The authors would like to thank all the academic experts and employees at the Specialized Hematology Laboratory, Department of Hematology, Division of Internal Medicine, University Medical Centre Ljubljana.

The study was approved by the Committee of Medical Ethics of the Republic of Slovenia. All procedures were in accordance with the Helsinki declaration.
1. Fluorescence-based quantitative real-time PCR (qPCR) and post-qPCR analysis by HRM
<ol>
	<li>Resuspend primers listed in the Table of Materials to 100 &#181;M with sterile, RNase and DNase free H2O (see Table of Materials). Make a 10 &#181;M working concentration primer. Primers used in the protocol were published before2.</li>
	<li>Quantitate granulocytes DNA by the fluorescence staining method following the manufacturer&#8217;s instruction11 (see Table of Materials). Prepare a 20 ng/&#181;L DNA solution using 10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0 (see Table of Materials).
	<ol>
		<li>In addition to DNA samples, prepare at least three DNA controls: two positive controls with the NM_004343.3 (CALR): c.1099_1150del52, p.(Leu367Thrfs*46) (52 bp deletion or type 1 mutation), and NM_004343.3 (CALR): c.1154_1155insTTGTC, p.(Lys385Asnfs*47) (5 bp insertion or type 2 mutation) genetic variant, as well as wild-type DNA and negative control as a non-template (NTC) control. 
		NOTE: Before performing the HRM setup, confirm that the qPCR instrument is calibrated for HRM experiments.</li>
	</ol>
	</li>
	<li>Prepare qPCR HRM Master Mix according to manufacturer&#8217;s protocol (see Table of Materials) as follows: 10 &#181;L of 2x qPCR Master Mix with Dye, 0.2 &#181;L of 10 &#181;M Forward Primer, 0.2 &#181;L of 10 &#181;M Reverse Primer, 8.6 &#181;L of sterile, RNase and DNase free H2O. Use the primers from the Table of Materials. Run three replicates for each DNA sample and control.
	<ol>
		<li>Prepare the qPCR HRM Master Mix according to the number of samples being processed. Include excess volume of at least 10% in the calculations to provide excess volume for the loss that occurs during reagent transfers.</li>
	</ol>
	</li>
	<li>Mix the reaction contents by gently tapping and inverting the tube and vortexing for 2-3 s. Collect all the scattered droplets from the wall of the tube to the bottom by a brief spin.</li>
	<li>Prepare a reaction plate appropriate for the instrument and HRM experiment (see Table of Materials). Transfer 19 &#181;L of the qPCR HRM Master Mix to the appropriate wells of the 96-well optical reaction plate.</li>
	<li>Pipet 1 &#181;L of the negative controls, positive controls, and samples into the appropriate wells of the optical reaction plate. For the NTC, transfer 1 &#956;L of sterile, RNase and DNase free H2O used for preparing the qPCR HRM Master Mix instead of DNA.</li>
	<li>Seal the reaction plate with the optical adhesive film. Do it firmly to prevent evaporation during the run. Check that the optical adhesive film is plane across all the wells in the reaction plate to ensure correct fluorescence detection.</li>
	<li>Spin the reaction plate at 780 x g at room temperature (RT) for 1 minute. Check that the liquid is at the bottom of the wells in the reaction plate. Proceed to run the assay. 
	NOTE: The protocol can be paused here. Store the plate at 4 &#176;C for no more than 24 h. Allow the plate stored at 4 &#176;C to warm to RT, and then spin the plate briefly before running it.</li>
	<li>According to the manufacturer&#8217;s instruction prepare and start the run to amplify and melt the DNA and to generate HRM fluorescence data in the qPCR instrument. In the instrument system software, assign the controls and samples to the appropriate wells.</li>
	<li>For the CALR genetic variant detection, change the default instrument amplification protocol and amplify the DNA using the following thermal cycling protocol: 95 &#176;C for 10 minutes, followed by 50 cycles of 95 &#176;C for 10 s and 62.5 &#176;C for 60 s.</li>
	<li>Perform the melt curve/dissociation (HRM) stage immediately after qPCR according to the manufacturer&#8217;s instructions12 as follows: 95 &#176;C for 10 s, 60 &#176;C for 1 min, and then a ramp rate of 0.025 &#176;C/s until 95 &#176;C. Hold the temperature at 95 &#176;C for 15 s and at 60 &#176;C for 15 s.</li>
	<li>The run ends automatically. First, review and verify the amplification plot (Figure 2).</li>
	<li>In the experiment menu of the instrument system software, select the amplification plot option. If no data are displayed, click the green button Analyze.
	<ol>
		<li>In the amplification plot tab, from the plot type drop-down menu, select the plot that displays the amplification data as the raw fluorescence readings normalized to the fluorescence from the passive reference (&#916;Rn) as a function of cycle number (&#916;Rn vs Cycle). In the plot color drop-down menu, select Sample.</li>
	</ol>
	</li>
	<li>From the graph type drop-down menu, select the linear amplification graph type. Show the baseline start and end cycle on the linear amplification graph by selecting the baseline start option. Verify that the baseline is set correctly. The end cycle should be set a few cycles before the cycle number where significant fluorescent signal is detected. The baseline is usually set from 3 to 15 cycles (Figure 2).</li>
	<li>From the graph type drop-down menu, select the log10 amplification graph type. Show the threshold line on the graph by selecting the threshold option. Adjust the threshold accordingly if not set correctly. The correctly set threshold means that the threshold line crosses the exponential phase of the qPCR curves (Figure 2).</li>
	<li>Verify that normal amplification curves are in all sample and positive control wells. Verify that there is no amplification in the NTC wells before cycle 40. A normal amplification plot shows an exponential increase in fluorescence that exceeds the threshold between cycles 15 and 35 (Figure 2). Exclude the sample wells from the analysis if there is no amplification in the well position. 
	NOTE: If the amplification plot looks abnormal or the NTC well indicates the amplification, identify and resolve the problem according to the manufacturer&#8217;s troubleshooting guide.</li>
	<li>Exclude the outlier with the quantification cycle (Cq) value that differs from replicates by more than 2 in the instrument system software12. The outliers may produce erroneous HRM results.</li>
	<li>In the derivative melt curves, review the pre- and post-melt regions/temperature lines. The pre- and post-melt regions should be within a flat area where there are no large peaks or slopes in the fluorescent levels (Figure 3). If needed adjust it accordingly. Set up the start and stop of the pre- and post-melt temperature lines approximately 0.5 &#176;C apart from each other (Figure 3). Restart the analysis if the parameters are adjusted by clicking on the Analyze button.</li>
	<li>In the difference plot tab, in the plot settings tab, choose one of the wild-type control (homozygote) replicates as the reference DNA and restart the analysis by clicking on the Analyze button.</li>
	<li>In the aligned melt curves tab, confirm that all positive controls have the correct genotype and NTC failed to amplify (Figure 4). From the well table, select the wells containing a positive control to highlight the corresponding melt curve in the analysis plots. Confirm that the color of the line corresponds to the correct genotype. Repeat steps for the wells containing the other positive controls and NTC.</li>
	<li>In the aligned melt curves tab, carefully review the plot displays for the unknown samples and compare them to the plot displays for controls (Figure 4). From the well table, select the wells containing the unknown sample replicates, check the color of the melt curve and align them with the controls in an ordered sequence by selecting the wells containing positive controls one by one. Repeat the process for all the unknown samples. 
	NOTE: The unknown sample contains the variant of one of the controls if its melting curve is tightly aligned to it. Different variant groups (different colors) could be displayed indicating that the unknown sample consists of an unknown variant, not corresponding to the controls (Figure 8). A low level of the somatic genetic variant can be present in the patient&#39;s sample. This could influence the interpretation of the HRM result, particularly at the detection limit of the assay (Figure 9). In these cases, the color or even the shape of the line could closely resemble that of the wild-type genotype.
	<ol>
		<li>When the result is inconclusive, combine the HRM results with the results of the agarose gel electrophoresis and sequencing methods. Retest the sample or request and retest a new sample if needed.</li>
		<li>Carefully review the data set for replicate curves and check that the alignment of each replicate within the group is tight. Exclude the replicate if it does not align tightly with the other samples in the group (outlier). Retest the sample if more outliers are present and the results are inconclusive. Be aware that the quantity and quality of the DNA sample influence the HRM results.</li>
	</ol>
	</li>
	<li>Analyze the result for the unknown sample in the Difference plot tab. Repeat the procedure described in the step 1.21 and verify that the results obtained for the unknown sample are the same.</li>
	<li>Then, run the qPCR HRM products on the agarose gel. 
	NOTE: The protocol can be paused here. Store the plate at 4 &#176;C for no more than 24 hours or at -20 &#176;C for a longer period time but no more than 7 days. Allow the plate stored at 4 &#176;C to warm to RT, and then spin the plate briefly before running it. Allow the plate stored at - 20 &#176;C to thaw and warm to RT, and then spin the plate briefly before running it.</li>
</ol>
2. Agarose gel electrophoresis
<ol>
	<li>Run qPCR HRM products on a 4% agarose pre-cast gel containing a fluorescent nucleic acid stain using the appropriate gel electrophoresis system (see Table of Materials, Figure 5). Run only one positive, negative, NTC and sample qPCR HRM replicate. 
	NOTE: Gloves should always be worn when handling gels. Any other gel electrophoresis system can fulfill this purpose if the equivalent agarose percentage, fluorescent nucleic acid stain and well format are chosen.</li>
	<li>Run the gel electrophoresis system according to the manufacturer&#8217;s protocol (see Table of Materials). Remove the pre-cast gel in the cassette from the package, remove the comb and insert it into the apparatus according to the manufacturer&#39;s instructions (see Table of Materials). 
	NOTE: Load the gel within 15 minutes of opening the package.</li>
	<li>Dilute a 10 &#956;L sample to 20 &#956;L with sterile, RNase and DNase free H2O. Load each well with 20 &#956;L of diluted sample.</li>
	<li>Dilute 3 &#956;L of DNA size standard solution to 20 &#956;L with sterile, RNase and DNase free H2O (see Table of Materials). Load the M well with 20 &#181;L of diluted DNA size standard solution (Figure 6).</li>
	<li>Fill any empty wells with 20 &#181;L of sterile, RNase and DNase free H2O.</li>
	<li>Immediately select the program according to the percentage of the gel being run and set the run time on the apparatus to 10 minutes.</li>
	<li>Start the electrophoresis within 1 min of loading sample by pressing the GO button. The electrophoresis time can be extended if insufficient resolution is obtained. 
	NOTE: Do not exceed the maximum recommended agarose electrophoresis running time according to manufacturer&#39;s instructions.</li>
	<li>When the electrophoresis is completed, visualize DNA in the gel using a blue light or UV transillumination. Visualizing, analyzing, and storing the electrophoresis images are mostly done by a gel imager with photo-documentation system (Figure 5).</li>
	<li>Analyze and interpret the visualized qPCR HRM products gel pattern by comparing the results of the unknown sample to positive controls (Figure 7 and Figure 8).</li>
	<li>If the unknown sample HRM and gel electrophoresis results indicates that an unknown genetic variant is present, sequence the qPCR HRM product using primers described in Table of Materials according to the protocol described13. 
	CAUTION: The agarose gels containing a fluorescent nucleic acid stain must be properly disposed of per institution regulations.</li>
</ol>

<ol>
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The authors have no conflicts of interest to disclose.

High-resolution melting of DNA is a simple solution for genotyping and genetic variant scanning14. It depends on sequence differences that result in heteroduplexes that change the shape of the melting curve. Different types of genetic variants with diverse frequencies can be observed in the gene specific for a certain group of patients with cancer1,2,15,16,<su...

Somatic genetic variants in the calreticulin gene (CALR) were recognized in 2013 in patients with myeloproliferative neoplasms (MPN) such as essential thrombocythemia and primary myelofibrosis1,2. Since then, more than 50 genetic variants in the CALR gene have been discovered, inducing a +1 (&#8722;1+2) frameshift3. The two most frequent CALR genetic variants are a 52 bp deletion (NM_004343.3 (CALR):c.1099_1150del52, p.(Leu367Thrfs*46)), also called type 1 mutation, and a 5 bp insertion (NM_004343.3 (CALR):c.1154_1155insTTGTC, p.(Lys385Asnfs*47)), also called type 2 mutation. These two genetic variants represent 80% of all CALR genetic variants. The other ones have been classified as type 1&#8211;like or type 2&#8211;like using algorithms based on the preservation of an &#945; helix close to wild type CALR4. Here, we present one of the highly sensitive and rapid methods for CALR genetic variant detection, the high resolution melting analysis method (HRM). This method enables the rapid detection of type 1 and type 2 genetic variants, which represent the majority of CALR mutations5. HRM was introduced in combination with real time &#187;polymerase chain reaction&#171; (qPCR) in 1997 as a tool to detect the mutation in factor V Leiden6. In comparison to Sanger sequencing that represents the golden standard technique, HRM is a more sensitive and less specific method5. The HRM method is a good screening method that enables a rapid analysis of a large number of samples with a great cost-benefit5. It is a simple PCR method performed in the presence of a fluorescent dye and does not require specific skills. Another benefit is that the procedure itself does not damage or destroy the analyzed sample that allows us to reuse the sample for electrophoresis or Sanger sequencing after the HRM procedure7. The only disadvantage is that it is sometimes difficult to interpret the results. Additionally, HRM does not detect the exact mutation in patients with non-type 1 or type 2 mutations8. In these patients, Sanger sequencing should be performed (Figure 1).
HRM is based on the amplification of the specific DNA region in the presence of saturating DNA fluorescent dye, which is incorporated in double-stranded DNA (dsDNA). The fluorescent dye emits light when incorporated in the dsDNA. After a progressive increase in temperature, dsDNA breaks down into single stranded DNA, which can be detected on the melting curve as a sudden decrease in fluorescence intensity. The shape of the melting curve depends on the DNA sequence that is used to detect the mutation. Melting curves of samples are compared to melting curves of known mutations or wild type CALR. Distinct melting curves represent a different mutation that is non type 1 or type 29.
The algorithm for the somatic genetic variant detection in the CALR gene by HRM, agarose gel electrophoresis and sequencing method (Figure 1) was used and validated in the retrospective study published before10.

<table><tbody><tr><td>E-Gel EX 4% Agarose</td><td>Invitrogen, Thermo Fischer Scientific</td><td>G401004</td><td></td></tr><tr><td>Fuorometer 3.0 QUBIT</td><td>Invitrogen, Thermo Fischer Scientific</td><td>Q33216</td><td></td></tr><tr><td>Invitrogen E-Gel iBase and E-Gel Safe Imager Combo Kit</td><td>Invitrogen, Thermo Fischer Scientific</td><td>G6465EU</td><td></td></tr><tr><td>MeltDoctor HRM MasterMix 2X</td><td>Applied Biosystem, Thermo Fischer Scientific</td><td>4415440</td><td>Components: AmpliTaqGold 360 DNA Polymerase, MeltDoctor trade HRM dye, dNTP blend including dUTP, Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results</td></tr><tr><td>MicroAmp Fast 96-well Reaction Plate (0.1 mL)</td><td>Applied Biosystems, Thermo Fischer Scientific</td><td>4346907</td><td></td></tr><tr><td>MicroAmp Optical adhesive film</td><td>Applied Biosystems, Thermo Fischer Scientific</td><td>4311971</td><td></td></tr><tr><td>NuGenius</td><td>Syngene</td><td>NG-1045</td><td>Gel documentation systems</td></tr><tr><td>Primer CALRex9 Forward</td><td>Eurofins Genomics</td><td></td><td>Sequence: 5&#039;GGCAAGGCCCTGAGGTGT&#039;3 (High-Purity, Salt-Free)</td></tr><tr><td>Primer CALRex9 Reverse</td><td>Eurofins Genomics</td><td></td><td>Sequence: 5&#039;GGCCTCAGTCCAGCCCTG&#039;3 (High-Purity, Salt-Free)</td></tr><tr><td>QIAamp DNA Mini Kit</td><td>QIAGEN</td><td>51306</td><td>DNA isolation kit with the buffer for DNA dilution.</td></tr><tr><td>Qubit Assay Tubes</td><td>Invitrogen, Thermo Fischer Scientific</td><td>Q32856</td><td></td></tr><tr><td>QUBIT dsDNA HS assay</td><td>Invitrogen, Thermo Fischer Scientific</td><td>Q32854</td><td></td></tr><tr><td>Trackit 100bp DNA Ladder</td><td>Invitrogen, Thermo Fischer Scientific</td><td>10488058</td><td>Ladder consists of 13 individual fragments with the reference bands at 2000, 1500, and 600 bp.</td></tr><tr><td>ViiA7 Real-Time PCR System</td><td>Applied Biosystems, Thermo Fischer Scientific</td><td>4453534</td><td></td></tr><tr><td>Water nuclease free</td><td>VWR, Life Science</td><td>436912C</td><td>RNase, DNase and Protease free water</td></tr></tbody></table>

genetic variant detection in the calr gene using high resolution melting analysis

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome&#8211;negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

The successfully amplified DNA region of interest with an exponential increase in fluorescence that exceeds the threshold between cycles 15 and 35 and very narrow values of the cycle of quantification (Cq) in all replicated samples and controls (Figure 2) is a prerequisite for the reliable identification of genetic variants by HRM analysis. This is achieved by using a precise determination of DNA with fluorescence staining and an equal amount of DNA in the qPCR HRM experiment (see step 1.2)....

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Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA ...

Tadej Paji&#269; 1,2, Tanja Bel&#269;i&#269; Miki&#269;1, Helena Podgornik1,3, Jurka Klun1, Sandra &#352;u&#263;urovi&#263;1,&#160;

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Research

JoVE Journal

Genetics

4.7K Views.  University Medical Centre Ljubljana. Tadej Pajič 1,2, Tanja Belčič Mikič1, Helena Podgornik1,3, Jurka Klun1, Sandra Šućurović1, 

Video: Genetic Variant Detection in the CALR gene using High Resolution Melting 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.

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

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Cancer Research

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

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.

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.

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

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.

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

Immunology and Infection

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Biology

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.

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 ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

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.

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

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

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