Name: technical demonstration of whole genome array comparative genomic hybridization
Uploaded: 2008-08-05T18:06:00.000+00:00
Duration: 16 min 37 s
Description: Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

We wish to thank members of the Wan Lam Lab BAC Array team especially Miwa Suzuki and Bryan Chi for the preparation of this article. This work was supported by funds from Canadian Institutes for Health Research, Genome Canada/Genome British Columbia, and NIH/NIDCR grant RO1 DE15965-01.

PROBE LABELING
Note: limit exposure of Cye Dyes to light at all times (this can be achieved by working in a darkened area or by shielding the tubes with a cover such as aluminum foil)
<ol>
<li>Combine: (Setup 1 reaction tube for reference and 1 reaction tube for sample) 
<ul>
<li>DNA (25-400 ng)</li>
<li>5 &micro;L of 5X random primers buffer (Final concentration: 5X Promega Klenow buffer and 7 &micro;g/ &micro;L random octamers)</li>
<li>Dilute to 17.0 &micro;L total volume with distilled H2O</li>
</ul>
</li>
<li>Boil for 10 minutes at 100&deg;C. Transfer immediately to ice for 1 minute.</li>
<li>Add 4 &micro;L of 10X dNTP mix (2mM dATP, dGTP, dTTP, 1.2mM dCTP).</li>
<li>Add CyeDyes: 
<ul>
<li>Add 2 &micro;L (2 nmoles) of Cy-3 labeled dCTP to Sample DNA </li>
<li>Add 2 &micro;L (2 nmoles) of Cy-5 labeled dCTP to Reference DNA (e.g., Novagen human genomic DNA)</li>
</ul>
</li>
<li>Add 2.5 &micro;L of Klenow (9 U/&micro;L, Promega ) and mix.</li>
<li>Incubate at 37&deg;C overnight* (~18 hours). *The overnight hybridization time can be adjusted between 14 to 24 hours without adversely affecting the labelling process to fit to a laboratory schedule.</li>
</ol>

SAMPLE CLEAN UP
(Combined probe clean-up and preparation for hybridization)
<ol>
<li>Using a Microcon YM-30 column: 
<ul>
<li>Add 100 &micro;L Cot-1 DNA (1&micro;g/&micro;L) to column. Do not touch membrane with pipette tip.</li>
<li>Pool the reactions (reference and sample) and add to column.</li>
<li>Place column in provided tube and spin at 13000 g for 10 minutes. </li>
<li>Add 200 &micro;L distilled H20 to membrane and repeat spin to wash.</li>
<li>Discard tube and add 45 &micro;L hybridization solution: (Roche DIG Easy)</li>
</ul>
Optional: add 5&micro;L sheared herring sperm DNA (10&micro;g/&micro;L unit, Promega) 
<ul>
<li>Invert Microcon, place in a new labeled tube, and spin at 3000 g for 3 minutes.</li>
</ul>
</li>
</ol>
CALCULATION OF INCORPORATIONS
<ol>
<li>Remove 1.5 &micro;L and measure Fluorophore incorporation using NanoDrop Spectrophotometer.&nbsp; Using your hybridization buffer as a blank (DIG Easy) follow the directions on the screen. (You can recover the sample from the pedestal, if necessary, after measurement.) (Note: Incorporations below 3.0 pmol/&micro;L in either channel have shown variable results.) </li>
<li>Denature at 85&deg;C for 10 minutes.</li>
<li>Place probe at 45&deg;C for 30 minutes to 1 hour (allows Cot-1 DNA annealing.)</li>
</ol>
ARRAY HYBRIDIZATION
<ol>
<li>Place 44 &micro;L probe solution onto the coverslip (22 mm x 60 mm) (Fisher Scientific). If available, place the coverslip on a slide warmer or heat block pre-warmed to 45&deg;C to maintain temperature.</li>
<li>Align slide over coverslip and probe solution, lower one edge of the slide allowing contact with the hybridization solution. Continue to lower until the coverslip is attached to the slide with surface tension. Invert slide.</li>
<li>Place the slide into hybridization cassette (Telechem), pre-warmed to 45&deg;C, and add 10 &micro;L of water in the lower groove (to control humidity during hybridization).</li>
<li>Seal cassette and incubate for 36&nbsp;- 40 hours at 45&deg;C.</li>
</ol>
ARRAY WASHING
Note: All wash solutions are at pH 7.0
Removal of the slide from the hybridization cassette is critical. DIG Easy quickly crystallizes at room temperature. The slide should be immediately immersed and the cover slip removed in the wash solution.
<ol>
<li>Add approximately 60 mL of 0.1XSSC, 0.1%SDS (pH 7.0, 45&deg;C) wash solution to a Coplin jar prior to opening the hybridization chamber.</li>
<li>Open chamber, add slide to wash solution and remove coverslip (it should slide off). </li>
<li>Wash the slide 3 times in 0.1XSSC + 0.1%SDS at 45&deg;C for 1 minute each with agitation. </li>
<li>Rinse the slide 3 times in fresh 0.1XSSC*. There should be no residual bubbles from the SDS wash visible. *The slides can remain in the final wash solution until ready to centrifuge and scan (~15 minutes).&nbsp; </li>
<li>Centrifuge the slide in a 50 mL Falcon tubes at 700 g for 3 minutes.</li>
<li>Immediately scan the slides (signal intensities will diminish over time.)</li>
</ol>

<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>

Poor quality DNA will not provide a good hybridization profile. It is essential to ensure that sample and reference DNA are free from contaminants such as phenol, RNA, salt, etc that may interfere with the random prime labeling step before starting a hybridization experiment. For example the resuspension of DNA in Tris - EDTA (TE) instead of water is not recommended as high salt concentration can inhibit the labeling reaction. We recommend assaying DNA quality using the Nanodrop spectrophotometer to measure both the D...

<table><tbody><tr><td>5X Klenow Buffer</td><td>Reagent</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>Random Octamers </td><td>Reagent</td><td>Alpha DNA</td><td></td><td></td></tr><tr><td>10X dNTP mix</td><td>Reagent</td><td>Promega Corp.</td><td></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></td><td></td></tr><tr><td>Cy-5 labeled dCTP</td><td>Reagent</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Human Genomic DNA Reference</td><td>Reagent</td><td>Novagen, EMD Millipore</td><td></td><td>Example of a possible reference</td></tr><tr><td>Klenow</td><td>Reagent</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>YM-30 Column</td><td>Reagent</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Cot-1 DNA</td><td>Reagent</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>DIG Easy</td><td>Reagent</td><td>Roche Group</td><td></td><td></td></tr><tr><td>Sheared Herring Sperm DNA</td><td>Reagent</td><td>Promega Corp.</td><td></td><td></td></tr><tr><td>Coverslip</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td>22mm x 60mm</td></tr><tr><td>Hybridization Cassette</td><td>Tool</td><td>Telechem</td><td></td><td></td></tr></tbody></table>

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.

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

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.

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 ...

technical-demonstration-whole-genome-array-comparative-genomic

Research

JoVE Journal

Biology

12.7K Views.  BC Cancer Research Centre. *The slides can remain in the 

Video: Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

Genetics

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.

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

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

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In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

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A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

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Neuroscience

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.

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

Competitive Genomic Screens of Barcoded Yeast Libraries

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

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DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

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DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

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In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

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

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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 diagnostic service laboratory.

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

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