Name: array comparative genomic hybridization (array cgh) for detection of genomic copy number variants
Uploaded: 2015-02-21T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

The authors have no acknowledgements.

1.标签反应<ol><li>在使用之前，预等分花青3和5标记dUTPs，未标记的核苷酸和随机引物根据制造商建议的浓度到96孔板中，并存储在-20℃以备使用。此协议，等分10.5微升适当标记的dUTP和10.5微升未标记的核苷酸和随机引物，以每个孔的目的。 </li><li>解冻的准备使用的96孔板核苷酸和引物，在4℃下进行约1小时，避光。 </li><li>一旦解冻，平衡该板在RT，至少30分钟，避光。 </li><li>平衡的DNA样品在60℃下至少15分钟。 </li><li>使用液体处理机器人，分配无核酸酶水到96孔板的每个孔中。 注:水的体积将取决于每个输入DNA样品的浓度，并应足以导致在50ng /微升的最终浓度。 </li><li>使用液体汉危及周围机器人，吸管1微克的DNA进96孔板的孔中（见1.5），以使总体积至20微升。 注意:也可以使用较小的DNA的量，虽然得到的数据的质量可能会受到影响（对珍贵的样品，输入DNA的数量下降到400纳克可以使用）。 </li><li>使用液体处理机器人，转移20微升平衡核苷酸和引物的成的稀释的DNA的板的各孔中。 </li><li>密封96孔板用条状盖（一个紧密的密封是至关重要的）。 </li><li>变性在99℃将DNA用于在PCR机器10分钟以加热盖，然后进行5分钟退火的引物捕捉在冰上冷却。 </li><li>使用液体处理机器人，加入10微升的克列诺外切DNA聚合酶到每个的DNA和吸管调匀。 注:总量应为50微升。 </li><li>密封96孔板，孵育在37℃下16小时。 注:4小时的潜伏期短足以但是一个O / N中cubation可以更方便。 </li><li>使用液体处理机器人，添加5微升的终止缓冲液到每个孔中。 注意:用于标记反应的试剂的概述显示于表1中 。 </li></ol> 2.去除未掺入的核苷酸<ol><li>除去使用基于硅胶膜离心柱和一个专用旋转柱处理机器人（自旋柱也可以被人工处理）未掺入的核苷酸。按照制造商的说明。 <ol><li>每个内容传送到井2毫升管;前期这些标签具有良好的ID。 注意:此步骤可手工或优选使用液体处理机器人。 </li><li>如果使用旋列处理机器人，然后装入2ml管，DNA的纯化离心柱和相关联的缓冲器的机器人。 </li><li>加入250微升高盐，DNA结合缓冲液（缓冲液PB）的标记反应的标记的DNA结合到二氧化硅膜。 </li><li>通过用500μl洗涤缓冲液（缓冲液PE）洗涤该膜两次除去杂质。 </li><li>添加15微升的低盐洗脱缓冲液（缓冲液EB）至膜以回收纯化的，标记的DNA中的〜12微升的体积。 </li></ol></li></ol> 3.结合HYB双<ol><li>结合适当的成对花青3和5标记的DNA的，COT-1DNA，一个制造商提供的阻挡混合物和使用液体处理机器人的生产商提供的杂交缓冲液。 注意:这些HYB对可以是一个样品和参考，或VS样品样品如果共享的CNV是不是一个问题（ 例如，我们经常配对的表型不匹配的试样，诸如肾发育不良患者VS一个颅患者，因为它们是非常不可能携带相同的临床显著CNV）。如果使用的是参照DNA，商用的DNA供给建议，它应该沿着样品进行处理。 <ol><li>用液体处理ロ机器人，添加COT-​​1DNA（3333毫微克/微升），将制造商提供的阻挡混合物和杂交缓冲液至96孔板的各孔中，使得每个孔含有1.1微升COT-1DNA，4.95微升阻断混合物和24.75微升杂交缓冲液。 </li><li>添加9.35微升适当花青3标记的DNA的每个孔中。预湿每个尖端，以提高注精度。 </li><li>添加9.35微升适当花青5标记的DNA的每个孔中。预湿每个尖端，以提高注精度。 </li><li>密封96孔板和涡旋1分钟。旋96孔板收集内容在每个孔的底部。 </li></ol></li></ol> 4.杂交<ol><li>预热杂交烘箱以65℃和prewarm 1背衬滑动和一个杂交腔室为每个阵列的幻灯片。 <ol><li>被施加到所述阵列之前变性在预杂交孵育标记的DNA。在旋转炉中进行杂交24小时。 </li><li>孵育96孔板在70℃下进行30分钟，然后在37℃下留至少5分钟。 </li><li>工作的一个加热平台在42℃，加载每个备份滑入使用前一个杂交腔室。确保垫圈滑动的透明部分对准所述杂交腔室的"加窗"的一部分。 </li><li>吸管42微升预先制备的杂交混合物（见第3节）到阵列支持滑上的适当位置。预湿每个尖端，以提高注精度。吸管慢慢走上中心每个位置，确保液体不接触橡胶圈的边界。 </li><li>一旦在支持幻灯片所有的位置都填满，小心地在上面的阵列幻灯片和组装的杂交室。确保与写在其上的阵列的一侧朝向背衬滑动。一定要完全拧紧杂交室螺丝。 </li><li>检查装配的杂交室。 Ë7.2.4有橡胶环边界之外无泄漏杂交混合物和每个气泡是〜4毫米的高度时，杂交室安置在其端部垂直。旋转杂交室，检查有没有被卡在位置不动气泡。如果有任何这些，给室内猛烈敲击板凳上。检查所有气泡移动。 </li><li>将杂交室进入旋转炉中在65℃下放置24小时。确保烤箱转子平衡;如果需要，可以使用其他的空室。 </li></ol></li></ol> 5.洗涤和扫描幻灯片<ol><li>洗阵列以除去过量的标记的DNA，然后进行扫描，测定各探针的荧光。 <ol><li>就拿杂交室出来烤箱和拆卸的。该阵列和垫片幻灯片将粘在一起;在洗涤缓冲液1淹没这些和用一双塑料，边缘平坦钳撬开他们分开。 </li><li>迪SCARD垫圈滑动件和放置在阵列滑入浸没在缓冲液1中的机架上。 </li><li>洗〜700毫升新鲜的洗涤缓冲液1中的阵列载玻片5分钟，剧烈搅拌（使用跳蚤和磁力搅拌器）。 </li><li>传输阵列滑动到〜700毫升洗涤缓冲液2和洗涤90秒，剧烈搅拌。轻轻提起阵列滑出的洗涤液2，他们应该出现干燥。 </li><li>加载阵列滑入扫描的幻灯片固定器，采用了滑盖保护。加载滑入阵列扫描仪和扫描后的阵列制造商的说明。 </li></ol></li></ol>

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	<li>Miller, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+statement:+chromosomal+microarray+is+a+first-tier+clinical+diagnostic+test+for+individuals+with+developmental+disabilities+or+congenital+anomalies.">Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.</a> Am. J. Hum. Genet. 86, 749-764 (2010).</li><li>Ahn, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+CGH+as+a+first+line+diagnostic+test+in+place+of+karyotyping+for+postnatal+referrals+-+results+from+four+years'+clinical+application+for+over+8,700+patients.">Array CGH as a first line diagnostic test in place of karyotyping for postnatal referrals - results from four years' clinical application for over 8,700 patients.</a> Mol. Cytogenet. 6 (16), 1755-8166 (2013).</li><li>Ahn, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+implementation+of+array+comparative+genomic+hybridisation+as+a+first+line+test+in+place+of+postnatal+karyotyping+for+genome+imbalance.">Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance.</a> Mol. Cytogenet. 3 (9), 1755-8166 (2010).</li><li>Beaudet, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+chromosomal+microarray+analysis+in+developmental+and+behavioral+pediatrics.">The utility of chromosomal microarray analysis in developmental and behavioral pediatrics.</a> Child Dev. 84, 121-132 (2013).</li><li>Park, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+implementation+of+whole-genome+array+CGH+as+a+first-tier+test+in+5080+pre+and+postnatal+cases.">Clinical implementation of whole-genome array CGH as a first-tier test in 5080 pre and postnatal cases.</a> Mol. Cytogenet. 4, 12 (2011).</li><li>Iafrate, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+large-scale+variation+in+the+human+genome.">Detection of large-scale variation in the human genome.</a> Nat. Genet. 36, 949-951 (2004).</li><li>Redon, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+variation+in+copy+number+in+the+human+genome.">Global variation in copy number in the human genome.</a> Nature. 444, 444-454 (2006).</li><li>Cafferkey, M., Ahn, J. W., Flinter, F., Ogilvie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+features+in+patients+with+15q11.2(BP1-BP2)+deletion:+Further+delineation+of+an+emerging+syndrome.">Phenotypic features in patients with 15q11.2(BP1-BP2) deletion: Further delineation of an emerging syndrome.</a> Am. J. Med. Genet. A. 164, 1916-1922 (2014).</li><li>Molin, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+microdeletion+syndrome+at+3q13.31+characterised+by+developmental+delay,+postnatal+overgrowth,+hypoplastic+male+genitals,+and+characteristic+facial+features.">A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.</a> J. Med. Genet. 49, 104-109 (2012).</li><li>Tropeano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-biased+autosomal+effect+of+16p13.11+copy+number+variation+in+neurodevelopmental+disorders.">Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders.</a> PLoS One. 8, e61365 (2013).</li><li>Bon, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+delineation+of+the+15q13+microdeletion+and+duplication+syndromes:+a+clinical+spectrum+varying+from+non-pathogenic+to+a+severe+outcome.">Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.</a> J. Med. Genet. 46, 511-523 (2009).</li></ol>

The authors have nothing to disclose.

阵列CGH不会检测平衡重排或倍体异常，如三倍体。此外，低水平的马赛克的不平衡可能不会被检测到。然而，阵列CGH对CNV检测比G-带状染色体分析，它已经取代了许多细胞遗传学实验室更高的分辨率。因此，目前的黄金标准的全基因组CNV检测。它可能是由下一代测序技术在未来被替换，但目前，它们的成本和技术复杂性与使用短相关读出用于检测CNV的意思，这还不是一个很好的适合临床使用。 这里所描述的协议是在我们的临床服务实验室的日常使用。每96个样品的两个运行每星期用液体处理机器人和一个专用的二氧化硅膜的自旋柱处理机器人处理。自动化强烈建议，以提高一致性和保证质量。从血液中提取DNA，唾液，产前样品（ 如绒毛膜绒毛或羊水）或组织样本就可以了，通常就是使用该协议。 臭氧是众所​​周知的降低菁染料，因此臭氧监测和保护建议。在臭氧含量的季节性变化也是常见的。我们的实验室监测和记录臭氧水平不断。壁挂式臭氧去除单元用于减少臭氧水平和该协议的特别敏感的部位（ 即，洗涤和扫描阵列）在无臭氧抽油烟机执行。如果可能的话，臭氧水平保持在低于5ppb的。 大多数试剂都是买来自制造商以有效外包的质量控制包;这已被证明是一种有效的策略。然而，这并不意味着不要求质量控制。事实上，质量指标都经过精心的任何异常情况监控:LOG2比（DLRS）的衍生标准偏差应Below 0.2，花青3和5的信号强度应大于500和花青3和5的信噪比应&gt; 30。这些度量产生为通过扫描软件中的扫描协议的一部分，并且被记录为长期监测。

它已被公知多年了缺失或染色体片段的额外拷贝会导致智力障碍，异形和congentical畸形，并在某些情况下会导致遗传综合征1。不过，可用，直到2000年代中期的全基因组检测这些变化的唯一技术是G-带状染色体分析，其中有四周5-10Mb的分辨率，这取决于不平衡的地区和性质，并不能检测异常染色体其中材料已经被替换的区域，从一个不同的染色体具有相同的带型。如荧光原位杂交和多重连接特异性探针扩增辅助细胞遗传学技术已经可以为特定位点的审讯，在涉嫌的具体综合征失衡的情况下，但它不是直到引进阵列比较基因组杂交（CGH阵列）进入常规临床诊断service 2-5的全基因组检测的拷贝数变异（CNV的），在大大提高分辨率（通常大约120KB）成为可能。临床服务工作一起研究的研究表明，CNV的某些区域是在正常人群6-7普遍，而其他的CNV，先前不可检测的，与neurodisabilities如孤独症和癫痫8-11相关联。 本文描述的协议，用于在我们的英国国民健康服务（NHS）临床诊断实验室;我们用新颖的杂交策略，批量测试和机器人，以减少在这个国家资助的服务成本。 之前下面详述的协议，高品质的DNA应该提取从适当的起始材料，通常血液，培养的细胞或组织样品。分光光度法，可用于测量浓度（应&gt; 50纳克/ UL），并检查260:280比例的吸光度（应BË1.8-2.0）。凝胶电泳可用于检查该DNA是高分子量无显著降解。 此协议是专为那些每个标签使用自动化液体处理机器人运行96个样品更高的吞吐量实验室。但是，它也可以适用于低吞吐量的实验室没有自动化。

<table><tbody><tr><td>CGH microarray 8 x 60K</td><td>Agilent</td><td>G4126</td><td></td></tr><tr><td>Array CGH wash buffers</td><td>Agilent</td><td>5188-5226</td><td></td></tr><tr><td>Array CGH hybridisation buffer</td><td>Agilent</td><td>5188-5380</td><td></td></tr><tr><td>Minelute purification kit</td><td>Qiagen</td><td>28006</td><td></td></tr><tr><td>Array CGH labelling kit</td><td>Enzo</td><td>ENZ-42672-0000</td><td></td></tr></tbody></table>

array comparative genomic hybridization (array cgh) for detection of genomic copy number variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

在杂交阵列，每个探针可视化为红色和绿色荧光染料的混合物（ 见图1）。红色到绿色荧光信号的每个探针的比值由扫描仪量化和关联的软件根据它们的基因组的位置绘出这些为log2比值，并确定区域的下降沿以外的预设边界。结果数组的痕迹让认定为基因组区域的不平衡解释。例如，从一个子带威廉斯综合征，反复出现微缺失综合征由低拷贝重复在7号染色体的近侧区介导的痕迹，示于图2。这种不平衡鉴定由软件和由红色线表示。 探测荧光日志比率应该紧紧围绕群集0， 如图2中 ，表示1的绿色/红色比:1的基因组的正常区域。散阵列的痕迹可能resul<html>吨，因而不能准确调用异常区域的，或无法确定的基因组失衡（ 见图3）。这类分散可由许多因素，包括DNA质量差，或臭氧在大气中的升高水平的存在而引起的。臭氧浓度监测建议，并在高浓度臭氧水平是有问题的，致力于臭氧排除橱柜可用;使用这些机柜通常导致显着改善阵列的品质。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51718/51718fig1highres.jpg" width="600" /> 数组下杂交图1.图像的白框显示放大的区域，其中红色探头和绿色的探头可以可视化（表示为失衡这些探头所代表的地区），对黄色探针的背景（表示平衡的基因组区域）。 ontent"FO:保持together.within页="总是"&gt; <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51718/51718fig2highres.jpg" width="600" /> 具有1.7MB缺失的样本，由-0.8连续43探针的平均比率示于图2（A）的实施例的跟踪对7号染色体。在这种情况下，被删除的区域被与威廉姆斯-博伊伦综合征，具有畸形特征，心脏缺陷和智障转诊指示一致相关联。上述（B）在USCS基因组浏览器上显示被删除的区域中，表示内被删除的基因地区。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51718/51718fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51718/51718fig3highres.jpg" width="600" /> 图3。差的杂交样品中的分散阵列跟踪的7号染色体的例子。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51718/51718fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 体积（微升 ） </td></tr><tr><td>引物和反应缓冲液</td><td align="right"> 20 </td></tr><tr><td> DNA（1微克）+水</td><td align="right"> 20 </td></tr><tr><td>克列诺核酸外切酶DNA聚合酶</td><td align="right"> 10 </td></tr></tbody></table> 表1.用于标记反应的试剂的综述。

Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

阵列CGH为基因拷贝数变异的检测已经取代G-带状核型分析。本文介绍了技术及其在诊断服务实验室的应用。

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

array-comparative-genomic-hybridization-array-cgh-for-detection

Universidad San Sebastian

Research

JoVE Journal

Biology

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

19.5K 次观看.  Guy's & St Thomas' NHS Foundation Trust. 阵列CGH为基因拷贝数变异的检测已经取代G-带状核型分析。本文介绍了技术及其在诊断服务实验室的应用。 

视频: Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Detection of Copy Number Alterations Using Single Cell Sequencing

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

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

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

应用实时 PCR 技术检测血清和血浆拷贝数

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

癌症研究

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全基因组阵列比较基因组杂交技术示范

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

生物学

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

基于半导体的下一代测序平台上非整倍体的植入前基因检测

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

通过有针对性的测序方法进行比较病变分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

行为学

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA微阵列：样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

DNA Microarrays

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica surfaces.
While DNA microarrays are used to monitor gene expression, protein microarrays detect biologically relevant interactions of a fluorescently labeled protein with other proteins, DNA, or small molecules.
DNA microarray slides are often called gene chips or DNA chips. DNA chips are widely used to profile thousands of genes parallelly. Other than gene expression, they are also used in studying microRNA expression, DNA copy numbers, and single-nucleotide polymorphism detection. Commercially, these chips are used in biotechnology, forensics, toxicology, drug discovery, and targeted therapy for infectious and genetic diseases.
The chemically stable nature of DNA probes makes DNA chips the most popular of the microarray technologies. The covalently bound single-stranded DNA probe retains its native interactions despite harsh chemicals and temperatures that are used during probe loading and chip preparation.
The most obvious shortcoming of DNA microarrays is the prerequisite of a known target sequence. Because DNA microarray probes are designed to complement the target cDNA sequence, the sequence of the mRNA from which the cDNA is copied must be known. This makes the technology unusable while looking for novel mRNA sequences. For such purposes, RNASeq is the preferred albeit expensive option.

DNA Microarrays: High-throughput Gene Expression Profiling

DNA 微阵列芯片

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in ...