We thank Erin Zoller, Jessica Bene, and Lindsey Hays for input and direction in protocol development. MTW was supported in part by NIH R21 HG008186 and a Trustee Award grant from the Cincinnati Children's Hospital Research Foundation. ZHP was supported in part by T32 GM063483-13.

1.解决方案和试剂的制备<ol><li>为了统一EMSA和DAPA使用定制的DNA寡核苷酸探针。 <ol><li>为了减少非特异性蛋白结合，设计短的寡核苷酸30，以及直接将感兴趣的变体在其17碱基对的内源性基因组序列侧接的中央（长度35-45碱基对（bp）之间）。对于EMSA寡核苷酸，加上5&#39;荧光。对于DAPA寡核苷酸，加上5&#39;生物素标记。 </li><li>同时订购有义链及其反向互补链。另外，为了双工（退火前）寡核苷酸。当命名的寡核苷酸，立足于建立的参考基因组的术语。 注:"风险"和"非风险"指定可以是疾病和具体项目，而"参考"和"非参考"更普遍相关。 </li><li>一旦寡核苷酸的到来，简要地在不含核酸酶的水含量和重悬降速至100μ的终浓度，M。储存于-20℃的悬浮股票。用铝箔包裹保护标记与光荧光团的寡核苷酸。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 名称 </td><td> 序列 </td></tr><tr><td> rs76562819_REF_FOR </td><td> GTAATGCCTTAATGAGAGAG 一个 GTTAGTCATCTTCTCACTTC </td></tr><tr><td> rs76562819_REF_REV </td><td> GAAGTGAGAAGATGACTAACŧCTCTCTCATTAAGGCATTAC </td></tr><tr><td> rs76562819_NONREF_FOR </td><td> GTAATGCCTTAATGAGAGAG 摹 GTTAGTCATCTTCTCACTTC </td></tr><tr><td> rs76562819_NONREF_REV </td><td> GAAGTGAGAAGATGACTAACÇCTCTCTCATTAAGGCATTAC </td></tr></tbody></table> 表1:实施例EMSA / DAPA寡核苷酸设计以测试SNP差分仓丁。"REF"代表参考等位基因，而"NONREF"代表非基准等位基因。 "FOR"表示正向链，而"REV"表示它的补。的SNP被认为是在红色。 <ol start="2"><li>制备胞质提取（CE）缓冲液有10mM HEPES（pH值7.9）的最终浓度，10毫KCl和0.1mM EDTA的在去离子水中。 </li><li>制备核提取（NE）用20mM HEPES（pH值7.9）的最终浓度，0.4M NaCl和1mM EDTA中在去离子水中缓冲。 </li><li>制备退火缓冲液的10mM Tris（pH值7.5-8.0）的终浓度，50mM的NaCl和1mM EDTA中在去离子水中。 </li></ol>从培养细胞核裂解液2.准备工作注意:此实验方案是使用B淋巴母细胞系进行了优化，但在其他几个无关的贴/悬浮细胞系已经测试和工程普遍。 <ol><li>丘尔重新罗斯韦尔园区纪念研究所（RPMI）1640的B-类淋巴母细胞有2mM L-谷氨酰胺，10％胎牛血清，并含有100单位/ ml青霉素，100微克/毫升链霉素的1×抗生素 - 抗真菌剂，和250ng的/ ml两性霉素B的<ol><li>种子在一系列200,000-500,000活细胞/ ml，并培育在37℃用5％二氧化碳培养瓶中与排出或松帽直立位置。 注意:当它们到达超过一百万个细胞/ ml的B-类淋巴母细胞的生长减缓。移液器向上和向下几次分手电池爆炸并返回细胞200,000-500,000细胞/ ml，以保持增长速度迅速。 </li></ol></li><li>洗涤培养的细胞，用10ml冰冷的磷酸盐缓冲盐水（PBS）洗涤两次，降速在4℃，300×g离心5分钟，并通过抽吸除去PBS中。 </li><li>计数每10 7个细胞使用血细胞计数器，重悬沉淀为1ml冷的PBS细胞。 注意:例如，如果溶解2×10 7 &lt;/ SUP&gt;细胞悬浮在2毫升PBS。 </li><li>等分试样1ml至1.5ml微量管中，使每个管含有10 7个细胞在PBS中。离心3300 XG为2分钟，4°C和吸掉PBS。 </li><li>在使用之前，添加1mM的二硫苏糖醇（DTT），1×磷酸酶抑制剂，和1x蛋白酶抑制剂的CE缓冲器的工作原液。用400μl的CE缓冲器的重悬细胞沉淀，并在冰上孵育15分钟。 </li><li>加入25微升10％的Nonidet P-40，并通过吹打混合。离心机在4℃，最高速度为3分钟。倒出并弃去上清液。 </li><li>在使用前，加入1毫摩尔DTT，1×磷酸酶抑制剂，和1x蛋白酶抑制剂对NE缓冲器的工作原液。与NE缓冲的30微升悬浮细胞沉淀，并震荡混合。 </li><li>在试管旋转器或在冰上10分钟温育在4℃。离心3300 XG 2分钟4℃。 </li><li>在存放之前收集上清液（核裂解液）和分装-806℃至避免可能降解蛋白多冻融。发表10微升等分试样使用bichoninic酸测定法（BCA）31来测量蛋白质浓度。 </li></ol> 3.电泳迁移分析（EMSA） <ol><li>准备工作寡糖股票及EMSA凝胶。 <ol><li>如果寡核苷酸在双工排序，解冻100μM的库存和稀释1:在退火缓冲液2000实现了50nM的工作原液。 </li><li>如果寡核苷酸定购单链的，解冻的100μM的股票和在退火缓冲液稀释1:10至达到100纳米工作的股票。在微量离心管中结合100微升彼此100nM的互补链溶液。 <ol><li>在一个热块放置在95℃下5分钟。关火块，并允许寡聚慢慢冷却到室温，使用前至少一小时。 </li></ol></li><li>预运行EMSA凝胶。 <ol><li>从预铸6％T取出滑动BE凝胶在去离子水冲洗数次，以便从井中去除任何缓冲。通过加入50毫升10×TBE的950毫升去离子水制备1升0.5×TBE缓冲液中。 </li><li>组装凝胶电泳仪和填充0.5X TBE缓冲液内室检查是否有泄漏。如果没有缓冲泄漏到外室，填充外室的方式大致三分之二。 </li><li> 60分钟前在运行100伏的凝胶。 </li><li>用200μl0.5×TBE缓冲液的冲洗每个孔中。 </li></ol></li></ol></li><li>准备结合缓冲液母液。 <ol><li>制备10×100毫摩尔Tris，500mM的氯化钾，10毫摩尔DTT的终浓度结合缓冲液;在去离子水的pH值7.5。 </li><li>在微量离心管中，创建一个主混合物由共同所有的反应（10微升10X结合缓冲液，10微升的DTT /聚山梨醇酯，5微升多聚d（IC）和2.5微升的鲑精DNA; 表2）的试剂。准备额外的10％考虑到由于移液体积损失。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 终浓度。 </td><td> RXN＃1 </td><td> RXN＃2 </td><td> RXN＃3 </td><td> RXN＃4 </td></tr><tr><td> 超纯水 </td><td> 20微升体积。 </td><td> 13.5微升</td><td> 11.98微升</td><td> 13.5μl </td><td> 11.98微升</td></tr><tr><td> 10X结合缓冲液 </td><td> 1X </td><td> 2微升</td><td> 2微升</td><td> 2微升</td><td> 2微升</td></tr><tr><td> DTT / TW-20 </td><td> 1X </td><td> 2微升</td><td> 2微升</td><td> 2微升</td><td> 2微升</td></tr><tr><td> 鲑鱼精子DNA </td><td> 500纳克/微升</tD&gt; <td> 0.5微升</td><td> 0.5微升</td><td> 0.5微升</td><td> 0.5微升</td></tr><tr><td> 1微克/微升多聚d（IC） </td><td> 1微克</td><td> 1微升</td><td> 1微升</td><td> 1微升</td><td> 1微升</td></tr><tr><td> 核提取物（5.26微克/微升） </td><td> 8微克</td><td> - </td><td> 1.52微升</td><td> - </td><td> 1.52微升</td></tr><tr><td> NE缓冲区 </td><td></td><td> 1.52微升</td><td> - </td><td> 1.52微升</td><td> - </td></tr><tr><td> 参考等位基因寡 </td><td> 50飞摩尔</td><td> 1微升</td><td> 1微升</td><td> - </td><td> - </td></tr><tr><td> 非参考等位基因寡 </td><td> 50飞摩尔</td><td> - </td><td> - </td><td> 1微升</td><td> 1微升</td></tr></tbody></table> 表2:实例EMSA反应SETU页表说明的例子EMSA测试的假设有基因型依赖性转录因子到特定的SNP的结合。 <ol start="3"><li>不含核酸酶的水添加到每个微量离心管中，使得在加入所有试剂的终体积为20微升。 </li><li>主混合物的适当量（5.5微升）添加到每个离心管中。 </li><li>添加8微克核裂解物到合适的离心管。包括含有低聚没有核提取物为阴性对照（ 如 表2，RXN＃1和＃RXN 3）管。 注意:每反应裂解物的最佳量必须通过实验滴定来确定。通常，滴定范围2-10微克裂解物就足够了。 </li><li>加入50 fmol的寡核苷酸到合适的离心管。轻弹混合和旋转简要内容到管的底部。孵育在室温下20分钟。 注意:如果attempti纳克一个超迁移，孵育用抗体的裂解物混合物在之前加入寡聚物室温20分钟。建议使用1微克的芯片级抗体以获得最佳效果。 </li><li>加入2微升10X橙样染料到每个离心管中。移液器上下混合。 </li><li>通过上下吹打混合，然后将每个样品排出到一个单独的井加载样本到运行前的6％TBE凝胶。在80 V上运行凝胶，直到橙色染料已迁移到2/3的一路下跌凝胶3/4。这大约需要60-75分钟。 </li><li>由于撬它用凝胶刀开除去从塑料盒中的凝胶，然后将凝胶中的0.5％TBE缓冲液的容器中，以防止其干燥。 </li><li>放置红外和化学发光成像系统的表面上的凝胶，作为确保消除，这将破坏图像的任何气泡或污染物。 </li><li>使用扫描系统软件，点击"采集"标签然后选择"绘制新"周围绘制相应于该凝胶位于扫描器的表面上的区域中的框。 </li><li>在"采集"选项卡中的"通道"部分中，选择相应的寡核苷酸的荧光标记的波长通道。在"扫描仪"部分中，单击"预览"来获得低质量的扫描预览。通过拖动周围所获取的预览图像向下到凝胶的部分蓝色框中调整扫描区域进行成像。 注:例如，如果使用标有700纳米荧光寡核苷酸，确保​​"700纳米"通道扫描之前选择。 </li><li>在"扫描控件"部分，选择"84μM"分辨率选项和"中等"质量选项。设置聚焦偏移到凝胶的厚度的一半。注意:例如，1毫米的凝胶会使用0.5mm的聚焦偏移。 </li><li>在"扫描仪"部分中，单击"开始"到begin扫描。 注意:在扫描过程中，亮度，对比度和颜色方案通常可以手动取决于扫描系统的制造商的调整。 </li><li>扫描完成后，选择"图像"选项卡，然后点击"旋转或翻转"中的"创建"部分来纠正方向。通过点击"导出"，在主菜单中保存图像文件，然后选择"单幅图像视图"。 </li></ol> 4. DNA亲和纯化实验（DAPA） <ol><li> 5μM寡工作股票的制备。 <ol><li>如果寡核苷酸双工下令，解冻100微米的股票，并在退火缓冲液稀释1:20至达到5微米工作的股票。 </li><li>如果寡核苷酸定购单链的，解冻的100μM的股票和在退火缓冲液稀释1:10至达到10微米的工作股票。结合10微升彼此10μM的互补链。在一个热块放置在95℃下5分钟。关火块，并允许寡聚慢慢冷却至在使用前室温。 </li></ol></li><li>之前开始，温暖的结合缓冲液，低严格洗涤缓冲液，高严格洗涤缓冲液和洗脱缓冲液至室温。 注:50毫微克的最终浓度/毫升多聚d（IC），可被加入到结合缓冲液，低严格洗涤缓冲液，并且高严格洗涤缓冲液，以降低蛋白质的寡聚物的潜在的非特异性结合。 </li><li>准备对每个变量绑定的混合物。 <ol><li>混合1体积核裂解物与2倍体积的结合缓冲液。 注:需要裂解液量必须通过实验，由于不同的转录因子的丰度来确定。核裂解物的100-250微克之间使用每列是在大多数情况下，足够的。 </li><li>添加1X磷酸酶抑制剂，1个蛋白酶抑制剂，和1x增强的结合（可选），轻弹管混匀数次。 注:100X结合增强包括750毫米氯化镁2毫米和300毫米氯化锌的。添加结合增强剂如果对TF的DNA的结合依赖于辅因子或还原剂。如果此信息不知道，添加结合增强剂。 </li><li>加10μl5μM生物素标记的DNA捕获（50皮摩尔）的各有关结合混合物。孵育在室温下20分钟。 注意:孵育时间和温度可以根据对TF变化。的最优值必须通过实验确定。 </li></ol></li><li>加入100μl链亲和素微球。孵育在室温下10分钟。 </li><li>对于所测试的每个寡探针，放置一个结合柱在磁分离器。直接放在离心管每个绑定列下，并应用100微升结合缓冲液冲洗柱子。 </li><li>吸取各结合混合物成单独的列的内容，并允许液体通过该柱完全流入微量在继续之前管。确保与已在混合物中的异体寡核苷酸来标记列。标记流过的样品，并用新的微量离心管更换收集低严格洗涤。 </li><li>应用100微升低严格洗涤缓冲液对柱;等到列水库是空的。重复洗4倍。标签低严格洗涤的样品，并与新的离心管代替收集高严格洗涤。 </li><li>应用100微升高严格性洗涤缓冲液对柱;等到列水库是空的。重复洗4倍。标签的高严格洗涤样品，并与新的离心管代替收集预洗脱。 </li><li>添加30微升天然洗脱缓冲液向柱，静置5分钟。标签洗脱前的样本及与新的离心管代替收集洗脱。 注意:此不洗脱结合蛋白;它洗剩余的高严格缓冲出列并用洗脱缓冲液代替它​​最大限度地洗脱的效率。 </li><li>添加一个额外的50微升本地洗脱缓冲液来洗脱结合转录因子。对于较高的产量，但不太集中洗脱液，增加额外的50微升天然洗脱缓冲液并收集流通。 注意:通过质谱分析洗脱的样品，以确定结合的转录因子32的身份。此后，验证通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），接着Western印迹33的蛋白质组的结果。如果质谱不可用，使用标准的技术，而不是一个Western印迹以确定表示基因型依赖性结合蛋白（S）的大小运行银染色。使用这些信息来缩小从介绍详细的计算方法预测课题组的列表。 </li></ol>

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作者什么都没有透露。

<html>虽然在测序和基因分型技术的进步大大提高了我们的能力，以找出与疾病相关的基因变异，我们了解这些变异影响的作用机制能力滞后。该问题的一个重要原因是，许多疾病相关变体位于在正对编码的基因组的区域，这有可能影响控制基因表达更难对预测的机制。在这里，我们提出了一种基于EMSA和DAPA技术，有价值的分子工具，用于识别基因型依赖TF绑定可能导致许多非编码变异体的功能活动的协议。虽然这两种技术已经在过去广泛使用，它们最近才适于结合的TF的基因变异分析。超越转录因子，EMSA也可用于分析遗传变异体上的RNA结合蛋白，只有轻微的调整，协议36的效果。 ove_content"&gt;本文中所呈现的协议是简单和容易执行;尽管如此，某些部分需要之前的实验首先附加考虑，重要的是通过执行严格的统计分析，以产生用于考虑变体的初始列表中这一步骤的错误。可以破坏所有下游的分析，因为很多变体可能差异绑定核裂解而不会造成疾病的风险。此外，在相关的细胞类型进行使用功能的基因组数据是至关重要的，因为不相关的细胞类型可以导致假阳性TF的产生约束力预测。目前，最广泛使用的功能基因组资源包括ENCODE 12和流程图表观基因组17。附加信息有关的细胞类型相关的感兴趣的疾病的基因表达水平可以从其他资源，如ImmGen 37，BioGPS 38来获得， 39，以及SNPsea 20。例如，这种RESOURCES可用于TF结合预测过滤，只包括那些在相关的细胞类型表达转录因子。 同样重要的是要考虑的体外实验如EMSA和DAPA的注意事项。特别是，它需要重复与单独的核裂解物制剂的实验，以减少假阴性。此外，一个成功的EMSA-超迁移可呈现在EMSA频带的进一步移位，其中抗体结合了的TF，或带的损失，在该抗体阻断TF的DNA结合结构域。在这两种情况下，包括向一个不同的TF同种型对照抗体和/或抗体是在确认超迁移的特异性是有用的。在执行超迁移的另一个考虑是，是否包括DTT /聚山梨酯反应。 DTT /聚山梨酯稳定样染料允许未结合的DNA更准​​确量化;然而，它也可以降低抗体的二硫键导致ING在EMSA-超迁移失败。它建议尝试有和没有DTT /聚山梨酯试图超迁移复杂时反应。多聚d（IC）和每个反应鲑鱼精子DNA的最佳量必须通过实验滴定来确定。通常，滴定范围1-6微克多聚d（IC）和50-500纳克的鲑精的是足够的。对DAPA一个有用的阳性对照包括使用共有序列具有很好的特点结合基序（从数据库如CIS-BP 23或40 Factorbook获得）一个TF和特定于该TF的抗体运行的西部。对于这两种测定法，加扰的寡可以用来表明任何观察到的结合是特异于感兴趣的寡核苷酸。 无论是EMSA和DAPA技术有实验的限制。例如，一个TF和DNA之间的结合亲和力在由缓冲条件很大程度上受到影响。理想情况下，缓冲条件应该模仿内源性conditio细胞核纳秒以允许最佳结合。对EMSA，不当缓冲条件可能会导致弱带或带的完全的损失。为DAPA，非理想的情况可能会导致对TF（县）在洗涤步骤中洗脱。因此，每个测定是仅在确定的TF-寡聚在一定的缓冲条件的结合有​​效的。最普遍有用的缓冲条件在上述协议提出的。第二个限制是，从EMSA和DAPA方法的实验结果提供了有关的机制，通过该转录因子结合至寡聚物的信息很少。转录因子可以结合寡核苷酸直接或受到其他因素的招募。因此，为了分析寡序列计算来预测转录因子可能结合至寡聚物和实验验证这些预测是重要的。例如，特异性结合的序列可以被突变或结合伴侣可以实验耗尽。最后，需要提取的用量要滴定每个实验吨Ø获得最佳的效果。太多的裂解液可能饱和的TF-寡约束力，任何掩盖风险差异和非风险等位基因之间的结合。对于其他故障排除，读者可以参考一些优秀的审查和方法的手稿30,41,42。 除了 ​​此处所描述的测定法，有多种可用于进一步研究变体的活细胞（ 图1，下图）内的作用在体内测定的。染色质免疫沉淀，随后通过等位基因特异性定量聚合酶链反应（芯片的qPCR）的研究可以进行，以确定是否在体外测定中的活细胞34被复制内结合所观察到的差。例如，如果使用杂对于感兴趣的变体的细胞，定量PCR实验可检测的TF免疫沉淀染色质两个等位基因之间的浓缩的差异。芯片需要芯片特定的等级抗体; howeveR，如果沉淀同类的TF抗体是不可用的，转染细胞用旗标记TF是一种有效的替代43。此外，差动对靶基因表达的结合的效果，可以通过在相关的细胞类型表达的数量性状基因座（eQTL）分析使用来自基因分型的细胞或公开可用的资源，如那些由Genevar 44编译本地收集表达数据分析。萤光素酶报道测定法也可用于探索到差分蛋白质的结合影响基因表达的程度。此类测定法可以被修改而不管变体是否位于一个启动子，增强子或阻抑区域45-47工作。最后，基因组编辑的技术，如CRISPR / Cas9 48，可用于产生只在一个单一的变体，它是用于确认因果重要不同细胞系。这种技术可以大大减少betw观察实验方差从遗传多样性受试者衍生的，因为官能读数分析基因表达或另一种疾病中间表型EEN细胞系可以在编辑的细胞系中进行，并与非编辑细胞系。 提出的策略的主要优点是，它使容易和快速检测基因型依赖性TF结合的。通过优先的关键遗传变异体，进一步实验可以被设计为确定其生物效应，并证明其因果关系。值得注意的是，这个协议可以应用于调查从GWAS或精细作图鉴定的任何疾病或表型相关联的变体。阿大，越来越多的基因数据和统计相关的遗传变异的名单宝库已经可用。在大多数情况下，驱动这些变体的统计关联的生物学机制尚不清楚。在这个提案中概述的战略使得许多非编码disea的准确诠释功能SE-相关变种。这种知​​识是用于驱动任何基于遗传疾病的分子机制的充分澄清至关重要。

测序和基因分型的基础研究，包括全基因组关联分析（GWAS），候选人基因研究和深测序研究，已经确定了在统计学上与疾病，性状或表型相关的许多基因变异。相反早期预测，大多数这些变体（85-93％）的分布在非编码区，并且不改变蛋白质1,2的氨基酸序列。解释这些非编码变体的功能和确定它们连接到相关的疾病的生物学机制，特征，或表型已被证明具有挑战性3-6。我们已开发，以确定链接变体的重要中间体的表型的分子机制的一般策略 - 基因表达。这条管线是专门设计来确定的TF由遗传变异体结合的调制。这一策略结合的目的是预测的计算方法和分子生物学技术在硅片的候选变体的生物学效应，并验证这些预测凭经验（ 图1）。 <img alt="图1" src="/files/ftp_upload/54093/54093fig1.jpg" /> 图1: 对于不包含在这个手稿以灰色阴影相关的详细协议的非编码基因变异步骤的分析战略方针 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在许多情况下，通过扩大变体的列表以包括与每个统计学关联变体在高联动不平衡（LD）的所有那些开始是很重要的。 LD是在两个不同的染色体位置的等位基因的非随机关联，可以由R 2统计7被测量的量度。 R 2是林的措施卡格两个变量之间的不平衡，有两个变量之间的R 2 = 1表示完全连锁。在高LD等位基因被发现在整个人群祖先染色体上共分离。当前的基因分型阵列不包括在人类基因组中所有已知的变体。相反，他们利用了人类基因组中的LD和包括公知的变体作为代理为LD 8的一特定区域内的其它变体的一个子集。因此，在没有任何生物后果的变体可以与特定疾病，因为它是在与一个有意义的生物效应的因果变体的变体的LD相关联。在程序上，建议转换的1000基因组最新发布的项目9项变量调用文件（VCF）与PLINK 10,11，用于全基因组关联分析的开放源代码工具兼容的二进制文件。随后，LD R 2&gt; 0.8所有其他遗传变异与每个输入遗传VAriant可确定为候选者。使用合适的参考人群该步进例如是很重要的，如果一个变种是在欧洲血统的科目，从类似的血统科目的数据应该用于LD扩展标识。 LD膨胀常常导致数十个候选变体，它是可能的，只有这些一小部分有助于疾病的机制。通常，它是不可行的实验逐一检查每个这些变体。因此，有用以利用数千公开可用功能基因组数据集作为过滤器的变体的优先次序。例如，编码财团12已经如DNA酶SEQ 13，ATAC进行数千描述转录因子的结合芯片起实验和辅因子，以及组蛋白标记在各种上下文，以及染色质辅助数据从技术-SEQ 14和FAIRE-SEQ 15。数据BASES和Web服务器如UCSC基因组浏览器16，路线图表观基因组17，蓝图表观基因组18，顺反组19，并重新映射20提供由这些和其他实验技术在广泛的细胞类型和条件产生的数据免费访问。当有太多的变体通过实验检验，这些数据可以被用来优先那些位于相关的细胞和组织类型的可能调控区之内。此外，在一个变型为特定蛋白的芯片起峰内的情况下，这些数据可以提供潜在线索作为对特定TF（S）或辅因子的结合可能影响。 接着，将所得的优先变体实验筛选，以验证预测的基因型依赖性蛋白使用EMSA 21,22结合。 EMSA测量的寡核苷酸上的非还原性TBE凝胶迁移的变化。荧光标记的寡核苷酸温育与核裂解物，以及核因子的结合将延缓在凝胶上的寡的运动。以这种方式，已经结合更多的核因子将呈现为对扫描更强的荧光信号寡。值得注意的是，EMSA不需要关于特定蛋白质的结合会受到影响的预测。 一旦变体被识别，它们位于预测的调节区之内，并且能够差动结合核因子中，采用计算的方法来预测具体TF（S），其结合它们可能会影响。我们优选使用CIS-BP 23,24，RegulomeDB 25，UniProbe 26和JASPAR 27。一旦候选人转录因子被确定，这些预测可以专门使用抗转录因子这些（EMSA-supershifts和DAPA-西部片）测试。一个EMSA-超迁移涉及增加了一个特定的TF-抗体与核裂解物和寡。阳性结果在EMSA-超迁移是再版esented如在EMSA频带的进一步转变，或带的损耗（在参考28中综述）。在互补DAPA，含有该变体和20个碱基对的侧翼核苷酸的5&#39;-生物素化的寡核苷酸双链体与来自相关的细胞类型（多个）核裂解物温育以捕获任何核因子特异性结合的寡核苷酸。低聚双链核因子复合物是通过在一个磁列链霉微珠固定。结合的核因子直接通过洗脱29,48收集。结合预测可以然后通过使用特异于所述蛋白质的抗体的Western印迹来评估。在没有明显的预测，或过多的预测，从DAPA实验的变体拉起伏的洗脱可被发送到一个蛋白质组学芯使用质谱来识别候选转录因子的情况下，其可以随后使用验证这些先前描述的方法。 在该articl剩余即，提供了一种用于遗传变异体的EMSA和DAPA分析的详细协议。

<table><tbody><tr><td>Custom DNA Oligonucleotides</td><td>Integrated DNA Technologies</td><td></td><td>http://www.idtdna.com/site/order/oligoentry</td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>BP366-500</td><td>KCl, for CE buffer</td></tr><tr><td>HEPES (1 M)</td><td>Fisher Scientific</td><td>15630-080</td><td>For CE and NE buffer</td></tr><tr><td>EDTA (0.5M), pH 8.0</td><td>Life Technologies</td><td>R1021</td><td>For CE, NE, and annealing buffer</td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td>NaCl, for NE buffer</td></tr><tr><td>Tris-HCl (1M), pH 8.0</td><td>Invitrogen</td><td>BP1756-100</td><td>For annealing buffer</td></tr><tr><td>Phosphate Buffered Saline (1x)</td><td>Fisher Scientific</td><td>MT21040CM</td><td>PBS, for cell wash</td></tr><tr><td>DL-Dithiothreitol solution (1 M)</td><td>Sigma</td><td>646563</td><td>Reducing agent</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Thermo Scientific</td><td>87786</td><td>Prevents catabolism of TFs</td></tr><tr><td>Phosphatase Inhibitor Cocktail&amp;nbsp;</td><td>Thermo Scientific</td><td>78420</td><td>Prevents dephosphorylation of TFs</td></tr><tr><td>Nonidet P-40 Substitute</td><td>IBI Scientific</td><td>IB01140</td><td>NP-40, for nuclear extraction</td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>For measuring protein concentration</td></tr><tr><td>Odyssey EMSA Buffer Kit</td><td>Licor</td><td>829-07910</td><td>Contains all necessary EMSA buffers</td></tr><tr><td>TBE Gels, 6%, 12 Wells</td><td>Invitrogen</td><td>EC6265BOX</td><td>For EMSA</td></tr><tr><td>TBE Buffer (10x)</td><td>Thermo Scientific</td><td>B52</td><td>For EMSA</td></tr><tr><td>FactorFinder Starting Kit</td><td>Miltenyi Biotec</td><td>130-092-318</td><td>Contains all necessary DAPA buffers</td></tr><tr><td>Licor Odyssey CLx</td><td>Licor</td><td></td><td>Recommended scanner for DAPA/EMSA</td></tr><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240-062</td><td>Contains 10,000 units/ml of penicillin, 10,000 &amp;micro;g/ml of streptomycin, and 25 &amp;micro;g/ml of Fungizone&amp;reg; Antimycotic</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>26140-079</td><td>FBS, for culture media</td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>22400-071</td><td>Contains L-glutamine and 25 mM HEPES</td></tr></tbody></table>

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.

在这一节中，关于裂解液的质量，其特征在于执行EMSA或dapA和变异当提供所期待的代表性结果。例如，已经提出，冻融蛋白样品多次可能导致变性。为了探索在这些"冻融"循环的上下文EMSA分析的再现性，二35沸点寡聚在同一遗传变异体不同，用核裂解物的单一批次解冻并重新冷冻用于指定量孵育。次图2表明，冷冻和解冻这个特定B淋巴细胞核提取物达5倍，对蛋白质的完整性似乎没有影响;然而，核蛋白质的稳定性通过样品而不同，并且因此，应在使用每个单独的细胞系测试。它也可能有不同批次prepar变异核裂解ations。是否该变化是由于之前的裂解，细胞传代次数，或其他因素，细胞周期的阶段，它使用裂解物的不同批次，以确保真正的结果是很重要的，以复制EMSA结果。 此外，以优化信噪比的EMSA是重要的。在这方面的一个重要的变量是寡聚浓度。寡（5-300飞摩尔）的量进行滴定，以评估不同量寡聚物如何影响该信号（ 图3）。未观察到条带的强度高达100 fmol的寡的增加。这一点后，信号高原提示100 fmol的是该EMSA反应的最佳寡量。 最后，从我们使用本手稿中描述的策略的一部分，已发表 ​​的研究中的一个有代表性的数字被提供（ 图4）。在日是研究34中，我们表明，rs6590330增加STAT1，在这两种协同激活和1型干扰素受体 ​​复合35的下游基因表达的抑制参与转录因子结合的狼疮相关风险等位基因。在这个例子中，STAT1首先由DAPA使用耦合到串联质谱（DAPA-MS）高效液相色谱法，随后通过蛋白质组分析，鉴定。一个DAPA-Western印迹被用来确认由蛋白质组学分析（STAT1）所确定的TF并确认STAT1的磷酸化形式被结合到非基准（狼疮风险）寡。该图示出了在此策略中的各种测定法如何可用于识别差分转录因子的结合的非编码的变体。 <img alt="图2" src="/files/ftp_upload/54093/54093fig2.jpg" /> 图2: 重复性分析和EMSA结果冻融循环的后果。含有遗传变体的参考（R）或非参考（NR）等位基因寡聚用于探测冻融的多个周期后的相同制备裂解物。 （赖氨酸:裂解液）。每个泳道含有的凝胶（蓝色箭头）的底部自由探针。的转录因子的寡核苷酸的结合可被看作是在凝胶（红色箭头）的上半部分的频带。含有凝胶（见支架）的下半部内的波段是不特定的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54093/54093fig3.jpg" /> 图 3: 不同的寡浓度的效果评估寡聚各种浓度的用于探测核裂解物的单一制剂。 Fluorescent信号具有增加量的寡增加，这表明寡是限制性试剂。在100-300 fmol的车道，其中蛋白质的量成为限制试剂的信号高原。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54093/54093fig4.jpg" /> 图4:rs6590330 的狼疮风险等位基因增加STAT1结合，由DAPA-MS STAT1和pSTAT1具有更高的结合相对于非风险等位基因含有rs6590330风险等位基因的寡核苷酸所评估。生物素标记的寡核苷酸用Epstein-Barr病毒转化的B细胞的核提取物温育。结合到寡聚蛋白用DAPA捕获。蛋白质然后通过SDS聚丙烯酰胺凝胶电泳分离和检测使用抗pSTAT1（A）或抗STAT1（B）。 M:标记物。 NR:含有rs6590330的非风险等位基因的寡; R:含有rs6590330的风险等位基因寡;突变体:含有破坏推定的STAT1结合位点rs6590330的下游寡;细胞裂解液:由B细胞核提取物。带的相对强度表示每个频带的下方。结果代表四次实验;而所有的实验证明增加的STAT1结合与风险等位基因的探针，在2/4的实验没有STAT1或来自非风险寡免疫沉淀中检测pSTAT1，而两者都在从风险寡免疫沉淀检测。这个数字已经从34参考修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54093/54093fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA at JoVE.com

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 ...

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Nanyang Technological University

Research

JoVE Journal

Biology

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

12.9K 次观看.  Cincinnati Children's Hospital. 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.

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

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

科研

生物化学

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

遗传学

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

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 ...

癌症研究

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

基因组规模筛选，用于识别和比较肿瘤中的治疗靶点

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

作者聚焦:通过基因组规模 shRNA 筛选寻找恶性周围神经鞘瘤的新治疗靶点 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

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

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

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

等位基因特异性基因表达检测，测试的遗传协会的功能基础

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

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）

摘要

探索更多视频

此视频中的章节

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

患者生物流体中肿瘤相关循环DNA的检测与监测

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

Naked-Eye Detection of Rare Point Mutations in DNA

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

等位基因特异性基因表达检测，测试的遗传协会的功能基础