Name: genetic variant detection in the calr gene using high resolution melting analysis
Uploaded: 2020-08-26T13:00:00
Description: Watch this Scientific Journal Video about Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis at JoVE.com

Os autores agradecem a todos os especialistas acadêmicos e colaboradores do Laboratório Especializado de Hematologia, Departamento de Hematologia, Divisão de Medicina Interna do Centro Médico Universitário Ljubljana.

O estudo foi aprovado pelo Comitê de Ética Médica da República da Eslovênia. Todos os procedimentos estavam de acordo com a declaração de Helsinque.
1. Análise quantitativa de PCR (qPCR) e pós-qPCR baseada em fluorescência e análise pós-qPCR pelo HRM
<ol><li>Primers resuspend listados na Tabela de Materiais a 100 μM com estéril, RNase e DNase livre H2O (ver Tabela de Materiais). Faça um primer de concentração de trabalho de 10 μM...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+melting+analysis+for+gene+scanning.">High resolution melting analysis for gene scanning.</a> Methods. 50, 250-261 (2010).</li><li>Bilbao-Sieyro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Resolution+Melting+Analysis:+A+Rapid+and+Accurate+Method+to+Detect+CALR+Mutations.">High Resolution Melting Analysis: A Rapid and Accurate Method to Detect CALR Mutations.</a> PLoS One. 9, 103511 (2014).</li><li>S&#322;omka, 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, 2316 (2017).</li><li>Li, X., 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=Comparison+of+three+common+DNA+concentration+measurement+methods.">Comparison of three common DNA concentration measurement methods.</a> Analytical Biochemistry. 451, 18-24 (2014).</li><li>Voytas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis.">Agarose Gel Electrophoresis.</a> Current Protocols in Immunology. 2, (1992).</li><li>Lee, P. 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O derretimento de alta resolução do DNA é uma solução simples para genotipagem e escaneamento de variantesgenéticas 14. Depende de diferenças sequenciais que resultam em heteroduplexes que mudam a forma da curva de fusão. Diferentes tipos de variantes genéticas com frequências diversas podem ser observadas no gene específico para um determinado grupo de pacientes com câncer1,2,15,<s...

As variantes genéticas somáticas do gene da calreticulina (CALR)foram reconhecidas em 2013 em pacientes com neoplasias mieloproliferativas (MPN), como trombocythemia essencial e mielófibrose primária1,2. Desde então, mais de 50 variantes genéticas no gene CALR foram descobertas, induzindo um quadro de 3 +1 (−1+2). As duas variantes genéticas CALR mais frequentes são uma exclusão de 52 bps (NM_004343,3 (CALR):c.1099_1150del52, p.(Leu367Thrfs*46)), também chamada de mutação tipo 1, e uma inserção de 5 bp (NM_004343,3 (CALR): c.1154_1155insTTGTC, p.(Lys385Asnfs*47)), também chamada de mutação tipo 2. Essas duas variantes genéticas representam 80% de todas as variantes genéticas CALR. Os outros foram classificados como tipo 1- like ou tipo 2 – como usando algoritmos baseados na preservação de uma hélice α perto do tipo selvagem CALR4. Aqui, apresentamos um dos métodos altamente sensíveis e rápidos para detecção de variantes genéticas CALR, o método de análise de fusão de alta resolução (HRM). Este método permite a detecção rápida de variantes genéticas tipo 1 e tipo 2, que representam a maioria das mutações CALR 5. O HRM foi introduzido em combinação com a reação em cadeia de polimerase em tempo real« (qPCR) em 1997 como uma ferramenta para detectar a mutação no fator V Leiden6. Em comparação com o sequenciamento Sanger que representa a técnica padrão dourada, o HRM é um método mais sensível e menos específico5. O método HRM é um bom método de triagem que permite uma análise rápida de um grande número de amostras com um ótimo custo-benefício5. É um método PCR simples realizado na presença de um corante fluorescente e não requer habilidades específicas. Outro benefício é que o procedimento em si não danifica ou destrói a amostra analisada que nos permite reutilizar a amostra para eletroforese ou sequenciamento de Sanger após o procedimento de HRM7. A única desvantagem é que às vezes é difícil interpretar os resultados. Além disso, o HRM não detecta a mutação exata em pacientes com mutações não tipo 1 ou tipo 28. Nesses pacientes, deve-se realizar o sequenciamento Sanger(Figura 1).
O HRM baseia-se na amplificação da região específica do DNA na presença de corante fluorescente de DNA saturado, que é incorporado em DNA de dupla cadeia (dsDNA). O corante fluorescente emite luz quando incorporado no dsDNA. Após um aumento progressivo da temperatura, o DSNA se divide em um único DNA encalhado, que pode ser detectado na curva de fusão como uma diminuição repentina da intensidade da fluorescência. A forma da curva de fusão depende da sequência de DNA que é usada para detectar a mutação. Curvas de fusão de amostras são comparadas com curvas de fusão de mutações conhecidas ou tipo selvagem CALR. Curvas de fusão distintas representam uma mutação diferente que não é tipo 1 ou tipo29.
O algoritmo para a detecção de variantes genéticas somáticas no gene CALR por HRM, o método de eletroforese e sequenciamento do gel agarose(Figura 1)foi utilizado e validado no estudo retrospectivo publicado antes do10.

<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&#39;GGCAAGGCCCTGAGGTGT&#39;3 (High-Purity, Salt-Free)</td>
		</tr>
		<tr>
			<td>Primer CALRex9 Reverse</td>
			<td>Eurofins Genomics</td>
			<td></td>
			<td>Sequence: 5&#39;GGCCTCAGTCCAGCCCTG&#39;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

A análise de derretimento de alta resolução (HRM) é um método poderoso para genotipagem e varredura de variações genéticas. A maioria das aplicações de HRM dependem de corantes de DNA saturados que detectam diferenças de sequência, e heterodúplexos que mudam a forma da curva de fusão. Excelente resolução de instrumentos e software especial de análise de dados são necessários para identificar as pequenas diferenças de curva de fusão que identificam uma variante ou genótipo. Diferentes tipos de variantes genéticas com diversas frequências podem ser observadas no gene específico para pacientes com uma doença específica, especialmente câncer e no gene CALR em pacientes com neoplasias mieloproliferativas nômodas negativas da Filadélfia. Alterações de nucleotídeos únicos, inserções e/ou exclusões (indels) no gene de interesse podem ser detectadas pela análise do HRM. A identificação de diferentes tipos de variantes genéticas baseia-se principalmente nos controles utilizados no ensaio qPCR HRM. No entanto, à medida que o comprimento do produto aumenta, a diferença entre curvas de tipo selvagem e heterozigoto torna-se menor, e o tipo de variante genética é mais difícil de determinar. Portanto, quando os indels são a variante genética predominante esperada no gene de interesse, um método adicional como a eletroforese de gel de agarose pode ser usado para o esclarecimento do resultado do HRM. Em alguns casos, um resultado inconclusivo deve ser re-verificado/re-diagnosticado pelo sequenciamento Sanger padrão. Neste estudo retrospectivo, aplicamos o método a pacientes JAK2 V617F negativos com MPN.

A região de interesse do DNA amplificado com sucesso com um aumento exponencial da fluorescência que excede o limiar entre os ciclos 15 e 35 e valores muito estreitos do ciclo de quantificação (Cq) em todas as amostras e controles replicados (Figura 2) é um pré-requisito para a identificação confiável de variantes genéticas pela análise do HRM. Isso é conseguido usando uma determinação precisa do DNA com coloração de fluorescência e uma quantidade igual de DNA no experimento...

Watch this Scientific Journal Video about Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis at JoVE.com

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

A análise de fusão de alta resolução (HRM) é uma solução sensível e rápida para detecção de variantes genéticas. Depende de diferenças sequenciais que resultam em heteroduplexes mudando a forma da curva de fusão. Ao pentear o HRM e a agarose gel eletroforesis, diferentes tipos de variantes genéticas, como indels, podem ser identificados.

Detecção de variantes genéticas no gene CALR usando análise de fusão de alta resolução

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

This method accurately defines calreticulin mutations in patients with suspected hematologic condition, especially where other diagnostic options are limited or there is an overlap with non-hematologic conditions. The main advantage of the high resolution mirror technique is detecting the common somatic genetic variants in the hotspot region of the calreticulin gene at the acceptable limit of detection and affordable cost. This technique enables rapid and noninvasive diagnosis of myeloproliferative disorders, especially essential thrombocythemia and primary myelofibrosis.Currently it has no effect on the choice of therapy in these patients. However, this may change in the future. Begin by preparing the qPCR HRM master mix as described in the text manuscript, making enough for three replicates of each DNA sample and control.Mix the reaction contents by gently tapping and inverting the tube and vortexing for two to three seconds, then briefly spin the tube to collect the scattered droplets. Transfer 19 microliters of the qPCR HRM master mix to each well of the 96 Well Optical Reaction Plate. Pipette one microliter of the negative controls, positive controls and samples into the appropriate wells of the plate for the no template control or NTC at one microliter of sterile RNAs and DNA's free water used for preparing the qPCR HRM master mix instead of DNA.Seal the reaction plate firmly with the optical adhesive film to prevent evaporation during the run. Then spin it at 780 times G at room temperature for one minute. Check that the liquid is at the bottom of the wells in the reaction plate and run the assay.Assign the controls and samples to the appropriate wells in the instrument system software and change the default instrument amplification protocol. Program the instrument as described in the text manuscript to perform the dissociation analysis immediately after qPCR. when the run end review and verify the amplification plot.In the experiment menu of the instrument system software select the amplification plot option. If no data are displayed, click the green analyze button. In the Amplification Plot tab from the plot type dropdown menu, select the plot that displays the amplification data as the fluorescence readings, normalized to the fluorescence from the passive reference as a function of cycle number, In the plot color dropdown menu, select sample.From the graph type dropdown menu, select the linear amplification graph type, verify that the baseline start and end cycle is set correctly. The end cycle should be set a few cycles before the cycle number where a significant fluorescent signal is detected. From the graph type dropdown menu, select the log 10 amplification graph type show the threshold line on the graph by selecting the threshold option and adjust the threshold.The threshold line should cross the exponential phase at the qPCR curves. Verify that all sample and positive control Wells have normal amplification curves and that there is no amplification in the NTC Wells. A normal amplification plot shows an exponential increase in florescence that exceeds the threshold between cycles 15 and 35.In the derivative melt curves review the pre and post melt regions or 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. If needed set the start and stop of the pre and post melt temperature lines, approximately 0.5 degrees Celsius apart from each other.Restart the analysis if the parameters are adjusted by clicking on the analyze button. In the plot settings tab of the difference pot tab, choose one of the wild type control replicates as the reference DNA and restart the analysis by clicking on the analyze button. In the aligned Melt curves tab, confirm that all positive controls have the correct genotype and that the NTC failed to amplify.From the well table, select a control well to highlight the corresponding melt curve in the analysis plots and confirm that the color of the line corresponds to the correct genotype. In the aligned melt curves tab carefully review the plot displays for the unknown samples and compare them to the plot displays for controls. Select the Wells containing the unknown sample replicates and align the melt curves with the controls in an ordered sequence by selecting the wells containing controls one by one.Analyze the result for the unknown sample in the Difference Plot tab to verify that the results obtained are consistent. Select the wells containing the unknown sample replicates and align the melt curves with the controls in an ordered sequence, selecting the Wells containing controls one by one. Run qPCR HRM products on a 4%agarose precast gel containing a fluorescent nucleic acid strain.Run only one positive negative NTC and sample qPCR HRM replicates. Remove the precast gel and the cassette from the package then remove the comb and insert the gel into the apparatus according to the manufacturer's instructions. Dilute a 10 microliter sample to 20 microliters with sterile RNA and DNA free water and load each well with 20 microliters of the diluted sample.Dilute three microliters of DNA size standard solution to 20 microliters and load it into the marker well. Fill any empty wells with 20 microliters of sterile RNA and DNA's free water. Immediately select the program according to the percentage of the gel being run and set the runtime on the apparatus to 20 minutes.Start the electrophoresis within one minute of loading the samples. When the electrophoresis is completed, visualize the DNA and the gel by using blue light or UV transillumination. And successfully amplified DNA region of interest with an exponential increase in fluorescents that exceeds the threshold between cycles 15 and 35 and very narrow values of the cycle of quantification in all replicates and controls is a prerequisite for the reliable identification of genetic variance by high resolution melting analysis.HRM analysis is performed immediately after qPCR, the active melt regions of the samples, the controls and the NTC are used to create their aligned melt curve plots. Correctly set pre and post melt regions are important for properly visualizing and identifying genetic variance in the samples. Aligned melt curves and different pots are shown here.The unknown samples are tightly aligned with one of the positive controls. Incorrectly set pre and post melt regions or temperature lines result in aligned melt curves and different plots where correct identification of the genetic variance is more difficult. The genetic variant in the sample can be identified by comparing the band pattern of the sample to the controls.And by combining the HRM and agarose gel electrophoresis. Correct genetic variant identification can only be done for the samples that contain the same genetic variant as one of the controls used in the HRM assay. Samples containing rare calreticulin genetic variants differ in the HRM result and electrophoresis band pattern.When attempting this protocol, it is important to carefully compare the aligned melt curve plot displays of the known and control samples.

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Genetics

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

Detection of Copy Number Alterations Using Single Cell Sequencing

Detecção de Copy Number Alterações Usando Sequencing única célula

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genética

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

PCR de fusão de alta resolução para receptor de complemento 1 polimorfismo de comprimento Genotípica: uma ferramenta inovadora para a avaliação da susceptibilidade genética da doença de Alzheimer

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

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Alta resolução hibridação fluorescente In Situ em embriões da drosófila e tecidos usando amplificação de sinal Tyramide

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

Promotor captura Oi-c: Alta resolução, todo o genoma caracterização das interações do promotor

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Comparação de alta resolução de frequências de conjugação bacteriana

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Identificação do elevado-throughput de sequências de genes regulatórios utilizando a próxima geração sequenciamento de conformação Circular cromossoma capturar (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Determinar a probabilidade de patogenicidade variante usando análise de sinal-ruído nível de aminoácido da variação genética

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

Um pipeline de bioinformática para analisar com precisão e eficiência os transcriptomas de MicroRNA em plantas

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

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

Identificando mutações por derretimento de alta resolução em uma população de lavando de arroz

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

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Transformação da Raiz do Tomate Seguida de Inoculação com Ralstonia Solanacearum para Análise Genética Direta da Doença de Wilt Bacteriano

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...