webpage powered by Implen  Array-CGH
An innovative screening method in molecular cytogenetics Microarray-based comparative genomic hybridization (array/matrix-CGH) is an improvement of conventional comparative genomic hybridization (CGH, Kallioniemi et al. 1992) and becomes more and more important as an innovative screening method in molecular cytogenetics. In combination with fluorescence in situ hybridization (FISH) new chances of an improved and more precise approach are developing within the scope of tumor cytogenetics and pre- and postnatal genetic analysis. Further applications of array-CGH and FISH are polar body and preimplantation genetic analysis. Array/matrix-CGH was developed in 1997 by Solinas-Toldo et al. at the group of Peter Lichter (DKFZ, Heidelberg, Germany). A grid of well-defined, genomically mapped DNA fragments (i.e. BAC clones, cDNA, oligonucleotides) immobilized on a glass surface replaces condensed metaphase chromosomes as hybridization target (Figure 1). Thereby, the resolution of copy number analysis could be increased dramatically and depends only on the number and distance of fragments represented. Currently whole genome arrays with resolutions from 1Mb to 150 kb or even in the range of single genes or exons are available. These high resolutions permit not only the detection of submicroscopic genomic imbalances, but also precise breakpoint determination of genomic aberrations. Therefore, array-CGH can complement the standard cytogenetic methods (chromosome analysis, subtelomere analysis, FISH) in prenatal, postnatal and tumorcytogenetic analysis. In addition, array-CGH enables the sequence based mapping of aberrations by the identification of chromosomal segments, showing copy number aberrations (deletion, duplication or amplification). | Metaphase chromosomes as hybridization targets are replaced by a grid of BAC-DNAs. “DIM” (CGH), “x1” (array-CGH) refers to a deletion in the patient’s DNA. “ENH” (CGH), “x3” (array-CGH) refers to a duplication in the patient’s DNA. Adapted with changes from Tönnies et al. (2002). | 
Figure 1: Comparison of conventional CGH and array-CGH
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The main requirements for cytogenetic applications are standardized and maximal reproducible experimental conditions with the highest possible sensitivity. Automation of hybridization and washing to a sensible degree guarantees standardized user independent experimental conditions with minimal variation, whilst offering an uncomplicated setup, ease of use and maintenance-free operation. In addition, the assay time is considerably reduced by the automation, making the implementation of array-CGH in the daily laboratory work feasible and calculable. Method To get an overview of the specific steps during an array-CGH experiment refer to the flow chart (Figure 2). The applied methods are adapted from protocols developed at the Max Planck Institute of Molecular Genetic with slight modifications concerning instruments and the sample amount (http://www.molgen.mpg.de/%7Eabt_rop/molecular_cytogenetics/ArrayCGH2006.html). Implen offers solutions for every single step speeding up the whole process due to automation and standardization. Implen offers solutions for every single step speeding up the whole process due to automation and standardization. | 
Figure 2: Flow Chart for the array-CGH process
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Labeling: Patient’s DNA and reference DNA (300-1000ng) are fluorescently labeled with Cy3 and Cy5, respectively, using a random priming protocol (e.g. Bioprime Array CGH Labeling Kit, Invitrogen). DNA concentration prior to labeling and efficiency of fluorescent dye incorporation is precisely measured using the NanoPhotometer™ (Implen, Munich, Germany). With its innovative optical pathway the NanoPhotometer™ enables spectrophotometrical analysis of submicroliter sample volumes (starting from 0.7 µl) and standard volumes (up to 3.5 ml). Differentially labeled DNAs (test = patient’s DNA versus reference DNA) are precipitated with CotI-DNA and dissolved in hybridization buffer (8% dextransulfate, 50% formamide, 2x SSC). Prior to hybridization the arrays are treated with blocking solution (25% formamid, 4x SSC, 0,1% SDS, BSA und herring sperm DNA).
Hybridization: After a short denaturation step, preannealing is carried out at 42°C for 2 hours. In the SlideBooster™ Hybridization Station (Implen, Munich, Germany) up to 8 arrays are hybridized in parallel with one unit (extension up to 32 arrays possible) under standardized conditions (42°C, 24h, pulse 5_5). LifterSlips™ (Implen, Munich, Germany) are used for hybridization. Hybridization volume for a slide surface of 25x 60 mm is 75µl.
Washing: Subsequent to hybridization the arrays are washed in the AdvaWash™ Universal Slide Washing Station (Implen, Munich, Germany). Three different washing buffers (wash buffer 1: 50% formamide, 2x SSC and 0,1% SDS; PN-buffer: 100mM sodium phosphate and 0,1% NP-40; PBS buffer) are used. The first washing step is at 42°C. The following steps are carried out at room temperature. The slides are dried by centrifuging prior to scanning (AdvaTube™, Implen, Munich, Germany).
Scanning: The resulting relative fluorescence intensities (probe to reference) are measured by a computer controlled microarray scanner (Figure 3).
Green Signal: microdeletion in patient’s DNA. Hybridization only by the green labeled reference DNA. Red Signal: microamplification in patient’s DNA. Hybridization only by the red labeled patient’s DNA. Yellow Signal: no changes in patient’s DNA. Red and green labeled DNA is hybridizing. | 
Figure 3 Scan of an array-CGH Slide
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Analysis: Following the initial image analysis the genomic profile is calculated using an appropriate analysis software like CGHPRO (Chen et al., 2005). Losses (deletions) and gains (duplications) are detected if the genomic profile exceeds predefined thresholds (log2ratio probe/reference -0.3 and 0.3, respectively). If desired array-CGH results can be confirmed by fluorescent in situ hybridization (FISH) using BAC DNA as probe (Figure 4). A: Conventional CGH profile of chromosome 8. The deletion in the region 8p is indicated by the arrow. An accurate classification of the breaking points is not possible. B: Array-CGH profile of chromosome 8. The log2ratio of patient to reference DNA is shown. The threshold values are marked with red and green lines (log2ratio sample/reference -0.3 and 0.3, respectively). Ratios below or above indicate deletions or duplications, respectively. The red bar indicates the deleted region on chromosome 8p. C: Confirmation of the array-CGH results by FISH analysis. red signal: BAC-probe RP11-395I14 localized at 8p21.2; green signal: control probe cep8 (Vysis, Downers Grove, IL) specific for the centromere of chromosome 8. The arrows mark chromosomes 8. | 
Figure 4: Interstitial deletion of chromosome 8p12-p21 (clinical case)
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Advantages Conventional cytogenetics has an average resolution of ~5-10 Mb. The use of array-CGH with a resolution of 1 Mb or higher allows the detection of submicroscopic genomic aberrations which are not detectable by conventional cytogenetics. Comparison between array-CGH and conventional CGH showed that the higher resolution of array-CGH allowed a better and more precise determination of aberration sizes and chromosomal breakpoints in 73% of the investigated cases. In addition the genotype-phenotype correlation and the identification of candidate genes for a certain phenotype can be improved by using array-CGH. The major prerequisite for the detection of aberrations in the submicroscopic range is a maximal stable base line with minimal variation (Figure 5). Two different DNAs labeled with Cy3 and Cy5 were hybridized on a BAC-array with app. 36000 clones. The log2ratio of cy3 to cy5 of the individual clones is shown (CGHPRO software: Chen et al., 2005). The threshold values are marked with red and green lines (log2ratio sample/reference -0.3 and 0.3, respectively). The detected difference in DNA quantity corresponds to DNA copy number polymorphisms present in the normal population with currently unknown pathogenetic relevance. The picture is a courtesy of Reinhard Ullmann and Fikret Erdogan, Max Planck Institute for Molecular Genetics, Berlin | 
Figure 5: Array-CGH profile
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Automation and standardization of the hybridization process using the SlideBoster™ Hybridization Station (Implen, Munich, Germany) and the AdvaWash™ Universal Slide Washing Station (Implen, Munich, Germany) guarantees a stable base line with minimal variation and maximal reproducible hybridizations. In addition, the hybridization geometry is flexible over the entire slide surface and the minimal required sample volume starts by 1µl. The analysis does not require a background correction and the overall deviation is minimal. Compared to manual protocols optimization and automation of the protocol leads to a reduction of hybridization and washing times. Applications for array-CGH - prenatal genetic analysis
- postnatal genetic analysis (mental retardation, dysmorphisms, congenital anomalies)
- tumorcytogenetics (hematology, tumorpathology)
- polar body analysis
- preimplantation genetic analysis
References Array-CGH Labeling and Hybridization Protocol of the Max Planck Institute of Molecular Genetics, Berlin, Germany >> Institute of Medical Genetics, Charité Universitätsmedizin Berlin >>
Array-CGH projects at the Institute of Medical Genetics, Charite Universitätsmedizin Berlin >>
Further Reading Albertson DG & Pinkel D, Genomic microarrays in human genetics and cancer, Human Molecular Genetics (2003)
Chen W et al., CGHPRO - A comprehensive data analysis tool for array CGH, BMC Bioinformatics (2005)
Kallioniemi A et al., Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors, Science 258: 818-821 (1992)
Rickman L et al., Prenatal diagnosis by array-CGH, European Journal of Medical Genetics (2005)
Shaffer LG & Bejjani BA, A cytogeneticist’s perspective on genomic microarrays, Human Reproduction Update (2004)
Solinas-Toldo S et al., Matrix-Based Comparative Genomic Hybridization: Biochips to Screen for Genomic Imbalances, Genes Chromosomes Cancer 20, 399-407 (1997)
Speicher M & Carter N, The new cytogenetics: blurring the boundaries with molecular biology, Nature Reviews Genetics, 6, 782-792 (2005)
Tönnies H et al., Modern molecular cytogenetic techniques in genetic diagnostics, Trends in Molecular Medicine (2002)
Wilton L, Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparatice genomic hybridization, Human Reproduction Update (2005) |
