Name: Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants (Video) | JoVE Journal
Uploaded: 2015-02-21T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Learn array comparative genomic hybridization to detect genomic copy number variants in patient samples. This method uses fluorescent labeling and hybridization to identify imbalances causing genetic syndromes.

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The authors have no acknowledgements.

1. Labelling Reaction
<ol>
	<li>Prior to use, pre-aliquot cyanine 3 and 5-labeled dUTPs, unlabeled nucleotides and random primers at the manufacturer-recommended concentrations into 96-well plates and store at -20 C ready for use. For the purposes of this protocol, aliquot 10.5 &#181;l of the appropriate labeled dUTP and 10.5 &#181;l unlabeled nucleotides and random primers to each well.</li>
	<li>Thaw the ready-to-use 96-well plate of nucleotides and primers at 4 &#176;C for about 1 hr, protected from light.</li>
	<li>Once thawed, equilibrate this plate at RT for at least 30 min, protected from light.</li>
	<li>Equilibrate DNA samples at 60 &#176;C for at least 15 min.</li>
	<li>Using a liquid handling robot, dispense nuclease-free water to each well of a 96-well plate. 
	NOTE: The volume of water will depend on the concentration of each input DNA sample and should be sufficient to result in a final concentration of 50 ng/ul.</li>
	<li>Using a liquid handling robot, pipette 1 &#181;g of DNA into a well of the 96-well plate (see 1.5) to bring the total volume to 20 &#181;l. 
	NOTE: Smaller quantities of DNA may be used although the quality of resulting data may be compromised (for precious samples, input DNA quantities down to 400 ng can be used).</li>
	<li>Using a liquid handling robot, transfer 20 &#181;l of the equilibrated nucleotides and primers into each well of the plate of diluted DNA.</li>
	<li>Seal the 96-well plate with strip caps (a tight seal is crucial).</li>
	<li>Denature the DNA at 99 &#176;C for 10 min in a PCR machine with a heated lid and then snap cool on ice for 5 min to anneal primers.</li>
	<li>Using a liquid handling robot, add 10 &#181;l of Klenow Exo DNA polymerase enzyme to each DNAs and pipette mix thoroughly. 
	NOTE: The total volume should be 50 &#181;l.</li>
	<li>Seal the 96-well plate and incubate at 37 &#176;C for 16 hr. 
	NOTE: A shorter incubation of 4 hr is sufficient however an O/N incubation can be more convenient.</li>
	<li>Using a liquid handling robot, add 5 &#181;l of stop buffer to each well. 
	NOTE: A summary of the reagents used for the labeling reaction is shown in Table 1.</li>
</ol>
2. Removal of Unincorporated Nucleotides
<ol>
	<li>Remove unincorporated nucleotides using silica-membrane based spin columns and a dedicated spin column processing robot (the spin columns may also be processed manually). Follow the manufacturer&#39;s instructions.
	<ol>
		<li>Transfer the contents of each well to a 2 ml tube; pre-label these with well ID&#8217;s. 
		NOTE: This step can be performed manually or preferably using a liquid handling robot.</li>
		<li>If using a spin column processing robot, then load the robot with 2 ml tubes, DNA purification spin columns and associated buffers .</li>
		<li>Add 250 &#181;l high salt, DNA binding buffer (buffer PB) to the labeling reaction to bind the labeled DNA to the silica membrane.</li>
		<li>Remove impurities by washing the membrane twice with 500 &#181;l wash buffer (buffer PE).</li>
		<li>Add 15 &#181;l low salt elution buffer (buffer EB) to the membrane to recover the purified, labeled DNA in a volume of ~12 &#181;l.</li>
	</ol>
	</li>
</ol>
3. Combine Hyb Pairs
<ol>
	<li>Combine the appropriate pairs of cyanine 3 and 5-labeled DNA, COT-1 DNA, a manufacturer-supplied blocking mix and a manufacturer-supplied hybridization buffer using a liquid handling robot. 
	NOTE: These hyb pairs can be a sample and a reference, or sample vs sample if shared CNVs are not an issue (e.g.,&#160;we routinely pair phenotype-mismatched samples, such as a renal dysplasia patient vs a craniosynostosis patient, as they are very unlikely to carry the same clinically significant CNV). If using a reference DNA, a commercial DNA supply is recommended, and it should be processed alongside the sample.
	<ol>
		<li>Using a liquid handling robot, add COT-1 DNA (3333 ng/&#181;l), the manufacturer-supplied blocking mix and hybridization buffer to each well of a 96-well plate so that each well contains 1.1 &#181;l COT-1 DNA, 4.95 &#181;l blocking mix and 24.75 &#181;l hybridization buffer.</li>
		<li>Add 9.35 &#181;l of the appropriate cyanine 3-labeled DNA to each well. Pre-wet each tip to enhance pipetting accuracy.</li>
		<li>Add 9.35 &#181;l of the appropriate cyanine 5-labeled DNA to each well. Pre-wet each tip to enhance pipetting accuracy.</li>
		<li>Seal the 96-well plate and vortex for 1 min. Spin the 96-well plate to collect contents at the bottom of each well.</li>
	</ol>
	</li>
</ol>
4. Hybridization
<ol>
	<li>Preheat a hybridization oven to 65oC and prewarm one backing slide and one hybridization chamber for each array slide.
	<ol>
		<li>Denature the labeled DNA in a pre-hybridization incubation before being applying to the arrays. Perform hybridization in a rotating oven for 24 hr.</li>
		<li>Incubate the 96-well plate at 70 &#176;C for 30 min and then leave at 37 &#176;C for at least 5 min.</li>
		<li>Working on a heated platform at 42 &#176;C, load each backing slide into a hybridization chamber prior to use. Ensure that the transparent part of the gasket slide aligns with the &#39;windowed&#39; part of the hybridization chamber.</li>
		<li>Pipet 42 &#181;l of the hybridization mix prepared previously (see section 3) onto the appropriate position on the array backing slide. Pre-wet each tip to enhance pipetting accuracy. Pipet slowly onto the center of each position, making sure the liquid does not touch the rubber ring boundary.</li>
		<li>Once all positions on the backing slide are filled, carefully lower the array slide on it and assemble the hybridization chamber. Make sure that the side of the array with writing on it is facing the backing slide. Be sure to tighten the hybridization chamber screw fully.</li>
		<li>Inspect the assembled hybridization chamber. Ensure that there is no leakage of hybridization mix outside the rubber ring boundaries and each air bubble is ~4 mm in height when the hybridization chamber is rested on its end vertically. Rotate the hybridization chamber and check that there are no air bubbles that are stuck in position and not moving. If there are any of these, give the chamber a sharp tap on the bench. Check that all air bubbles are moving.</li>
		<li>Place the hybridization chamber into the rotating oven at 65 &#176;C for 24 hr. Make sure the oven rotor is balanced; use other empty chambers if necessary.</li>
	</ol>
	</li>
</ol>
5. Washing and Scanning Slides
<ol>
	<li>Wash arrays to remove excess labeled DNA and then scan to measure fluorescence of each probe.
	<ol>
		<li>Take hybridization chambers out of oven and disassemble. The array and gasket slides will be stuck together; submerge these in wash buffer 1 and use a pair of plastic, flat edged forceps to pry them apart.</li>
		<li>Discard the gasket slide and place the array slide into a rack submerged in buffer 1.</li>
		<li>Wash the array slide in ~700 ml of fresh wash buffer 1 for 5 min, stirring vigorously (use a flea and a magnetic stirrer).</li>
		<li>Transfer the array slide to ~700 ml wash buffer 2 and wash for 90 sec, stirring vigorously. Gently lift the array slides out of wash buffer 2, they should emerge dry.</li>
		<li>Load the array slide into a slide holder of the scanner, using a slide protector. Load the slide into the array scanner and scan following the array manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
</ol>

<ol><li>Miller, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Consensus+statement%3A+chromosomal+microarray+is+a+first-tier+clinical+diagnostic+test+for+individuals+with+developmental+disabilities+or+congenital+anomalies%5BTitle%5D)+AND+%22Am.+J.+Hum.+Genet%22%5BJournal%5D)">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/pubmed?term=((Array+CGH+as+a+first+line+diagnostic+test+in+place+of+karyotyping+for+postnatal+referrals+-+results+from+four+years%27+clinical+application+for+over+8%2C700+patients%5BTitle%5D)+AND+%22Mol.+Cytogenet%22%5BJournal%5D)">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/pubmed?term=((Validation+and+implementation+of+array+comparative+genomic+hybridisation+as+a+first+line+test+in+place+of+postnatal+karyotyping+for+genome+imbalance%5BTitle%5D)+AND+%22Mol.+Cytogenet%22%5BJournal%5D)">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/pubmed?term=((The+utility+of+chromosomal+microarray+analysis+in+developmental+and+behavioral+pediatrics%5BTitle%5D)+AND+%22Child+Dev%22%5BJournal%5D)">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/pubmed?term=((Clinical+implementation+of+whole-genome+array+CGH+as+a+first-tier+test+in+5080+pre+and+postnatal+cases%5BTitle%5D)+AND+%22Mol.+Cytogenet%22%5BJournal%5D)">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/pubmed?term=((Detection+of+large-scale+variation+in+the+human+genome%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">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/pubmed?term=((Global+variation+in+copy+number+in+the+human+genome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Phenotypic+features+in+patients+with+15q11.2%28BP1-BP2%29+deletion%3A+Further+delineation+of+an+emerging+syndrome%5BTitle%5D)+AND+%22Am.+J.+Med.+Genet.+A%22%5BJournal%5D)">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/pubmed?term=((A+novel+microdeletion+syndrome+at+3q13.31+characterised+by+developmental+delay%2C+postnatal+overgrowth%2C+hypoplastic+male+genitals%2C+and+characteristic+facial+features%5BTitle%5D)+AND+%22J.+Med.+Genet%22%5BJournal%5D)">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/pubmed?term=((Male-biased+autosomal+effect+of+16p13.11+copy+number+variation+in+neurodevelopmental+disorders%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Further+delineation+of+the+15q13+microdeletion+and+duplication+syndromes%3A+a+clinical+spectrum+varying+from+non-pathogenic+to+a+severe+outcome%5BTitle%5D)+AND+%22J.+Med.+Genet%22%5BJournal%5D)">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.

Array CGH will not detect balanced rearrangements or ploidy abnormalities such as triploidy. Furthermore, low level mosaic imbalances may not be detected. However, array CGH has a higher resolution for CNV detection than G-banded chromosome analysis which it has replaced in many cytogenetics laboratories. It is therefore the current gold standard for genome-wide CNV detection. It may be replaced by next-generation sequencing technologies in the future but currently, their cost and the technical complexities associated wi...

It has been known for many years that deletions or extra copies of chromosomal segments cause intellectual disability, dysmorphism and congentical malformations, and in some cases cause genetic syndromes1. However, the only technology available until the mid-2000s for the genome-wide detection of these changes was G-banded chromosome analysis, which has a resolution of around 5-10Mb, depending on the region and nature of the imbalance, and which cannot detect abnormal chromosomes where material has been replaced by a region from a different chromosome with the same banding pattern. Ancillary cytogenetic techniques such as fluorescence in situ hybridization and multiplex ligation-specific probe amplification have been available for the interrogation of specific loci, in cases of suspected specific syndromic imbalance, but it was not until the introduction of array comparative genomic hybridization (array CGH) into routine clinical diagnostic service2-5 that genome-wide detection of copy number variants (CNVs) became possible at a greatly increased resolution (typically around 120kb). Clinical service work alongside research studies have shown that CNVs for some regions are widespread in the normal population6-7, whilst other CNVs, previously undetectable, are associated with neurodisabilities such as autism and epilepsy8-11.
The protocol described in this paper is used in our UK National Health Service (NHS) clinical diagnostic laboratory; we use novel hybridization strategies, batch testing and robotics to minimize cost in this state-funded service.
Prior to the protocol detailed below, high quality DNA should be extracted from the appropriate starting material, commonly blood, cultured cells or tissue samples. Spectrophotometry can be used to measure concentration (should be &#62;50 ng/ul) and check 260:280 absorbance ratios (should be 1.8-2.0). Gel electrophoresis can be used to check that the DNA is of high molecular weight without significant degradation.
This protocol is designed for higher throughput laboratories that are labeling 96 samples per run using automated liquid handling robotics. However, it may be adapted for lower throughput labs without automation.

<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 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.

Each probe on a hybridized array is visualized as a mixture of red and green fluorochromes (see Figure 1). The ratio of red to green fluorescent signal for each probe is quantified by the scanner and the associated software plots these as log2 ratios according to their genomic position, and identifies regions falling outside preset boundaries. The resulting array traces allow interpretation of regions identified as genomically unbalanced. For instance, the trace from a child with Williams syndrome, a rec...

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.

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

The overall goal of this procedure is to array comparative genomic hybridization to identify imbalances in the genome that can lead to a wide variety of genetic diseases. This is accomplished by first labeling the patient's DNA with a fluorescent dye. In the second step, the patient, DNA and a differently labeled reference DNA, are hybridized to the array.Next, the array is scanned to measure the amount of the patient and reference DN abound to each probe. In the final step, the scanned image is processed to identify the regions of the genome where the patient's DNA has more or less material than the reference DNA ultimately array. Comparative genomic hybridization is used to determine whether the patient carries a copy number variant that results in a genetic syndrome.Visual demonstration of this method is critical as assembly of the array slide can be difficult and it is easy for the hybridization. Mix the spill out of the chamber Before beginning the labeling reaction. Thaw the ready-to-use 96 well plate of nucleotides and primers at four degrees Celsius and protected from light for about an hour.Once thawed, equilibrate the covered plate for another 30 minutes at room temperature, equilibrate the DNA samples for 15 minutes at 60 degrees Celsius at this time as well. While the samples are warming, use a liquid handling robot to dispense enough nuclease free water into each well of a new 96 well plate. Then when the DNA is ready transfer one microgram of sample per well to the 96 well plate of water.Now transfer 20 microliters of the equilibrating nucleotides and primers to each one of the plate containing the diluted DNA samples and seal the plate with strip caps taking care to make a tight seal. Next, denature the DNA at 99 degrees Celsius in a PCR machine with a heated lid after 10 minutes and knee the primers by snap cooling the plate on ice for five minutes. Then use the liquid handling robot to add 10 microliters of clean out exo DNA polymerase enzyme to each sample.Mix the samples by pipette and then seal and incubate the plate at 37 degrees Celsius for 16 hours. The next day terminate the reaction with five microliters of stop buffer per well Then remove the unincorporated nucleotides. Transfer the contents of each well to individual pre-labeled two milliliter tubes.Load the samples and DNA purification spin columns into a spin column processing robot, as well as the appropriate associated buffers. According to the manufacturer's instructions, the spin column processing robot will bind the labeled DNA to the silica membrane with 250 microliters of high salt DNA binding buffer per tube, after which the impurities will be removed from the membranes with two washes of 500 microliters of wash buffer each. The robot will then add 15 microliters of low salt solution buffer to the membranes to recover the purified labeled DNA samples in approximate 12 microliter volumes to hybridize the samples.Next preheat a hybridization oven to 65 degrees Celsius and prewarm the backing slides and hybridization chambers. To prepare the hybridization mix, combine 1.1 microliters of cot 1D NA 4.95 microliters of the manufacturer supplied blocking mix, and 24.75 microliters of hybridization buffer. Use the liquid handling robot to allocate this mix into each well of a new 96 well plate pre wetting each tip to enhance the transfer accuracy.Next pipette 9.35 microliters of the appropriate signing, three labeled DNA, followed by 9.35 microliters of the appropriate signing, five labeled DNA. To each well seal the plate, fourex the samples for one minute, and give the plate a quick spin to collect the contents at the bottom of each. Well denature the labeled DNA in a pre hybridization incubator.First for three minutes at 95 degrees Celsius, followed by 30 minutes at 37 degrees Celsius. Then working on a 42 degrees Celsius heated platform. Place the backing slide into the hybridization chamber, ensuring that the transparent part of the gasket aligns with the windowed part of the hybridization chamber Using pre-wet tips.Now slowly pipette 42 microliters of the hybridization mix into the center of the appropriate position of the array backing slide. Taking care that the liquid does not touch the rubber ring boundary. Once all the positions are filled carefully lower the array slide onto the backing slide and assemble the hybridization chamber.Taking care that the side of the array with writing is facing the backing slide, then tighten the hybridization chamber screw fully and inspect the assembled hybridization chamber, confirming that there is no leakage of hybridization. Mix outside of the rubber ring boundaries and that each air bubble is approximately four millimeters in height. When the hybridization chamber is rested on its end vertically, rotate the hybridization chamber to check this.All the air bubbles in each position are moving. If any of the bubbles are stuck, give the chamber a sharp tap on the bench. Then place the hybridization chambers into the rotating oven at 65 degrees Celsius for 24 hours.The next day submerge the hybridization chambers in wash buffer one, and use a pair of plastic flat edged forceps to pry the slides apart. Then discard the gasket slides and place the array slides into a rack submerge in fresh buffer one. Wash the array slides in approximately 700 milliliters of wash buffer for one to five minutes, stirring vigorously with a flea and a magnetic starr.Then transfer the array slides to around 700 milliliters of wash buffer, two for 90 seconds with more vigorous stirring at the end of the second wash. Gently lift the array slides outta the buffer. They should emerge dry.Then load the array slides into the slide holders at the scanner along with the slide protectors, and then scan the samples. According to the array manufacturer's instructions, each probe on a hybridized array is visualized as a mixture of red and green fluorochromes. The ratios of red to green fluorescent signal for each probe are quantified by the scanner and the associated software lots them as log two ratios according to their genomic position.The resulting array traces allow interpretation of regions identified as genomically unbalanced. For example, in this trace from a child with Williams syndrome, a recurring micro deletion syndrome mediated by low copy repeats in the proximal region of chromosome seven, the genomic imbalance was identified by the software with a red line. The prop fluorescent log ratio should cluster closely around zero, indicating a green to red ratio of one to one for normal regions of the genome.Scattered array traces as observed in this scan, may result in inaccurate calling of the abnormal regions, or a failure to identify the genomic imbalance and may be caused by a number of factors, including poor DNA quality, or the presence of rays, levels of ozone in the atmosphere sphere. After watching this video, you should have a good understanding of how to process patient samples for testing by A CGH and to produce good quality data for downstream analysis and detection of copy number variation.

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JoVE Journal

Biology

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Learn array comparative genomic hybridization to detect genomic copy number variants in patient samples. This method uses fluorescent labeling and hybridization to identify imbalances causing genetic syndromes.

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

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