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>

<ol>
	<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。 ...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CGH microarray 8 x 60K</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">G4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH wash buffers</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH hybridisation buffer</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Minelute purification kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">28006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH labelling kit</td>
			<td style="width:71px;">Enzo</td>
			<td style="width:119px;">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?...

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

Research

JoVE Journal

Biology

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

19.4K 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

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

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 ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性的微型驱动器阵列在体内录音：驱动器的制造

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

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, ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

罕见的基因变异检测从集资使用斯普林特测序

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...