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

The authors have no acknowledgements.

1. Mærkningen Reaktion <ol><li> Inden brug pre-alikvot cyanin 3 og 5-mærkede dUTPs, umærkede nukleotider og tilfældige primere ved producenten anbefalede koncentrationer i 96 brønde plader og opbevares ved -20 C klar til brug. Med henblik på denne protokol, portion 10,5 pi af den passende mærket dUTP og 10,5 pi umærkede nukleotider og tilfældige primere til hver brønd. </li><li> Optø klar-til-brug 96-brønds plade af nukleotider og primere ved 4 ° C i ca. 1 time, beskyttet mod lys. </li><li> Når optøet, ...

<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>

Array CGH vil ikke registrere afbalancerede rearrangementer eller ploidi abnormiteter såsom triploidy. Endvidere kan lavt niveau mosaik ubalancer ikke påvises. Men matrix CGH har en højere opløsning for CNV detektion end G-banded kromosom analyse, som den har erstattet i mange cytogenetik laboratorier. Det er derfor den aktuelle gold standard for hele genomet CNV detektion. Det kan erstattes af næste generation sekventering teknologier i fremtiden, men i øjeblikket, deres omkostninger og de tekniske kompleksitet f...

Det har været kendt i mange år, at deletioner eller ekstra kopier af kromosomale segmenter forårsager intellektuelle handicap, dysmorphism og congentical misdannelser, og i nogle tilfælde forårsage genetiske syndromer 1. Den eneste teknologi, der findes indtil midten af ​​2000&#39;erne for hele genomet detektion af disse ændringer var imidlertid G-banded kromosom analyse, som har en opløsning på omkring 5-10 MB, afhængigt af region og arten af ​​den ubalance, og som ikke kan opdage unormale kromosomer hvor materialet er blevet erstattet af en region fra et andet kromosom med samme banding mønster. Accessoriske cytogenetiske teknikker såsom fluorescens in situ hybridisering og multiplex ligation-specifik probe forstærkning har været til rådighed for afhøring af specifikke loci, i tilfælde af mistanke om specifik syndromisk ubalance, men det var ikke før indførelsen af matrix komparativ genomisk hybridisering (array CGH) i rutinemæssig klinisk diagnostisk service 2-5 at genom-dækkende påvisning af kopi nummer varianter (CNVs) blev mulig på et stærkt forøget opløsning (typisk omkring 120 KB). Klinisk servicearbejde sideløbende undersøgelser har vist, at CNVs for nogle regioner er udbredt i den normale population 6-7, anden CNVs mens tidligere målbart, er forbundet med neurodisabilities som autisme og epilepsi 8-11. Den i dette papir protokol bruges i vores UK National Health Service (NHS) klinisk diagnostisk laboratorium; vi bruger nye hybridiseringsmetoder strategier, batch test og robotteknologi til at minimere omkostningerne i denne statsfinansieret tjeneste. Forud for protokollen beskrevet nedenfor, bør høj kvalitet DNA ekstraheres fra det passende udgangsmateriale, almindeligvis blod, dyrkede celler eller vævsprøver. Spektrofotometri kan anvendes til at måle koncentrationen (bør være&gt; 50 ng / ul), og kontroller 260: 280 absorbans forhold (bør be 1,8-2,0). Gelelektroforese kan anvendes til at kontrollere, at DNA&#39;et er af høj molekylvægt uden væsentlig nedbrydning. Denne protokol er beregnet til højere gennemløb laboratorier, som er mærkning 96 prøver pr run hjælp af automatiserede håndtering af flydende robotteknologi. Det kan imidlertid være indrettet til lavere gennemløb labs uden automation.

<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.

Hver probe på en hybridiseret array visualiseres som en blanding af røde og grønne fluorochromer (se figur 1). Forholdet mellem rød til grøn fluorescerende signal for hver probe kvantificeres ved scanneren og tilhørende software plotter disse som log2 nøgletal efter deres genomiske position, og identificerer områder, der falder uden forudindstillede grænser. De resulterende array-spor tillader fortolkning af regionerne identificeret som genomisk ubalanceret. For eksempel spor fra et barn med Wi...

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

Array CGH til påvisning af genomiske kopital varianter har erstattet G-banded karyotype-analyse. Dette papir beskriver den teknologi og dens anvendelse i en diagnostisk tjeneste laboratorium.

Array Comparative Genomisk hybridisering (Array CGH) til påvisning af genomiske Kopiér nummer Varianter

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

Research

JoVE Journal

Biology

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

Isolation of Genomic DNA from Mouse Tails

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 ...

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

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. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

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 ...

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, ...

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 ...

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 ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

Detektion af sjældne genomiske varianter fra poolet sekventering under anvendelse Splinter

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

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Genomisk Transformation af Picoeukaryote<em&gt; Ostreococcus Tauri</em

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