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Comparative Genomic Hybridization Bioarray™ CGH Labeling System with Cyanine-3 and Cyanine-5 Dutp

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Comparative Genomic Hybridization Bioarray™ CGH Labeling System with Cyanine-3 and Cyanine-5 Dutp Data Sheet Enzo Life Sciences Microarray Analysis Comparative Genomic Hybridization BioArray™ CGH Labeling System with Cyanine-3 and Cyanine-5 dUTP Comparative Genomic Hybridization Test DNA Digest DNA Random Prime Label (CGH) is the method of choice for vs. with two DNA Samples determining additions, deletions Reference DNA Restriction Enzyme with the Enzo System; and chromosomal abnormalities one with Cyanine-3 and the other with across the genome. Addressing Cyanine-5-dUTP the critical need for a complete Hybridize to Block Labeled random primed cyanine labeling DNA Arrays, Repetitive Sequences system, the BioArray™ CGH Wash and Scan with Cot-1 DNA Labeling System is specially for- mulated to provide the end-user with all the components necessary Analyze Ratio of Identify Genomic to label genomic DNA for compar- Fluorescent Signals Aberrations ative genomic microarray analysis. and developmental disorders and out the genome with high sensitivity. The table, at top right, displays an for developing diagnostic and thera- The simple, rapid protocol yields overview of the standard CGH pro- peutic targets. efficient and reproducible fluores- cedure. In comparative genomic cent DNA labeling that results in hybridization, labeled DNA from test strong signals with low background. cells is directly compared with Proven Performance This is demonstrated by the data labeled DNA from normal cells and The Enzo BioArray™ CGH Labeling represented in the figures on the fol- most commonly hybridized to DNA System for the preparation of lowing pages. fragments on BAC microarrays. Cyanine-3 and Cyanine-5 labeled Fluorescent ratios are calculated for DNA provides all necessary The new Enzo BioArray™ CGH each spot on the array enabling reagents for random primed label- Labeling System provides a consis- detection of regions of the genome ing including primers and optimized tent and standardized labeling that are amplified or deleted in the cyanine-3 and cyanine-5 deoxynu- method for CGH array analysis that test tissue. cleotide mixes. enables the end-user to easily dis- cern duplicated and deleted regions Array based CGH measures copy The BioArray™ CGH Labeling of a chromosome. number variations at multiple loci System is a robust and consistent simultaneously, thus providing an system that enables detection of important tool for studying cancer chromosomal alterations through- Applications: + Comparative genomics + Whole genome analysis + Pharmacogenomics + Evolutionary science + Toxicogenomics + Medical genetics + Pre-natal genetic defects + Study of somatic cell genomes in cancer + Post-natal evaluation of genetic disease + Evaluation of effective drug treatments in oncology Advantages: + Achieves high reproducibility and specificity in + Maximizes yield and sensitivity with strong signal detecting chromosomal abnormalities for both stan- intensity and low background for both cyanine-3 and dard and dye swap experiments. cyanine-5-dUTP. + Saves time and money by offering all components, + Performs well with a broad range of commercially including cyanine-labeled nucleotides in one system. available slide surface chemistries. + Simplifies array CGH labeling and allows for com- + Standardizes the CGH labeling protocol and elimi- plete fluorescent labeling in approximately 2-4 hours. nates the need for home brew methods. 42670v01 04/01/2005 BioArray™ CGH Labeling System Figure 1: Detection of Chromosomal Changes A series of CGH microarray experiments were conducted Male vs. Female Genomic DNA Comparison comparing pooled normal male Y and pooled normal female • Male vs. Female (minus 1) • Female vs. Male (plus 1) genomic DNA. The Enzo • Female vs. Female BioArray™ CGH Labeling System was utilized to label male and female DNA with cya- autosomal X nine-3 and cyanine-5, respec- tively. The samples were mixed and hybridized for 72 hours at 37°C to a high density human BAC microarray. (Cyanine-5/Cyanine-3) 2 In parallel, a second BAC array Log was hybridized in a dye swap- ping experiment (male = cya- nine-5/female = cyanine-3). The arrays were washed, scanned and analyzed to generate the 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 10 11 11 12 12 13 14 15 16 17 18 19 20 21 22 X Y graph shown in Figure 1. The Chromosome graph demonstrates the predict- ed result that male and female DNA was compared to itself. As (log2). This represents an average genomes differ only on the X and Y expected, no significant deviation no change +/-1.2 fold. In summary, chromosome. The dye switching from 0 (log2) was detected. In this this tight baseline value facilitates shows the cyanine-3/cyanine-5 example, the standard deviation detection of gains and losses in intensity ratio inverts when the dyes around 0 (-0.011) was +/- 0.110 chromosomal material. are reversed. Autosomal chromo- somes show no changes. Average Log2 Cy5/Cy3 Ratio +/- SD Autosomes +/- SD X Y To assess the reproducibility of the Male Cy3 Female Cy5 -0.017 +/- 0.143 0.473 -1.156 labeling system, normal female Female Cy3 Male Cy5 -0.010 +/- 0.172 -0.423 1.255 Female Cy3 Female Cy5 -0.011 +/- 0.110 -0.002 0.019 Figure 2: Specificity These CGH BAC arrays represent a specific sub-grid of the full genome array displaying portions of the Y chro- mosome. Figure 2A Figure 2B Figure 2C Male Cy3, Female Cy5 Male Cy5, Female Cy3 Female Cy3, Female Cy5 Figure 2A: Male DNA was labeled Figure 2B: A dye swap experiment Figure 2C: Female DNA was labeled with Cyanine-3 (green) and female was performed with male DNA with either Cyanine-3 or Cyanine-5 DNA was labeled with Cyanine-5 labeled with Cyanine-5 and female and compared with itself. The three (red). Figure 2A displays strong DNA labeled with Cyanine-3. The Y-chromosome BAC clones display green fluorescence on three spotted same three BAC clones display very weak yellow fluorescence. BAC clones that represent the three strong red fluorescence while the This result is consistent with the different regions of the Y chromo- other spots remain yellow. BAC clones reported on the Y chro- some. All other BAC clones con- mosome. taining regions of autosomal chro- mosomes are yellow. Figure 3: Sensitivity The figure at right displays a typical hybridization of the Enzo BioArray™ CGH Labeling System across a large section of a whole genome BAC array. The high signal-to-noise ratio is the key attribute that increases the high sensitivity of the Enzo system. Figure 4: Validation A Full Genome 3 Chromosome 12 2 2.5 region of interest 2 1.5 region of interest 1.5 1 1 } 0.5 0.5 0 0 -0.5 -0.5 -1 } -1.5 -1 (Cyanine-5/Cyanine-3) Median Intensities Median (Cyanine-5/Cyanine-3) (Cyanine-5/Cyanine-3) Median Intensities Median (Cyanine-5/Cyanine-3) 2 -2 2 -1.5 Lo g -2.5 Lo g -3 -2 1 1 1 2 2 2 3 3 3 4 4 5 5 6 6 6 7 7 8 8 9 9 10 11 11 12 12 13 14 14 15 16 17 17 18 19 20 21 22 X Y 1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 106 113 120 127 134 141 148 155 Chromosome BAC Clone Position Figure 4: Validation Experiment A A major cancer research institution provided Enzo with DNA samples from a normal patient and from a biopsied tumor. The biopsied tumor DNA had known changes on chromosome 12. The DNA samples were digested with Hae III restriction enzyme and purified. Using the Enzo BioArray™ CGH Labeling system, 850 nanograms of each sample was labeled with either Cyanine-3 or Cyanine-5. Replicate samples were labeled in the opposite dye configuration for the dye swap analysis. The BioArray™ CGH Labeling System detected the known alterations in chromosome 12, as shown by figure 4. Figure 5: Validation B Chromosome 8 Chromosome 16 2 1.5 1 0.5 0 -0.5 -1 (Cyanine-5/Cyanine-3) Median Intensities Median (Cyanine-5/Cyanine-3) 2 (Cyanine-5/Cyanine-3) Median Intensities Median (Cyanine-5/Cyanine-3) 2 -1.5 Lo g Lo g -2 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 1 7 13 19 25 31 37 43 49 55 62 68 74 80 86 92 98 104 110 116 122 128 134 BAC Clone Position BAC Clone Position Figure 5: Validation Experiment B A collaborator at a prominent research institution provided Enzo with a DNA sample previously characterized with known alterations on chromosomes 8 and 16. Chromosome 8 displays a known heterozygous deletion at BAC clone position 133, while the deletion at BAC clone position 60 indicates a new finding. On chromosome 16, a known 16p heterozygous deletion is identified on the first two BAC clones of the chromosome. Arrays using Enzo’s BioArray™ CGH Labeling System verify the known alterations on chromosomes 8 and 16. BioArray™ CGH Labeling System For more information or to place an order, contact your Enzo Life Sciences Alliance Manager at 1.800.221.7705 or 1.631.694.7070 or visit our web site at www.enzolifesciences.com. Ordering Information >> Catalog Number Description Quantity 42670 BioArray™ CGH Labeling System 2 x 10 labeling reactions The BioArray™ CGH Labeling System contains all the necessary components for effective cya- nine labeling of genomic DNA in dual sample comparative genomic hybridization arrays. Product Components >> Reagent Primers/Reaction Buffer Cyanine-3-dUTP Nucleotide Mix Cyanine-5-dUTP Nucleotide Mix Klenow DNA Polymerase Stop Buffer Nuclease-free Water Patents: This product and the use of this product is covered by one or more claims of Enzo patents, including but not lim- ited to the following: U.S.
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