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

作者要感谢卢布尔雅那大学医学中心血液学系血液学系，内科专业血液学实验室的所有学术专家和员工。

该研究得到了斯洛文尼亚共和国医学伦理委员会的批准。所有程序都符合《赫尔辛基宣言》。
1. 基于荧光的定量实时荧光聚合酶链（qPCR）和HRM的qPCR后分析
<ol><li>将 材料表中 列出的引物重悬至100μM，不含无菌，RNase和DNase的H2O（见 材料表）。制作10μM工作浓度底漆。该协议中使用的引物在2之前发表。</li>
	<li>按照制造商的?...

<ol>
	<li>Nangalia, 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=Somatic+CALR+mutations+in+myeloproliferative+neoplasms+with+nonmutated+JAK2.">Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.</a> New England Journal of Medicine. 369, 2391-2405 (2013).</li><li>Klampfl, 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=Somatic+mutations+of+calreticulin+in+myeloproliferative+neoplasms.">Somatic mutations of calreticulin in myeloproliferative neoplasms.</a> New England Journal of Medicine. 369, 2379-2390 (2013).</li><li>Vainchenker, W., Kralovics, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+basis+and+molecular+pathophysiology+of+classical+myeloproliferative+neoplasms.">Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms.</a> Blood. 129, 667-679 (2017).</li><li>Pietra, D., 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=Differential+clinical+effects+of+different+mutation+subtypes+in+CALR-mutant+myeloproliferative+neoplasms.">Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.</a> Leukemia. 30, 431-438 (2016).</li><li>Lim, K. H., 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=Rapid+and+sensitive+detection+of+CALR+exon+9+mutations+using+high-resolution+melting+analysis.">Rapid and sensitive detection of CALR exon 9 mutations using high-resolution melting analysis.</a> Clinica Chimica Acta. 440, 133-139 (2015).</li><li>Lay, M. J., 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=Real-time+fluorescence+genotyping+of+factor+V+leiden+during+rapid-cycle+PCR.">Real-time fluorescence genotyping of factor V leiden during rapid-cycle PCR.</a> Clinical Chemistry. 43, 2262-2267 (1997).</li><li>Vossen, R. H. A. M., Aten, E., Roos, A., Den Dunnen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+melting+analysis+(HRMA)+-+More+than+just+sequence+variant+screening.">High-resolution melting analysis (HRMA) - More than just sequence variant screening.</a> Human Mutation. 30, 860-866 (2009).</li><li>Barbui, T., Thiele, J., Vannucchi, A. M., Tefferi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationale+for+revision+and+proposed+changes+of+the+WHO+diagnostic+criteria+for+polycythemia+vera,+essential+thrombocythemia+and+primary+myelofibrosis.">Rationale for revision and proposed changes of the WHO diagnostic criteria for polycythemia vera, essential thrombocythemia and primary myelofibrosis.</a> Blood Cancer Journal. 5, 337-338 (2015).</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, 1748-1754 (2004).</li><li>Belcic Mikic, T., Pajic, T., Sever, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CALR+mutations+in+a+cohort+of+JAK2+V617F+negative+patients+with+suspected+myeloproliferative+neoplasms.">CALR mutations in a cohort of JAK2 V617F negative patients with suspected myeloproliferative neoplasms.</a> Scientific Reports. 9, 19838 (2019).</li><li>Invitrogen, Thermo Fischer Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QubitTM dsDNA HS Assay Kits. Manual (MAN0002326, MP32851, Revision: B.0). , (2015).</li><li>Applied Biosystems, Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High Resolution Melt Module for ViiA TM 7 Software v1. Getting Started Guide. Part Number 4443531 Rev. B. , (2011).</li><li>Applied Biosystems, Thermo Fischer Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High-Resolution Melt Analysis for Mutation Scanning Prior to Sequencing. Application Note. , (2010).</li><li>Reed, G. H., Kent, J. O., 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+DNA+melting+analysis+for+simple+and+efficient+molecular+diagnostics.">High-resolution DNA melting analysis for simple and efficient molecular diagnostics.</a> Pharmacogenomics. 8, 597-608 (2007).</li><li>Gonzalez-Bosquet, 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=Detection+of+somatic+mutations+by+high-resolution+DNA+melting+(HRM)+analysis+in+multiple+cancers.">Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.</a> PLoS One. 6, 14522 (2011).</li><li>Van Der Stoep, N., 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=Diagnostic+guidelines+for+high-resolution+melting+curve+(HRM)+analysis:+An+interlaboratory+validation+of+BRCA1+mutation+scanning+using+the+96-well+LightScanner.">Diagnostic guidelines for high-resolution melting curve (HRM) analysis: An interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner.</a> Human Mutation. 30, 899-909 (2009).</li><li>Singdong, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Prognosis+Significance+of+JAK2+(V617F),+MPL,+and+CALR+Mutations+in+Philadelphia-Negative+Myeloproliferative+Neoplasms.">Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms.</a> Asian Pacific Journal of Cancer Prevention. 17, 4647-4653 (2016).</li><li>Erali, M., 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+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. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <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+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> Journal of Visualized Experiments. , e3923 (2012).</li><li>Helling, R. B., Goodman, H. M., Boyer, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Endonuclease+R&#183;EcoRI+Fragments+of+DNA+from+Lambdoid+Bacteriophages+and+Other+Viruses+by+Agarose-Gel+Electrophoresis.">Analysis of Endonuclease R&#183;EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis.</a> Journal of Virology. , 1235-1244 (1974).</li></ol>

DNA的高分辨率熔化是基因分型和遗传变异扫描的简单解决方案14。它取决于导致异质双面体改变熔化曲线形状的序列差异。在特定癌症患者群体1、2、15、16、10的基因特异性中可以观察到不同频率的不同类型的遗传变异。目的基因中的缺失或插入可以通过HRM?...

钙质素基因（CALR）的体细胞遗传变异在2013年在骨髓增殖性肿瘤（MPN）患者中得到认可，如原发性血小板增多症和原发性骨髓纤维化1，2。从那时起，在CALR基因中发现了50多个遗传变异，诱导了+1（−1 + 2）帧移位3。两个最常见的CALR遗传变异是52 bp缺失（NM_004343.3（CALR）：c.1099_1150del52，p.（Leu367Thrfs*46）），也称为1型突变，以及5 bp插入（NM_004343.3（CALR）：c.1154_1155insTTGTC，p.（Lys385Asnfs*47）），也称为2型突变。这两个遗传变异代表了所有CALR遗传变异的80%。其他的已被归类为1型或2型相似，使用基于保存接近野生型CALR4的α螺旋的算法。在这里，我们介绍了一种用于CALR遗传变异检测的高灵敏度和快速方法，即高分辨率熔解分析方法（HRM）。该方法能够快速检测1型和2型遗传变异，这些变异代表了大多数CALR突变5。HRM于1997年与实时"聚合酶链反应"（qPCR）相结合引入，作为检测因子V Leiden6突变的工具。与代表黄金标准技术的Sanger测序相比，HRM是一种更灵敏且特异性更低的方法5。HRM方法是一种很好的筛选方法，可以快速分析大量样品，并具有巨大的成本效益5。这是一种在荧光染料存在下进行的简单PCR方法，不需要特定的技能。另一个好处是，该过程本身不会损坏或破坏分析的样品，这使我们能够在HRM程序7之后重复使用样品进行电泳或Sanger测序。唯一的缺点是有时很难解释结果。此外，HRM不能在非1型或2型突变的患者中检测到确切的突变8。在这些患者中，应进行Sanger测序（图1）。
HRM基于在饱和DNA荧光染料存在下对特定DNA区域的扩增，其掺入双链DNA（dsDNA）中。荧光染料在掺入dsDNA时会发光。在温度逐渐升高后，dsDNA分解成单链DNA，可以在熔融曲线上检测到荧光强度的突然降低。熔化曲线的形状取决于用于检测突变的DNA序列。将样品的熔化曲线与已知突变或野生型CALR的熔融曲线进行比较。不同的熔融曲线代表非类型1或类型29的不同突变。
通过HRM、琼脂糖凝胶电泳和测序方法检测 CALR 基因中体细胞遗传变异的算法（图1）在10之前发表的回顾性研究中得到了应用和验证。

<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

高分辨率熔解分析（HRM）是基因分型和遗传变异扫描的强大方法。大多数HRM应用依赖于检测序列差异的饱和DNA染料，以及改变熔化曲线形状的异质双链体。需要出色的仪器分辨率和特殊的数据分析软件来识别识别变异或基因型的微小熔化曲线差异。在针对特定疾病（尤其是癌症）患者的特异性基因中，以及在费城染色体阴性骨髓增殖性肿瘤患者的 CALR 基因中，可以观察到具有不同频率的不同类型的遗传变异。目标基因中的单核苷酸变化，插入和/或缺失（indels）可以通过HRM分析检测到。不同类型遗传变异的鉴定主要基于qPCR HRM测定中使用的对照。然而，随着产物长度的增加，野生型和杂合子曲线之间的差异变小，遗传变异的类型更难确定。因此，当indels是目标基因中预期的流行遗传变异时，可以使用琼脂糖凝胶电泳等其他方法来澄清HRM结果。在某些情况下，不确定的结果必须通过标准 Sanger 测序重新检查/重新诊断。在这项回顾性研究中，我们将该方法应用于患有MPN的 JAK2 V617F阴性患者。

成功扩增的DNA感兴趣区域，荧光指数增加，超过周期15和35之间的阈值，并且所有重复样品和对照的定量周期（Cq）值非常窄（图2）是通过HRM分析可靠地鉴定遗传变异的先决条件。这是通过在qPCR HRM实验中使用荧光染色和等量DNA精确测定DNA来实现的（参见步骤1.2）。 图2 显示了目标DNA区域的成功扩增，其中所有样品和对照的Cq值都处于非常窄的间隔内。NT...

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

高分辨率熔解分析（HRM）是遗传变异检测的灵敏快速解决方案。这取决于导致异质双链体改变熔化曲线形状的序列差异。通过梳理HRM和琼脂糖凝胶电泳，可以鉴定不同类型的遗传变异，如indels。

使用高分辨率熔解分析检测 CALR 基因中的遗传变异

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.

Research

JoVE Journal

Genetics

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

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

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

科研

遗传学

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

高分辨率融合PCR用于补体受体1长度多态性基因分型：阿尔茨海默病基因易感性评估的创新工具

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

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

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

启动子捕获高分辨率, 全基因组的启动子交互分析

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

细菌结合频率的高分辨率比较

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

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

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

遗传变异的氨基酸级信噪比分析确定变异致病性的可能性

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

生物信息学管道，用于准确、高效地分析植物中的微RNA转录

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

通过高分辨率熔融在水稻种群中识别突变

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

番茄根转化后与拉子托尼亚索拉纳卡鲁姆接种，直接遗传分析细菌性威尔特病

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