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>按照制造商的说明11 通过荧光染色方法定量粒细胞DNA（见 材料表）。使用10mM Tris-Cl，0.5mM EDTA，pH 9.0制备20ng / μL DNA溶液（见 材料表）。<ol>
<li>除DNA样本外，还准备至少三种DNA对照：两个阳性对照，NM_004343.3（CALR）：c.1099_1150del52，p.（Leu367Thrfs*46）（52 bp缺失或1型突变），和NM_004343.3（CALR）：c.1154_1155insTTGTC，p.（Lys385Asnfs*47）（5 bp插入或2型突变）遗传变异，以及野生型DNA和阴性对照作为非模板（NTC）对照。 注意：在执行HRM设置之前，请确认qPCR仪器已针对HRM实验进行了校准。</li>
	</ol></li>
	<li>根据制造商的方案（见 材料表）制备qPCR HRM预混液如下：10μL2x qPCR预混料与染料，0.2μL10μM正向底漆，0.2μL10μM反向底漆，8.6μL无菌，RNase和DNase游离H2O. 使用 材料表中的引物。对每个DNA样本和对照运行三次重复。<ol>
<li>根据正在处理的样品数量准备qPCR HRM预混液。在计算中包括至少10%的过量体积，以提供试剂转移过程中发生的损失的过量体积。</li>
	</ol></li>
	<li>通过轻轻敲击并倒置管并涡旋2-3 s来混合反应内容物。通过短暂的旋转收集从管壁到底部的所有散落液滴。</li>
	<li>准备适合仪器和HRM实验的反应板（见 材料表）。将19μL qPCR HRM预混液转移到96孔光学反应板的适当孔中。</li>
	<li>将1μL阴性对照，阳性对照和样品移液到光学反应板的适当孔中。对于NTC，转移1μL无菌，RNase和DNase游离H2O，用于制备qPCR HRM预混液而不是DNA。</li>
	<li>用光学粘合膜密封反应板。用力防止在运行过程中蒸发。检查光学胶膜是否在反应板中的所有孔上都是平面的，以确保正确的荧光检测。</li>
	<li>在室温（RT）下以780×g旋转反应板1分钟。 检查液体是否在反应板中孔的底部。继续运行测定。 注意：协议可以在此处暂停。将板在4°C下储存不超过24小时。让储存在4°C的板温升至室温，然后在运行之前短暂旋转板。</li>
	<li>根据制造商的说明准备并开始运行以扩增和熔化DNA，并在qPCR仪器中生成HRM荧光数据。在仪器系统软件中，将控制装置和样品分配到适当的孔中。</li>
	<li>对于 CALR 遗传变异检测，更改默认的仪器扩增方案并使用以下热循环方案扩增DNA：95°C持续10分钟，然后在95°C下循环50次，持续10秒，62.5°C持续60秒。</li>
	<li>在qPCR之后立即按照制造商的说明12 执行熔融曲线/解离（HRM）阶段，如下所示：95°C持续10秒，60°C持续1分钟，然后升温速率为0.025°C / s，直到95°C。 将温度保持在95°C15秒，在60°C下保持15秒。</li>
	<li>运行将自动结束。首先，查看并验证扩增图（图2）。</li>
	<li>在仪器系统软件的实验菜单中，选择扩增图选项。如果未显示任何数据，请单击绿色按钮 "分析"。<ol>
<li>在扩增图选项卡中，从图类型下拉菜单中，选择显示扩增数据的图，该图显示的原始荧光读数作为被动参考（ΔRn）的荧光归一化为来自被动参考（ΔRn）的荧光，作为周期数（ΔRn vs Cycle）的函数。在打印颜色下拉菜单中，选择" 示例"。</li>
	</ol></li>
	<li>从图形类型下拉菜单中，选择线性放大图形类型。通过选择基线开始选项，在线性放大图上显示基线开始和结束周期。验证基线设置是否正确。在检测到明显荧光信号的周期数之前，应将结束周期设置为几个周期。基线通常设置为3至15个周期（图2）。</li>
	<li>从图表类型下拉菜单中，选择log10 放大图表类型。通过选择阈值选项在图表上显示阈值线。如果未正确设置，请相应地调整阈值。正确设置的阈值意味着阈值线穿过qPCR曲线的指数相位（图2）。</li>
	<li>验证所有样品和阳性对照孔中是否有正常的扩增曲线。验证周期 40 之前 NTC 孔中没有扩增。正常的扩增图显示荧光的指数增长超过了周期15和35之间的阈值（图2）。如果孔位置没有扩增，则从分析中排除样品孔。 注：如果扩增图看起来异常或NTC信号孔指示扩增，请根据制造商的故障排除指南识别并解决问题。</li>
	<li>排除定量周期（Cq）值与仪器系统软件12中的重复值相差2以上异常值。异常值可能会产生错误的 HRM 结果。</li>
	<li>在导数熔化曲线中，查看熔化前和熔化后区域/温度线。熔化前和熔后区域应在荧光水平中没有大峰或斜率的平坦区域内（图3）。如果需要，请相应地进行调整。设置熔化前和熔化后温度管路的启动和停止，彼此相距约0.5°C（图3）。如果通过单击"分析"按钮调整了参数，请重新启动 分析 。</li>
	<li>在差异图选项卡的图解设置选项卡中，选择一个野生型对照（纯合子）复制作为参考DNA，然后单击" 分析 "按钮重新开始分析。</li>
	<li>在对齐的熔体曲线选项卡中，确认所有阳性对照具有正确的基因型并且NTC未能扩增（图4）。从孔表中，选择包含正对照的孔，以突出显示分析图中的相应熔融曲线。确认品系的颜色对应于正确的基因型。对含有其他阳性对照和NTC的孔重复步骤。</li>
	<li>在对齐的熔融曲线选项卡中，仔细查看未知样品的图显示，并将其与对照的图显示进行比较（图4）。从孔表中，选择包含未知样品重复的孔，检查熔体曲线的颜色，并通过逐个选择包含阳性对照的孔，按顺序将它们与对照对齐。对所有未知样本重复该过程。 注：如果其中一个对照的熔化曲线与其紧密对齐，则未知样品包含其变体。可以显示不同的变体组（不同的颜色），表明未知样本由未知变体组成，与对照不对应（图8）。患者样本中可能存在低水平的体细胞遗传变异。这可能会影响HRM结果的解释，特别是在测定的检测限下（图9）。在这些情况下，线条的颜色甚至形状可能与野生型基因型的颜色非常相似。<ol>
<li>当结果不确定时，将HRM结果与琼脂糖凝胶电泳和测序方法的结果相结合。重新测试样品或请求，并在需要时重新测试新样品。</li>
		<li>仔细检查数据集中的仿行曲线，并检查组中每个仿行的对齐是否紧密。如果重复与组中的其他样本（异常值）不紧密对齐，则将其排除。如果存在更多异常值并且结果不确定，请重新测试样本。请注意，DNA样本的数量和质量会影响HRM结果。</li>
	</ol></li>
	<li>在 "差值 "图选项卡中分析未知样本的结果。重复步骤1.21中描述的过程，并验证为未知样品获得的结果是否相同。</li>
	<li>然后，在琼脂糖凝胶上运行qPCR HRM产物。 注意：协议可以在此处暂停。将板在4°C下储存不超过24小时，或在-20°C下储存更长的时间但不超过7天。让储存在4°C的板温升至室温，然后在运行之前短暂旋转板。让储存在-20°C的板解冻并加热到室温，然后在运行之前短暂旋转板。</li>
</ol>2. 琼脂糖凝胶电泳
<ol><li>使用适当的凝胶电泳系统在含有荧光核酸染色的4%琼脂糖预浇注凝胶上运行qPCR HRM产物（见 材料表， 图5）。仅运行一个阳性、阴性、NTC 和样品 qPCR HRM 重复。 注意：处理凝胶时应始终佩戴手套。如果选择等效的琼脂糖百分比，荧光核酸染色和孔格式，任何其他凝胶电泳系统都可以实现这一目的。</li>
	<li>根据制造商的协议运行凝胶电泳系统（见 材料表）。从包装中取出盒中的预制凝胶，取出梳子并按照制造商的说明将其插入设备（见 材料表）。 注意：在打开包装后15分钟内加载凝胶。</li>
	<li>用无菌，RNase和DNase游离的H 2 O将10μL样品稀释至20μL，用20μL稀释的样品加载每个孔。</li>
	<li>将3μLDNA大小标准溶液稀释至20μL，不含无菌，RNase和DNase的H2O（见材料表）。用20μL稀释的DNA大小标准溶液加载M孔（图6）。</li>
	<li>用20μL无菌，RNase和DNase游离H2O填充任何空孔。</li>
	<li>立即根据正在电泳的凝胶百分比选择程序，并将设备上的电泳时间设置为10分钟。</li>
	<li>通过按下 GO 按钮在加载样品后1分钟内开始电泳。如果获得不充分的分辨率，电泳时间可以延长。 注：根据制造商的说明，不要超过建议的最长琼脂糖电泳运行时间。</li>
	<li>电泳完成后，使用蓝光或紫外线透射可视化凝胶中的DNA。可视化，分析和存储电泳图像主要由带有照片记录系统的凝胶成像仪完成（图5）。</li>
	<li>通过将未知样品的结果与阳性对照的结果进行比较来分析和解释可视化的qPCR HRM产物凝胶图谱（图7和图8）。</li>
	<li>如果未知样品HRM和凝胶电泳结果表明存在未知的遗传变异，则使用材料表中描述的引物根据所述方案对qPCR HRM产物进行测序13。 注意：含有荧光核酸染色剂的琼脂糖凝胶必须根据机构法规进行适当处理。</li>
</ol>

<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>

作者没有利益冲突需要披露。

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 ...

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;

genetic-variant-detection-calr-gene-using-high-resolution-melting

Research

JoVE Journal

Genetics

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

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

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

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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 ...

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.

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#039;s Disease Gene Susceptibility Assessment

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.

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

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 tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

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

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

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

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

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

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

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)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

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

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

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

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

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

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

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

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).

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 ...

Methods Article

使用高分辨率熔解分析检测 CALR 基因中的遗传变异