Name: technical demonstration of whole genome array comparative genomic hybridization
Uploaded: 2008-08-05T18:06:00
Description: Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

我们要感谢为编写本文运临实验室的BAC阵列队，特别是三轮铃木和布赖恩志的成员。这项工作是支持由来自加拿大研究所的资金卫生研究院，加拿大基因组/基因组不列颠哥伦比亚省和美国国立卫生研究院/ NIDCR授予RO1 DE15965 - 01。

探针标记 注：限制接触CYE染料在任何时候都光（这可以通过在黑暗的领域工作，或屏蔽如铝箔盖管） <ol><li>结合： （安装1个反应管样品，以供参考和1个反应管） <ul><li> DNA（25-400纳克） </li><li> 5μL5X缓冲随机引物（终浓度：5X Promega公司的Klenow缓冲和7微克/μL，随机八聚体） </li><li>稀释到17.0μL，用蒸馏水H 2 O的总体积</li></ul></li><li>煮沸10分钟，在100...

<ol>
	<li>Lockwood, W. W., Chari, R., Chi, B., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+array+comparative+genomic+hybridization+technologies+and+their+applications+in+human+genetics.">Recent advances in array comparative genomic hybridization technologies and their applications in human genetics.</a> European Journal of Human Genetics. 14, 139-1x`48 (2006).</li><li>Ishkanian, A. S., Malloff, C. A., Watson, S. K., deLeeuw, R. J., Chi, B., Coe, B. P., Snijders, A., Albertson, D. G., Pinkel, D., Marra, M. A., Ling, V., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiling+resolution+DNA+microarray+with+complete+coverage+of+the+human+genome.">A tiling resolution DNA microarray with complete coverage of the human genome.</a> Nature Genetics. 36, 299-303 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Ishkanian, A. S., Malloff, C. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+high+throughput+validation+of+amplified+fragment+pools+of+BAC+DNA+for+constructing+high+resolution+CGH+arrays.">Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.</a> BMC Genomics. 5, 6 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Horsman, D. E., Squire, J. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetically+balanced+translocations+are+associated+with+focal+copy+number+alterations.">Cytogenetically balanced translocations are associated with focal copy number alterations.</a> Human Genetics. 120, 795-805 (2007).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeGH+--+a+software+tool+for+visualization+of+whole+genome+array+comparative+genomic+hybridization+data.">SeeGH -- a software tool for visualization of whole genome array comparative genomic hybridization data.</a> BMC Bioinformatics. 5, 13 (2004).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., Ng, R. T., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MD-SeeGH:+a+platform+for+integrative+analysis+of+multi-dimensional+genomic+data.">MD-SeeGH: a platform for integrative analysis of multi-dimensional genomic data.</a> BMC Bioinformatics. 9, 243 (2008).</li><li>Coe, B. P., Ylstra, B., Carvalho, B., Meijer, G. A., Macaulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+resolution+of+array+CGH.">Resolving the resolution of array CGH.</a> Genomics. 89, 647-653 (2007).</li><li>Garnis, C., Coe, B. P., Lam, S. L., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+array+CGH+increases+heterogeneity+tolerance+in+the+analysis+of+clinical+samples.">High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.</a> Genomics. 85, 790-793 (2005).</li></ol>

质量差的DNA，将不会提供一个良好的杂交个人资料。它是必不可少的，以确保样品和参照DNA污染物，如苯酚，RNA，盐等可能与干预的随机总理标签步骤之前开始杂交实验。例如再悬浮的DNA在Tris - EDTA代替水（TE）是不建议高盐浓度能抑制标记反应。我们建议使用Nanodrop分光光度计测量DNA的数量以及吸光度曲线的整体形状和260/280，二百三十分之二百六十零比例化验DNA的质量。 洗衣机：从杂交盒中删除的幻灯片?...

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5X Klenow Buffer</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Random Octamers </td>
<td>Reagent</td>
<td>Alpha DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X dNTP mix</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>2mM dATP, dGTP, dTTP, 1.2mM dCTP</td>
</tr>
<tr>
<td>Cy-3 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Genomic DNA Reference</td>
<td>Reagent</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>Example of a possible reference</td>
</tr>
<tr>
<td>Klenow</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>YM-30 Column</td>
<td>Reagent</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cot-1 DNA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG Easy</td>
<td>Reagent</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sheared Herring Sperm DNA</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>22mm x 60mm</td>
</tr>
<tr>
<td>Hybridization Cassette</td>
<td>Tool</td>
<td>Telechem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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technical demonstration of whole genome array comparative genomic hybridization

阵列比较基因组杂交（阵列CGH）技术是一种检测DNA片段或基因的基因组中的1剂量的收益和损失的方法。这项技术的最新进展，使高分辨率的整个基因组的遗传变异在癌症和其他遗传性疾病 2鉴定比较。小组Megabase分辨率平铺设置阵列（SMRT）阵列是由一组细菌人工染色体（BAC），跨越2〜100个碱基对的人类基因组（KB）段的克隆约三万重叠。这些BAC的目标是独立的合成和发现重复单一载玻片 2-4 。阵列比较基因组杂交的基础上有竞争力的杂交的原则。样品和参照DNA差异与菁- 3标记和荧光染料菁- 5，和合作的阵列杂交。潜伏期后，未​​结合的样品从幻灯片冲洗成像阵列。 SeeGH（www.flintbox.ca）免费提供的自定义软件包是用来处理大量的数据收集 - 一个单一的实验产生53892个数据点。 SeeGH可视化之间在每个BAC目标的2样品与5,6染色体的位置，这是垂直对齐的log2信号强度的比值。 SMRT的阵列，可以检测高达50 KB的大小 7小的改动。 SMRT的阵列，可以检测多种，包括DNA的收益，损失，扩增和纯合性缺失的DNA重组事件。 SMRT的阵列的一个独特优势是，可以使用从福尔马林固定石蜡包埋样品中分离DNA。当与非放大的DNA（25 - 100ng）的低投入的要求相结合 ，这使得如7,8显微切割生产的珍贵样品的分析。这要归功于大尺寸每个BAC杂交的目标，让约束力，以生产足够的标记的样品进行检测的信号。这个平台的另一个优势是组织异质性的耐受性，减少繁琐的组织显微切割8的需要。此视频的协议是一个标签输入DNA，通过对整个基因组的瓦片路径SMRT的阵列信号收购一步一步的教程。

Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

这个视频是一个全基因组平铺路径阵列比较基因组杂交，扫描整个人类基因组只用25-100纳克的DNA，可以从多种来源，包括档案福尔马林固定材料隔离​​技术示范的杂交协议。

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 ...

Research

JoVE Journal

Biology

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

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

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

使用全山原位杂交分子和有机体生物学链接

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

DNA Microarrays: Sample Quality Control, 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, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Competitive Genomic Screens of Barcoded Yeast Libraries

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

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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

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

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 ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...