the plate, seal the plate with an optical PCR plate seal. Perform the PCR with the appropriate cycling parameters to generate thermal melt profiles following the parameters described in the manuscript, then examine the melt profiles by assigning wild-type control to the reference cluster. Mark the different clusters with corresponding colors on the 96-well template.Next, select the individuals with curves of interest and remove the selected individuals from the tubes to back cross. Blood feed the made it females, followed by collecting G2As. In the representative analysis, the sequence analysis of the Aedes aegypti's ZIP11 mutant is shown.The electropherogram from Aedes aegypti ZIP11 heterozygous mutants indicated the nucleotide position where the indel occurred. The indel is represented by a shift from single to double peaks as polymorphic positions show both nucleotides concomitantly. At the end of the run, the number of deleted or inserted base pairs was calculated by counting the single peaks.Mosquitoes containing mutations in the genes Aedes aegypti ZIP11 and myo-fem were genotyped and sequenced verified using the high-resolution melt analysis of HRMA. The fluorescent signals of the samples were normalized to relative values of one to zero. Additionally, each curve was subtracted from the wild-type reference and denoted the corresponding magnification.A failed automatic cluster assignment is shown in the representative results, where a proper distinction between the groups was not made. Further, each sample was analyzed individually and assigned to the correct groups based on the similarity to reference samples of heterozygotes, homozygotes, and transheterozygotes. After performing HRMAs, sequencing the DNA from the mutated individuals is necessary to analyze the indels and identify the other mutations in the targeted sequence.

indel-detection-following-crisprcas9-mutagenesis-using-high

Research

JoVE Journal

Genetics

3.1K Views.  Texas A&M University. Questo protocollo è un modo molto semplice ed efficiente per rilevare i mutanti generati dalla tecnologia CRISPR / Cas9 da numerosi individui. I due principali vantaggi di questa tecnica sono che può essere fatto in poche ore e che è applicabile a una moltitudine di organismi. La progettazione del primer può essere impegnativa per alcuni geni e possono essere necessari più cicli di progettazione e verifica del primer per rendere efficiente l'HRMA.Per eseguire una PCR sfumata, iniz...