Name: methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria
Uploaded: 2015-11-10T13:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria at JoVE.com

作者感谢比尔和梅林达·盖茨基金会的支持技术开发和培训。

注意:此协议包括体积和浓度为96孔板，8联管和毛细管为基础的系统（10微升反应体积）;然而，HRM，也可以在384孔平板的系统通过缩放所有卷相应进行了500微升反应体积。检测对于大多数系统的范围是10至第10纳克模板DNA。 1.准备模板<ol><li>直接从参与野外现场研究中的患者获得与全血储存血液，沉淀的红细胞，或在滤纸上获得的恶性疟原虫的样品。 注:样品也可以培养适应体外生长;一些标准实验室菌株进行分析可以从疟疾研究和参考试剂资源中心（MR4排序，样品可以直接从沉淀红血细胞或培养使用或从全血，红细胞，或滤纸萃取。 </li><li>从提取的DNA滤纸或培养适应株 注:人力资源管理是盐浓度敏感;由于可变提取方法浓度差异可能显著影响熔化温度。虽然具体最终缓冲区并不成功的显著的因素，使用标准的共同洗脱或悬浮缓冲液（ 即水 ，1个特[&#39;小特"或"DNA悬挂缓冲":10毫摩尔Tris-CL，0.10毫摩尔EDTA]或1×TE [10mM的三Cl，1mM EDTA中]）对于所有样品，以避免异常的或不一致的熔融曲线。 <ol><li>准备模板稀释10微克-10纳克每微升的标准特，TE，或水，每10微升反应。 </li></ol></li><li>从沉淀的红血细胞的直接放大<ol><li>确定红细胞原虫使用超薄涂片显微镜 注:可从美国疾病控制和预防中心（CDC）的方法来确定受感染的红血细胞的原虫:HTTP:<html> //www.cdc.gov/dpdx/diagnosticProcedures/blood/microexam.html </li><li>在PCR级水，TE，或碲400的稀释:parasitemias 0.5％以上1。使用3微升为每5〜10微升反应。 </li></ol></li><li>控制 注意:HRM可用于基因扫描，以检测在一个人口12序列变体;但是，准确的基因分型要求的标准序列验证，以包含SNP被审问。质粒构建或疟疾菌株与已知的基因型都可以使用。对于大多数应用，3D7（MR4 MRA-151）用作野生型对照。由于运行之间的变化，总是包括阳性和阴性对照。 <ol><li>阳性对照:准备10微克 - 10纳克每质粒1μl的稀释或标准将序列经过证明的SNP基因型。 </li><li>阴性对照:用PCR级水作为一种无模板对照（NTC）的扩增反应。 </li></ol></li></ol>ve_title"&gt; 2。PCR扩增<ol><li>准备反应混合物<ol><li> （可选）矿物油覆盖。注意:一些人力资源管理系统是独立熔化平台，并没有热盖，以防止扩增过程中凝聚和产品的蒸发。 <ol><li>加入20微升的轻质矿物油，每孔装版的反应混合物之前。 </li></ol></li><li>准备好所有的化验工作10倍的股票<ol><li>请参阅表1为推荐的10倍的股票工作浓度以及终浓度为阻止探针为基础的人力资源管理的基因分型。 </li></ol></li><li>准备反应混合物<ol><li>向每个孔中板或管中，加入1微升10X正向工作原液和反向引物和探针，4.0 - 5.0微升2.5倍或2.0倍的人力资源管理主混合物，1μl的模板，PCR级水为10微升的总反应体积。 </li></ol></li><li>盖板或试管<ol><li>使用光学薄板Seals的板为基础的放大，光纤帽带管为基础的系统，及塑料瓶盖毛细管为基础的系统。要确保覆盖已经完成，板和盖完全密封，并固定，防止扩增过程中蒸发。 </li></ol></li><li>旋转样品<ol><li>旋板3分钟，在1800×g离心以除去气泡，如果矿物油用于分离油相和水相。 </li></ol></li></ol></li><li>扩增<ol><li>将反应板或管的热循环仪。运行标准的阻塞的探针或MAAB扩增方案（ 表2和3，分别）。注意在这些方法之间的退火温度的差异。 </li></ol></li></ol> 3.人力资源管理分析<ol><li>熔化<ol><li> PCR扩增后，前往熔炼分析。对于不具有内置放大的独立熔炼系统 （如，BioFire防御LightScanner系统），再从热循环仪，并装在人力资源管理工具移动放大板。从系统软件接口，熔化板40 - 80℃下同时生产探针和扩增子的熔融峰。 注意:为了节省熔化和分析时间，熔化窗口可被缩短以覆盖温度范围只是探针或扩增子熔化区域。熔化后，将板和管可以再熔融如有必要并保存在-20℃或4℃。商店玻璃管仅在4℃，以防止管从开裂。 </li></ol></li><li>熔融分析<ol><li>删除负面样本<ol><li>在分析软件，从进一步的分析样本未扩增或有锯齿熔融峰删除。注意:选择分析模式针对单独运行文件后，仪器自动组的正和负样本。前基因型每个子集，用户可以手动改变呼叫。 <ol><li>无论是从融曲线或熔融峰认为，在曲线上右键单击要删除，选择它们。选择错误分类样本后，进入"新呼叫"，选择相应的组，然后单击"应用更改"。 </li></ol></li></ol></li><li>规范化<ol><li>从标准化荧光的负导数相对于温度（-dF​​ / dT）的（&#39;差分曲线"）视图，移动正常化棒以包围探针熔化区域。 注意:探针熔融区域是两个熔融区域的较低的温度。正常化杆应该在的熔融峰的碱。 </li></ol></li><li>计算组<ol><li>标准化后，选择"计算组的自动分配样本组。 如果需要的话，手动重新分配样品到特定组。 注:有些分析软件已经不能改变高灵敏度阈值;在这些情况下，作者oftware可以分配样本，以在视觉上属于另一个不同的组。 </li></ol></li><li>指定基因型<ol><li>基于标准（阳性对照），每个组分配的基因型。 <ol><li>在用户确认所计算的群体是正确的（如果不是，那正确的修改过的分组选项卡下选择错误分类样本，并从"新呼叫"旁边的下拉框中选择正确的组），选择"编辑组名"下的"分组"选项卡。 </li><li>输入的标准基因型为相应的彩色框（例如，如果标准3D7有一个已知的A基因型，是红豆集团，红豆集团拥有基因型A）。按"确定"按钮，保存结果。 </li></ol></li></ol></li><li>导出结果<ol><li>出口型称作为一个电子表格做进一步样本人群的分析。 </li></ol></li></ol></li></ol>

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作者什么都没有透露。

连续人力资源管理是一个后-PCR分析步骤;因此，样品制备和分析的设置是类似于标准PCR方案，以用于跟踪从过渡的荧光嵌入染料的额外掺入双链期间高分辨率熔化步骤到单链DNA。对于成功的人力资源管理数据的一个最重要的因素是一个强大的PCR产物。分析设计是关键，可用于人力资源管理的分析设计进行优化的多种工具，商业和在线13。的80-150个碱基对伴随〜20个碱基对的扩增子的长度阻止探针集中于与SNP或感兴趣的工作最好的SNP区分SNP位点以及多个SNP的单元型的探针区域内。探针可以使用一个3&#39;C3垫片或2个碱基对错配的3&#39;端，这防止扩增期间延伸被阻止。探针可被设计为匹配沃森或克里克模板链;选择要看实践的检验，以确定哪些探头设计，可以接受的熔融峰。过量正向或反向引物被用于产生单链扩增产物退火到阻止探头。如果探针包含正向链序列，然后反向引物过量使用，典型地以1:5的比例，反之亦然为匹配反向链是探针。 同样，人力资源管理效果最好有足够的和强大的PCR产物。 PCR反应优化需要梯度PCR和琼脂糖凝胶或生物分析仪分析，以确定最佳退火温度。模板浓度可使用的方法，例如预扩增，使用低引物浓度和循环数，以增加模板浓度13的所有目标偏置多路复用扩增来增加。 只有极少数的仪器都与MAAB兼容。玻璃毛细管管的热性能用我ñ这些系统有助于此方法。毛细管的小内径以及通过玻璃的传热迅速提供更严格的温度控制比标准PCR的塑料制品，其具有由于塑料的绝热性能退火和变性循环之间较慢转移速率。用这种方法，检测灵敏度可以从2-5％减少到一个突变等位基因的小于1％的野生型和突变等位基因10的混合物。 HRM为SNP基因分型一个轻便，高效且经济的工具中的各种设置和许多应用，从传染病监测到扫描癌基因的变体。其他一些工具，如罗氏Nano和的LightCycler系统（480和96），以及生态实时荧光定量PCR系统提供HRM除了标准扩增，实时，和拷贝数的功能。使用高通量的机器，人力资源管理条码费莱总比$ 0.50元法在我们手中，包括所有的试剂和耗材。 在这里描述的背景下，基于现场的应用程序允许实时监测人口与疟疾防治工作，包括降低种群的多样性，检测进口寄生虫种类，并降低药物的敏感性相关的SNP的出现和传播相关的变化。改进的方法提供额外的灵敏度，以便向该疟疾消灭和根除的进展非常有用。

尽管疟疾控制和消除新的兴趣，疟疾仍然是一个世界性的负担，有可能感染全球近一半的人口，每年超过55万的死亡，在撒哈拉以南非洲1特别是儿童。 这些新的控制和根除方案已经支持的基因组文艺复兴时期，有大量测序和分析以减少的药物敏感性相关的基因突变疟原虫，毒力增强，以及人口2,3特性。单核苷酸多态性（SNP）是最常见的确定遗传变异体4-7之间。 便携式SNP基因分型方法提供现场实时监测人口与跟踪8。除了指纹个体寄生虫中，"分子条形码&#39;也被用来检测等位基因频率戏剧性颞移以及方差有效群体大小和感染9的复杂性。 虽然这组信息的SNP很容易适应多种基因分型平台，高分辨率熔解（HRM）分析，特别适合基于场的研究，其中灵敏，简便的操作和检测的低成本新的突变相比，排序等方法是在资源贫乏地区的吸引力。 HRM开始用纳入荧光染料标准聚合酶链式反应（PCR）。 PCR后熔炼分析确定峰值扩增子的熔化温度;在很短的扩增子的单个SNP差可能会导致大量峰值熔融温度（Tm）的差异。 一些改进这种方法提供更好的基因分型的分辨率来区分单核苷酸多态性，包括IV级（AT）的SNP，并检测轻微的突变等位基因与目前多个等位基因的样品（polygenomic感染）。首先，将测定法整合集中于与SNP区域的短探针除了向前和反向引物中存在不同浓度。这些封端的探针在PCR过程中没有放大，而是结合在过量产生铸坯由于不对称的引物浓度是增加生产探针 - 模板产物。这些独立的双链扩增子由探头势必多余的模板链是〜20-30基点;相比整个扩增子（80-150碱基）导致具有单个或多个碱基探针-模板关联错配10大得多的Tm差异其显著降低长度。 第二，突变等位基因扩增偏压（MAAB）降低反应的退火温度，以偏向存在于在polygenomic感染低比率的突变等位基因的反应。退火温度设为完全匹配（野生型）的Tm值和错配（突变体）的探针之间秒。在此温度下，野生型等位基因的结合是足够稳定相比，它的失配等效的扩增受到阻碍，从而偏压朝当两者都存在于单一样品 10的突变等位基因的扩增。 有了这些人力资源管理的改进，这种技术允许起源跟踪，同时在南美11疫情的感染和检测的新的基因突变，位于序列彼此紧邻多个SNP位点的差异，和突变有关寄生虫和身份出现在不到1 ％polygenomic样品10的等位基因。 增加的敏感性是地区接近预消除状态和那些在危险中为新兴的耐药性显得尤为重要。快速，轻松地找出与现场的敏感性降低有关进口的寄生虫和SNP的能力通知有关监测工作和控制方案它们的实现和识别热点增加根除疟疾和消除工作的成效。本协议描述的方法来阻止探针为基础的MAAB以​​提高人力资源管理的基因分型的敏感性。

<table><tbody><tr><td>LightScanner Master Mix</td><td>BioFire Defense</td><td>HRLS-ASY-0003</td><td></td></tr><tr><td>Light mineral oil</td><td>Sigma</td><td>M5904</td><td>for BioFire Defense plate-based HRM systems</td></tr><tr><td>TE</td><td>Teknova</td><td>T0226</td><td></td></tr><tr><td>Te</td><td>Teknova</td><td>T0221</td><td>TE buffer with reduced EDTA</td></tr><tr><td>PCR grade water</td><td>Teknova</td><td>W3330</td><td></td></tr><tr><td>Plasmodium falciparum standards</td><td>MR4</td><td>MRA-151, 156, 205, 330</td><td>MR4.org offers free genomic DNA</td></tr><tr><td>Light Scanner Primer Design Software</td><td>BioFire Defense</td><td></td><td>commercial sofware for HRM assay design</td></tr><tr><td>LightCycler 480 High Resolution Melting Master Mix</td><td>Roche</td><td>4909631001</td><td>For use in LightCycler-480, -96, and nano. 2x concentration</td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td>G2938A</td><td>Agilent 2100 bioanalyzer</td></tr><tr><td>LightCycler 2.0</td><td>Roche</td><td>3531414001</td><td>Capillary-based system for MAAB</td></tr><tr><td>LightCycler Nano</td><td>Roche</td><td>6407773001</td><td>Strip tube-based HRM and amplification</td></tr><tr><td>LightCycler 96</td><td>Roche</td><td>5815916001</td><td>Strip-tube and plate-based HRM and amplification</td></tr><tr><td>LightCycler 480</td><td>Roche</td><td>5015278001</td><td>plate-based HRM (96 and 384-well plates) and amplification</td></tr><tr><td>LightScanner-96</td><td>BioFire Defense</td><td>LSCN-ASY-0011</td><td>plate-based HRM (96-well)&amp;nbsp;</td></tr><tr><td>LightScanner-384</td><td>BioFire Defense</td><td>LSCN-ASY-0001</td><td>plate-based HRM (96-well)</td></tr><tr><td>96-well plates</td><td>Roche</td><td>4729692001</td><td>96-well plates for HRM (LightCycler</td></tr><tr><td>384-well plates</td><td>Roche</td><td>4729749001</td><td>384-well plates for HRM (LightCycler)</td></tr><tr><td>96-well plates</td><td>Bio-Rad</td><td>HSP-9665&amp;nbsp;</td><td>96-well plates for HRM (LightScanner)</td></tr><tr><td>384-well plates</td><td>Bio-Rad</td><td>HSP-3865&amp;nbsp;</td><td>384-well plates for HRM (LightScanner)</td></tr><tr><td>LightCycler 8-Tube Strips (clear)</td><td>Roche</td><td>6327672001</td><td>strip tubes for HRM (Light Cycler 96 and Light Cycler Nano)</td></tr><tr><td>LightCycler Capillaries (20 &amp;mu;l)</td><td>Roche</td><td>4929292001</td><td>capillaries for HRM</td></tr><tr><td>Optical plate seals</td><td>Roche</td><td>4729757001</td><td>other brands also work</td></tr></tbody></table>

methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria

尽管几十年来根除努力，疟疾仍是一个全球性的负担。近期在区域消灭和根除全球新的兴趣一直陪伴左右疟原虫寄生物种造成疟疾，包括地理种群的敏感性降低有关的抗疟疾药物的特征以及变化增加了基因组信息。 一个常见的​​遗传变异，单核苷酸多态性（SNP），提供有吸引力的目标寄生虫的基因分型。这些标记不仅为跟踪药物抗性标记也用于跟踪使用不根据药物或其他选择压力标记寄生虫种群是有用的。 SNP基因分型方法提供了跟踪耐药性以及指纹个别寄生虫人口监测，特别是在应对疟疾防治工作的地区接近eliminat的能力离子状态。 虽然单核苷酸多态性信息已经确定是不可知的特定基因分型技术，高分辨率熔解（HRM）分析，尤其适合到外地为基础的研究。相比，需要在一个单一的标记探针个体的SNP，并提供最好的10％的灵敏度检测的SNP在包含多个基因组（polygenomic）样品的标准荧光探针为基础的方法，人力资源管理，提供2-5％的敏感性。修改HRM，如阻止探针和不对称引物浓度以及扩增的退火温度的优化偏压PCR朝向次要等位基因的扩增，进一步增加HRM的灵敏度。而改善灵敏度取决于具体的实验中，我们已经增加检测灵敏度的次要等位基因的不到1％。 在地区接近根除疟疾，尽早发现新出现的或进口的耐药性是至关重要的公关的OmpT反应。同样，检测polygenomic感染和区分神秘的地方水塘进口寄生虫类型的能力可以通知控制程序。 这份手稿描述修改高分辨率熔解技术，进一步提高其灵敏度，以确定患者样品中polygenomic感染。

数据可以被可视几种不同的方式，根据分析软件和仪器。通常情况下，标准化荧光的负导数的相对于温度（-dF​​ / dT）的对温度的结果的曲线图是最直接的可视化熔融峰以及确定基因型。 全熔化窗口（40℃-80℃）下，将导致在两种扩增子和探针熔解区域。 图1示出了归一化的熔融峰为两个区域的一个例子。分析窗口设置为只是在探头区域中的结果对应于特定的SNP峰明确分化（ 图2）。 基于探测的分析清楚地显示了两个等位基因与纯合匹配的SNP的峰（ 图3），该熔融温度的单个峰的存在。 图4表明 progressively减少期间扩增（MAAB）的结果，退火温度在朝向突变型等位基因（左侧峰）（图4A）的偏置，结果在混合等位基因（图4B）的群体突变等位基因的敏感性增加。 <img alt="图1" src="/files/ftp_upload/52839/52839fig1.jpg" /> 图1.探针和扩增子熔化的区域。绘制归一化的荧光的负导数相对于温度在宽的熔化窗口的结果在这两个探针（较低的温度）和扩增子的熔解峰可以被分析。 （改编自丹尼尔斯等人DOI:10.1128 / AAC.05737-11） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52839/52839fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 图2.探针熔解峰。完美匹配（通常是野生型等位基因）带来更高的熔融峰（红色），而SNP错配具有较低的熔融温度（灰色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52839/52839fig2large.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/52839/52839fig3.jpg" /> 图3. Polygenomic熔化峰。当两个等位基因是存在于样品中，基于探针的分析HRM表示两个等位基因作为两个峰曲线（橙色）与匹配的单等位基因样品（红色和灰色），该峰。 （改编自丹尼尔斯等人DOI:10.1128 / AAC.05737-11） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52839/52839fig3large.jpg" target="_blank">请点击这里查看大v</a>版为这一数字的。 <img alt="图4" src="/files/ftp_upload/52839/52839fig4.jpg" /> 图4.突变等位基因扩增偏差（MAAB）。A.渐进地降低扩增退火温度偏压朝向对应于突变体（较低的温度）等位基因在一个或polygenomic polyallelic样品峰的熔解峰反应。 B. MAAB导致人力资源管理灵敏度以检测polygenomic或polyallelic样品中存在低于1％的次要等位基因。 （B部分改编自丹尼尔斯等人DOI:10.1128 / AAC.05737-11） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52839/52839fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 表1.建立了基于探针的高分辨率熔解扩增反应。 <table><tbody><tr><td>零件</td><td>每重卷动作（96孔板和毛细管） </td><td>每个反应体积（384孔板） </td><td>最终浓度</td></tr><tr><td> HRM预混</td><td> 4-5微升</td><td> 2-2.5微升</td><td> 1个</td></tr><tr><td> 10×过量引物</td><td> 1微升</td><td> 0.5微升</td><td> 0.50μM </td></tr><tr><td> 10 x其它底漆</td><td> 1微升</td><td> 0.5μL </td><td> 0.1μM </td></tr><tr><td> 10X探头</td><td> 1微升</td><td> 0.5微升</td><td> 0.40μM </td></tr><tr><td> PCR级水</td><td> 1-2微升</td><td> 0.5-1微升</td><td></td></tr><tr><td>模板DNA </td><td> 1微升</td><td> 0.5微升</td><td> 0.01-10纳克/微升</td></tr><tr><td></td><td> 10微升</td><td> 5微升</td><td></td></tr></tbody></table> 表2. STANDARD高高分辨率熔解扩增条件。 <table><tbody><tr><td>温度</td><td>时间</td></tr><tr><td>保持</td><td> 95℃ </td><td> 120秒</td></tr><tr><td rowspan="2"> 45个循环</td><td> 95℃ </td><td> 30秒</td></tr><tr><td> 68°C * </td><td> 30秒</td></tr><tr><td rowspan="2"> 1周期</td><td> 95℃ </td><td> 30秒</td></tr><tr><td> 28°C </td><td> 30秒</td></tr></tbody></table> *该温度是扩增子依赖性 表3.扩增条件突变等位基因扩增偏见。 <table><tbody><tr><td>温度</td><td>时间</td><td>周期</td><td>斜坡率</td><td>采集方式</td><td>分析模式</td></tr><tr><td>变性</td><td> 95℃ </td><td> 30秒</td><td> 1 </td><td> 20 </td><td>无</TD&gt; <td>无</td></tr><tr><td rowspan="2">周期</td><td> 95℃ </td><td> 2秒</td><td rowspan="2"> 55 </td><td> 20 </td><td>无</td><td rowspan="2">定量</td></tr><tr><td> 56°C * </td><td> 15秒</td><td> 20 </td><td>单</td></tr><tr><td rowspan="3">熔化</td><td> 40℃ </td><td> 0秒</td><td rowspan="3"></td><td rowspan="3"> 0.3 </td><td>无</td><td>无</td></tr><tr><td> 45°C * </td><td> 0秒</td><td rowspan="2">连续</td><td rowspan="2">熔化</td></tr><tr><td> 90°C * </td><td> 0秒</td></tr></tbody></table> *该温度是扩增子依赖性和表演MAAB时将减少

Watch this Scientific Journal Video about Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria at JoVE.com

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

虽然高分辨率熔解分析提供了一个异质群体的单核苷酸多态性区分的能力，突变等位基因扩增偏置可提高其检测存在于样品中相对较低的百分比等位基因的能力。本协议描述的改进，提高了高分辨率熔解分析的灵敏度。

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 ...

methods-to-increase-sensitivity-high-resolution-melting-single

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.5K 次观看.  Harvard University. 虽然高分辨率熔解分析提供了一个异质群体的单核苷酸多态性区分的能力，突变等位基因扩增偏置可提高其检测存在于样品中相对较低的百分比等位基因的能力。本协议描述的改进，提高了高分辨率熔解分析的灵敏度。 

视频: Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

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 ...

科研

遗传学

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

免疫与感染

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 ...

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

数字液滴PCR检测转基因按蚊种群中的Indels突变

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

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 ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

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

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

通过焦磷酸测序对线粒体基因组中的单核苷酸多态性进行基因分型

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

医学

Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature

Source: Tsujimoto, H. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63008">Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti.</a> J. Vis. Exp. (2021).
This video demonstrates the gradient polymerase chain reaction for optimization of the annealing temperature of primers, which sets gradient annealing temperature along wells. The effect of different annealing temperatures is determined by the yield of amplified products that helps to optimize the reaction condition.

梯度聚合酶链式反应，用于确定最佳退火温度

Source: Tsujimoto, H. et al., Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti. J. Vis. ...