We thank the Ligue contre le Cancer de C&#244;te d&#8217;Or and the Centre Georges-Fran&#231;ois Leclerc for their financial support. We thank Philip Bastable for the editing of the manuscript.

基因组DNA 1。评估（基因组DNA）产量<ol><li>量化新鲜提取基因组DNA的制备图书馆前。使用荧光估产方法量化完整的基因组DNA（避免使用分光光度计进行基因组DNA的产量评估）。测量（二百八十零分之二百六十nm）的比率，并确保它是在1.8和2注：50纳克基因组DNA的需要进行实验。 </li></ol> 2，基因组DNA富集：第1天，上午<ol><li>开始之前<ol><li>从DNA富集试剂盒（见表材料/设备），解冻DNA缓冲液（TD）和冰的DNA酶（TDE1）解决方案。至少30分钟，在使用前，使纯化的磁珠和停止目标（ST）的缓冲液至室温（RT）。每个样品中添加20微升基因组DNA样品在2-2.5纳克/微升直接放入96孔板中，1个井。重要提示：使用阳性对照，以验证协议（与已知序列变异样本）。 </li></ol></li><li> Tagmentation <ol><li> M九，所有试剂彻底颠倒5倍，后短暂离心。在室温，加入25微升的TD缓冲液到每个孔中含有50的gDNA纳克（20微升），然后取5μl的TDE1缓冲器。将吸管50微升，轻轻吸取整个音量上下10倍。 </li><li>覆盖板用粘接膜和离心机在280 XG 1分钟，在20℃。然后，将板在热循环仪（盖在100℃加热），然后运行下面的程序：55℃5分钟，10℃，无时间限制。 </li><li>在此培养后，制备与实验管理器软件的样品片材，以定义将要使用的索引。 </li></ol></li><li> Tagmentation净化 注意：该步骤是关键的。要非常小心。 <ol><li>检查无沉淀净化磁珠和ST缓冲器。如果沉淀物存在，热身手中的解决方案。解冻再悬浮缓冲（RSB）冰上。 </li><li>在RT下，涡街溶液和添加15μl到每个孔中含有的样品。将吸管65微升，轻轻吸取整个音量上下10倍。孵育5分钟，在室温。覆盖板和离心机在280 XG 1分钟，在20℃。 </li><li>加入52微升的预先混合纯化的磁珠在每个孔中。将吸管117微升，轻轻吸取整个音量上下10倍。下室温温育10分钟。覆盖板和离心机在280 XG 1分钟，在20℃。 </li><li>除去粘合膜。放置在96孔板上的磁性支架在室温2分钟。确保上清出现彻底清除。 </li><li>制备新鲜的80％乙醇溶液（24个样品，加入2毫升蒸馏水至8毫升乙醇中）。取出并弃去上清液，注意不要打扰珠。 </li><li>同时使板在磁性支架，加入200微升新鲜制备的80％乙醇中，以每孔，而不会干扰珠孵育该板为至少30秒在室温下进行。弃上清，重复此洗了第二次。确保乙醇已被删除。让干燥，在室温下搅拌10分钟或直到乙醇完全蒸发。 </li><li>从磁底座上卸下板并添加22.5微升RSB缓冲。移液器设置为22.5微升，轻轻吸取整个体积上下10倍。放置在96孔板上的磁性支架并孵育2分钟。确保上清出现彻底清除。轻轻的转移在一个新的96孔板20μL上清液。 注意：任选地，通过使用一个片段分析器确定tagmented样本的大小。而不是上面提到的步骤，使用高分辨率的丙烯酰胺凝胶电泳。片段应为150个基点至1000个基点之间。 </li></ol></li><li>第1次PCR扩增<ol><li>解冻PCR扩增预混含有聚合酶（LP-PMM），索引1引，索引2引物溶液在冰上。匹配组合Ø˚F指标与实验管理器示例表。对于多路，准备从索引1（I7，N7xx）对每个样品不同I7。 </li><li>在每孔加入20微升的LP-PMM，5微升指数1和5微升指数2将吸管50微升，轻轻吸取整个音量上下10倍。覆盖板用粘合剂微密封膜。离心机在280 XG 1分钟，在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序1（ 表1）。 注：该过程可以停止在这一点上与该反应过夜储存在热循环或2至8℃下2天。 </li></ol></li></ol> 3，基因组DNA富集：第1天，下午<ol><li>开始之前<ol><li>融寡聚物（CSO），捕获目标缓冲器1（NCT1），并在冰上RSB的解决方案。至少30分钟，在使用前，使纯化的磁珠至室温。 </li></ol></li><li> PCR纯化<ol><li>从步骤2.4离心盘，持续1分钟，在280 XG在20℃。 </li><li>在RT下，在各孔中加入45微升混合纯化的磁珠。移液器设置为95微升，轻轻吸取整个体积上下10倍。下室温温育10分钟。放置在96孔板上的磁性支架在室温2分钟。 </li><li>确保上清出现彻底清除。制备新鲜的80％乙醇溶液（24个样品，加入2毫升蒸馏水至8毫升乙醇中）。弃去上清液，注意不要打扰颗粒。 </li><li>同时使板在磁性支架，加入200微升新鲜制备的80％乙醇中，以每孔，而不会干扰珠孵育该板进行30秒，在室温下进行。弃上清，重复此洗了第二次。确保乙醇已被删除。让干燥，在室温下搅拌15分钟。 </li><li>从磁底座上卸下板加入40μl的RSB缓冲。设置管TTE至40微升，轻轻吸取整个音量上下10倍。 </li><li>放置在96孔板上的磁性支架并孵育2分钟。上清应该出现彻底清除。轻轻转移38微升上清液到新的96孔板中。 </li><li>测定各样品的通过荧光法的产率。如果所评估的产率是30至50纳克/微升之间的协议的第一部分将被验证。 </li></ol></li><li> 1号杂交<ol><li>普尔高达每池12个样本在这个阶段通过将500ng的每种样品中一种新的96孔板中。确保每个池的最终体积不超过40微升。 注：如果需要的话，减少池的体积通过使用真空浓缩装置​​在RT（注意：DNA的浓度而不能加热进行修改，即使在30℃下加热使DNA降解）。通过添加RSB溶液调整最终体积至40微升。 </li><li>充分混匀NCT1解决方案。对于每个孔含有40微升合并的DNA样品中，加50微升NCT1的，和10μl的CSO。移液器设置为100微升，轻轻吸取整个体积上下10倍。密封该板，并离心1分钟，在280 XG在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序2（ 表1）。 </li></ol></li></ol> 4，基因组DNA富集：2日，上午<ol><li>开始之前<ol><li>从DNA富集试剂盒（见表材料/设备）解冻2 N氢氧化钠（HP3），目标洗脱缓冲液1（ET1），目标洗脱缓冲液1 ET2，链亲和素磁珠（SMB），洗涤液1（WS1），洗解决方案2（WS2），洗涤液3（WS3），CSO和NCT1解决方案，在室温。 </li></ol></li><li> 1号杂交洗<ol><li>从该热循环和离心分离1分钟，在280 XG取出96孔板在20℃。取下粘合盖很carefuLLY。 </li><li>从平板转移100μl的反应混合物到新的MIDI 96孔板中并加入250微升以及涡旋SMB溶液。移液器设置为350微升，轻轻吸取整个体积上下10倍。密封该板，并在室温下孵育30分钟。 </li><li>离心机在280 XG 1分钟，在20℃下，除去了粘合剂密封，并放置在盘上的磁性支架，持续2分钟。确保上清出现彻底清除。弃去上清液，并从磁性底座上卸下盘。 </li><li>加入200μl的良好混合WS1溶液进入井含珠。移液器设置为200微升，轻轻吸取的整个体积上下15X避免气泡/泡沫形成。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。弃去上清液，并从磁性底座上卸下盘。 </li><li>加入200μl的良好混合WS2溶液进入井其所含进不去珠。移液器设置为200微升，轻轻吸取的整个体积上下15倍，避免形成气泡/泡沫。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。弃去上清液，并从磁性底座上卸下盘。 </li><li>加入200μl的良好混合WS2溶液到含有珠的孔中。移液器设置为200微升，轻轻吸取的整个体积上下15x连接。 </li><li>传送在新的96孔板的整个体积。密封该板，并将该板在热循环仪（盖加热至100℃）。在42℃下启动热循环30分钟。 </li><li> Imediately放置盘上的磁性支架，持续2分钟。确保上清出现彻底清除。取下密封并立即丢弃所有上清液。然后，从磁底座上卸下盘。 </li><li>加入200μlWS 2成含珠孔。将吸管200微升，轻轻吸取的整个体积上下15倍，避免形成气泡/泡沫。密封该板，并将该板在热循环仪（盖加热至100℃）。在42℃下启动热循环30分钟。 </li><li>立即将板在磁性支架，持续2分钟。确保上清出现彻底清除。取下密封并立即丢弃所有上清液。然后，从磁底座上卸下盘。 </li><li>加入200μl的良好混合WS3溶液到含有珠的孔中。移液器设置为200微升，轻轻吸取的整个体积上下15x连接。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。丢弃所有上清液，并从磁性底座上卸下盘。 </li><li>重复此WS3洗了第二次。 </li><li>除去上清液后，密封板，并短暂离心​​拉下残留上清。在放置板磁性支架，持续2分钟。丢弃剩余的上清液。 </li><li>在0.2 ml管中，混匀28.5微升ET1和1.5微升HP3解决方案。这样的搭配是1池，若再池准备，以备池的数量乘以这些卷。 </li><li>从磁底座上卸下板。加入23微升上述制备的混合物。移液器设置为23微升，轻轻吸取的整个体积上下15x连接。密封该板，并留下5分钟，在室温。离心机在280 XG 1分钟，在20℃。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。除去粘合膜和转移21微升上清液到新的96孔板中。 </li><li>加入4微升ET2溶液到每个孔中。移液器设置为25微升，轻轻吸取的整个体积的上下10倍和密封板。 注：该过程可以停止在这一点上与反应贮存7天，在-15℃下。如果板被冻结，启动第二杂交之前完全解冻。 </li></ol></li><li> 第二杂交<ol><li>离心盘，持续1分钟，在280 XG在20℃。除去粘合膜，并添加50μl的NCT1，10微升的CSO，与15微升的PCR级水至25μl的库。移液器设置为100微升，轻轻吸取的整个体积上下10倍。 </li><li>密封该板，并离心1分钟，在280 XG在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序2（ 表1）。 </li></ol></li></ol> 5，基因组DNA富集：2日，下午<ol><li>开始之前<ol><li>解冻ET1，ET2，SMB，WS1，WS2，WS3和HP3溶液在RT杂交洗涤。从DNA富集试剂盒，解冻PCR扩增预混含有聚合酶（TC-PMM），PCR引物鸡尾酒（PPC），和RSB的解决方案，在冰上进行PCR扩增。 </li></ol></li><li> 第二杂交洗<ol><li>按照完全相同的协议为4.2 - 1号杂交洗。 </li></ol></li><li> PCR扩增<ol><li>在新的96孔板中，混合20微升洗脱的来自步骤5.2中，将25μl的TC-PMM的，和5μl的PPC。移液器设置为50微升，轻轻吸取的整个体积上下10倍。密封该板，并离心1分钟，在280 XG在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序3（ 表1）。 </li></ol></li></ol> 6，基因组DNA富集：3天<ol><li>开始之前<ol><li>冰解冻RSB的解决方案。至少30分钟，在使用前，使纯化的磁珠至室温。 </li></ol></li><li> PCR纯化<ol><li>离心反应板从步骤5.3，持续1分钟，在280 XG在20℃，并除去粘合剂覆盖。 </li><li>在室温下，加入90微升的previously混合纯化的磁珠在每个孔中。移液器设置为140微升，轻轻吸取整个体积上下10倍。孵育15分钟，在室温。放置在96孔板上的磁性底座5分钟，在室温。确保上清出现彻底清除。 </li><li>制备新鲜的80％乙醇溶液（2样品中，加入200微升的蒸馏水，以800微升乙醇）。弃去上清液，注意不要打扰颗粒。 </li><li>同时使板在磁性支架，加入200微升新鲜制备的80％乙醇中，以每孔，而不会干扰珠孵育该板进行30秒，在室温。弃上清，重复此清洗一次。确保乙醇已被删除。让干燥在室温进行15分钟或直到乙醇完全蒸发，而该板是在磁性支架上。 </li><li>从磁底座上卸下板，加入30微升的RSB缓冲。移液器设置为30微升，并轻轻吸取吨他整个音量上下10倍。将培养板在室温下2分钟。 </li><li>放置在96孔板上的磁性支架并孵育5分钟，或直至上清液出现完全清楚。轻轻转移28微升上清液到新的96孔板（板类型：TCY）。 注：该过程可以停止在这个阶段，并存储在-15℃下进行长达7天的反应。 </li></ol></li><li>库验证和定量<ol><li>通过使用片段分析仪确定库的质量。高分辨率的聚丙烯酰胺凝胶电泳可以用来代替上述的步骤，但不推荐使用。片段应为150个基点至1000个基点之间。 注：推荐：通过qPCR定量库测序过程中获得集群的最佳数量。 </li><li>作为参考，使用经过验证库（2米）。如果第一个库正在准备中，使用规定的sequenc校准噬菌体PhiX的DNA（10纳米）荷兰国际集团的设备。 </li><li>制备阳性对照的系列稀释液，得到7个点，20，16，8,6，4,2，和下午1点在EBT（EB洗脱缓冲液+0.1％吐温20）。淡化利益图书馆在1/1000〜1/2000。每一点必须以一式三份进行分析。 </li><li>制备下列混合物（乘法卷所使用的孔的数目）：对于1个孔中，添加10微升的SYBR Green含有定量PCR主混合物（2×），0.2微升正向引物（10μM，AATGATACGGCGACCACCGAGAT），0.2微升反向引物的（10μM，CAAGCAGAAGACGGCATACGA），和7.6微升的PCR级水。 </li><li>混合后，废除在96孔板中，18微升混合液到每个孔中，并加入2μl的阳性对照和文库稀释。密封该板，并离心1分钟，在280 XG在20℃。然后，把平板的定量PCR热循环和运行程序4（ 表1）。 </li><li>分析结果如常规绝对量化，得到每个升的产率ibrary。定量PCR的优点是，它仅量化的DNA，将与流动池进行交互。 注：请不要使用的DNA荧光定量由于DNA分子模拟在试剂中的一个存在。这种量化方法将高估图书馆产量，从而低估了集群得分。 </li></ol></li><li>排序下水<ol><li>稀释每个库在4纳米的30微升的洗脱缓冲液（EB）的最终体积，并集中所有的图书馆，拌匀。 </li><li>准备在EB缓冲液新鲜的0.2N的NaOH溶液，搅拌10微升该NaOH溶液与10微升汇集库。搅拌均匀，准确地孵育5分钟在室温。 </li><li> 5分钟后，立即加入980μL杂交液（从测序盒）拌匀。然后，混合400μL与600μL杂交液这种混合物。拌匀并注入了8转600微升到300周期盒（2×150）PM。启动顺序。 </li></ol></li></ol> 7，数据分析<ol><li>一旦设备处理的数据中，.bam和.vcf文件转移到新电脑上。 注：转座子碎片结合的脚印。因此，软件会自动修剪这些数据。 </li><li>收集与所述设备分析所获得的遗传变异。 </li><li>通过按照制造商的指示进行分析导出的文件（.bam，的.vcf）与另一软件（阿拉穆特）。然后，比较用两种分析中得到的遗传变异。唯一的变化检测两次都被认为存在。 </li></ol> 表1 PCR条件 <table><tbody><tr><td> 节目 </td><td> 温度 </td><td> 时间 </td><td> 民。重复 </td></tr><tr><td rowspan="7"> 1 </td><td> 72℃ </td><td> 3分钟</td><td> 1 </td></tR&gt; <tr><td> 98℃ </td><td> 30秒</td><td> 1 </td></tr><tr><td> 98℃ </td><td> 10秒</td><td rowspan="3"> 10 </td></tr><tr><td> 60℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 10℃ </td><td>无限</td><td></td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="19"> 2 </td><td> 95℃ </td><td> 10分钟</td><td> 1 </td></tr><tr><td> 93℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 91℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 89℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 87℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 85°C </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 83℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 81℃ </tD&gt; <td> 1分钟</td><td> 1 </td></tr><tr><td> 79℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 77℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 75℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 71℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 69℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 67℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 65℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 63℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 61℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 59℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 58℃ </td><td> 1分钟</td><td> 16-18小时</td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="6"> 3 </td><td> 98℃ </td><td> 30秒</td><td> 1 </td></tr><tr><td> 98℃ </td><td> 10秒</td><td rowspan="3"> 10 </td></tr><tr><td> 60℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 10℃ </td><td>无限</td><td></td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="3"> 4 </td><td> 95℃ </td><td> 10分钟</td><td> 1 </td></tr><tr><td> 95℃ </td><td> 15秒</td><td rowspan="2"> 40 </td></tr><tr><td> 60℃ </td><td> 1分钟</td></tr></tbody></table>

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The authors have no conflicts of interest to declare.

广泛使用的NGS的设备和技术已经在癌症和遗传疾病的研究提供新的机会。除了全基因组测序和RNA测序，产生大量的无数的患者选择的基因组DNA序列的分析同时提供了诊断的前景十分广阔。在这里，我们开发了使用Nextera技术，同时研究11完整的基因在24例中等通量测序装置（表材料/设备）的特定设计（随叫随到）。该协议允许快速生成的数据，使患者的错误的低风险的担忧快速反应。如示于?...

2010年，有近150万人（主要是女性）开发的全球乳腺癌。据估计，5％至10％的这些情况是遗传的。大约20年前，BRCA1和BRCA2被认定为参与遗传性乳腺癌和卵巢癌1。自从大约15年前，BRCA1和BRCA2基因的编码区已被测序，以确定遗传倾向的乳腺癌和卵巢癌。在BRCA1和BRCA2的变化在10％至20％选家庭2表明这些区域的分析是不够有效的筛查检测。近日，BRCA1和BRCA2基因的非编码序列（启动子，内含子，3&#39;UTR）的分析，强调了新的基因突变/变异可能与乳腺癌3-6的风险较高。 BRCA1和BRCA2蛋白参与同源重组修复（HHR），它是由众多的合作伙伴7完成。而在BRCA1或BRCA2基因的改变诱发的DNA修复缺陷，其他伙伴也可能会影响到乳腺癌的危险性。这个假设似乎因为BRIP1 8已被验证，PALB2 9对宫颈癌和乳腺癌的一个行之有效的影响，分别为。此外，还有两个其他的“中度风险”的乳腺癌易感基因，ATM和CHEK2，也可以定期研究10。 从这些研究之后，我们决定开发一个协议分析11个基因（ 自动取款机，BARD1，BRCA1，BRCA2，BRIP1，CHEK2，PALB2，RAD50，RAD51C，RAP80和TP53），24例同时使用一个非常简单且相对基于转座技术快速的协议，以充实和测序中等吞吐量的设备上。谢谢该技术，我们测序完成基因的启动子的3&#39;-UTR的端部的起动，除了RAP80，对于其中的2500 bp的内含子区域没有被覆盖的（CHR 5：176,381,588-176,390,180）。这代表共约1000300 BP研究与2,734探头。通常，BRCA1和BRCA2的外显子序列由Sanger测序，这需要1.5个月时间少于20名患者进行分析。本协议（ 图1），在相同的时间，11个完整的基因为大于75的病人可以进行分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MiSeq</td>
			<td>Illumina</td>
			<td>SY-410-1001</td>
			<td>Sequencing/medium throughput device</td>
		</tr>
		<tr>
			<td>Nextera Enrichment kit</td>
			<td>Illumina</td>
			<td>FC-123-1208</td>
			<td>Transposase based technology</td>
		</tr>
		<tr>
			<td>300 cycle cartridge</td>
			<td>Illumina</td>
			<td>15033624</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Magnetic purification beads</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Alpaqua</td>
			<td>A32782</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Life Technologies</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr>
			<td>MIDI 96-well plates</td>
			<td>Biorad</td>
			<td>AB0859</td>
			<td></td>
		</tr>
		<tr>
			<td>Microseal A</td>
			<td>Biorad</td>
			<td>MSA-5001</td>
			<td>This seal is necessary only for PCR amplification. Other standard seals can be used throughout the experiment</td>
		</tr>
		<tr>
			<td>MiSeq</td>
			<td>Illumina</td>
			<td></td>
			<td>Provided with the sequencing device 
			Experiment Manager software</td>
		</tr>
		<tr>
			<td></td>
			<td>Illumina</td>
			<td></td>
			<td>Internet adress: http://designstudio.illumina.com/NexteraRc/project/new&#62; 
			Manufacturer website tool</td>
		</tr>
	</tbody>
</table>

gdna enrichment by a transposase-based technology for ngs analysis of the whole sequence of brca1, brca2, and 9 genes involved in dna damage repair

广泛使用的新一代测序开辟了为癌症研究和诊断的新途径。农工商会带来癌症大量的新数据，尤其是癌症遗传学。目前的知识和未来发现将使得有必要研究一种巨大的基因可能涉及遗传因素对癌症的数目。在这方面，我们开发了一个Nextera设计研究11参与DNA损伤修复完整的基因。该协议的开发是为了安全地同时学习11个基因（ATM，BARD1，BRCA1，BRCA2，BRIP1，CHEK2，PALB2，RAD50，RAD51C，RAD80和TP53）从启动到3&#39;-UTR 24例。这个协议中，根据转座技术和基因组DNA富集，给出了一个很大的优点的时间为基因诊断由于采样复用条件。这个协议可以安全地使用与血液的gDNA。

样品QC成果这种方法来确定靶基因的序列的能力是基于基因组DNA（ 图2A）的质量和tagmentation步骤的质量。如果tagmentation是不够（ 图2B，上图），测序将不会令人满意。如上面所提到的，tagmentation纯化后，将基因组DNA的应tagmented成片段从150 100bp到1000bp的多数大约300 bp的片段（ 图2B，下图）。 在文库制备结束，库质量是通?...

Watch this Scientific Journal Video about gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair at JoVE.co

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

gdna-enrichment-transposase-based-technology-for-ngs-analysis-whole

Research

JoVE Journal

Biology

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

12.1K 次观看.  Centre Georges-François Leclerc. gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair. 

视频: gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫荧光DNA修复蛋白相互作用的可视化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

An Integrated Approach for Microprotein Identification and Sequence Analysis

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model organisms. However, despite this wealth of sequence information, comprehensive gene annotation efforts have proven challenging, especially for small proteins. Notably, conventional protein annotation methods were designed to intentionally exclude putative proteins encoded by short open reading frames (sORFs) less than 300 nucleotides in length to filter out the exponentially higher number of spurious noncoding sORFs throughout the genome. As a result, hundreds of functional small proteins called microproteins (&#60;100 amino acids in length) have been incorrectly classified as noncoding RNAs or overlooked entirely.
Here we provide a detailed protocol to leverage free, publicly available bioinformatic tools to query genomic regions for microprotein-coding potential based on evolutionary conservation. Specifically, we provide step-by-step instructions on how to examine sequence conservation and coding potential using Phylogenetic Codon Substitution Frequencies (PhyloCSF) on the user-friendly University of California Santa Cruz (UCSC) Genome Browser. Additionally, we detail steps to efficiently generate multiple species alignments of identified microprotein sequences to visualize amino acid sequence conservation and recommend resources to analyze microprotein characteristics, including predicted domain structures. These powerful tools can be used to help identify putative microprotein-coding sequences in noncanonical genomic regions or to rule out the presence of a conserved coding sequence with translational potential in a noncoding transcript of interest.

The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

一种用于微蛋白鉴定和序列分析的综合方法

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model ...