Name: genetic variant detection in the calr gene using high resolution melting analysis
Uploaded: 2020-08-26T13:00:00.000+00:00
Duration: 8 min 46 s
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>

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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&#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

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

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.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

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

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

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.

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 ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

癌症研究

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

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 ...

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.

使用埃及伊蚊的高分辨率熔融分析进行CRISPR/Cas9诱变后的Indel检测

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

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

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.

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

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

免疫与感染

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.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

生物学

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.

利用电泳迁移分析（EMSA）和筛选功能的非编码基因变异DNA的亲和沉淀试验（DAPA）

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