Comparative Genomic Hybridization

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Information about Comparative Genomic Hybridization
Science-Technology

Published on April 27, 2014

Author: aglinantony

Source: authorstream.com

Comparative Genomic Hybridization: Comparative Genomic Hybridization Introduction: Introduction Detection of chromosomal copy number changes without the need for cell culturing In 1992, University of California, San Francisco Chromosomal gains and losses throughout the whole genome of a tumor Pathogenesis or progression of tumors 1) Metaphase Slide Preparation: 1) Metaphase Slide Preparation Phytohaemagglutinin -- peripheral blood lymphocytes from a karyotypically normal man or woman Preferable to use metaphase spreads from women (XX, Y doesn’t contain much information) Cells incubated for 4 days Cells are arrested in mitosis stage by adding colchicine Fixed in 3:1 methanol/acetic acid Prepare slides Dry overnight (RT 24°C and a relative humidity of 60–70%) Several batches of donors have to be checked 1) Metaphase Slide Preparation: 1) Metaphase Slide Preparation Metaphase Little cytoplasm Minimal overlapping of the chromosomes Low cell density paired with a high mitotic index Adequate length (400–550 bands) and not contain separated chromatids 2) DNA Isolation from Tumor Tissue: 2) DNA Isolation from Tumor Tissue Fresh or frozen tissue -- high molecular size (not degraded) Formalin fixed, paraffin wax embedded tissue – partially degraded (300–20 000 bp ) or cross linked Neutral buffered formalin (pH 7.0) and fixation for no longer that 24 hr Cross linking of DNA caused by the fixative can hamper the function of the enzymes, DNA polymerase and Dnase Sodium thiocyanate , lysis buffer , high concentrations of proteinase K Labeling process Nick translation Nucleotides with digoxigenin , biotin, or fluorochrome labelled nucleotides 3) Contamination: 3) Contamination Contamination (or dilution) of the tumor DNA with normal DNA or from stromal and inflammatory cells Cell sorting techniques Tumor tissue can be microdissected from the sections by demarcating with a marker, scraping it off with a surgical blade, and collecting it in Eppendorf tubes 0.5–1 μ g of DNA Degenerate oligonucleotide primed polymerase chain reaction -- DOP-PCR Results not reliable 4) Reference DNA Labeling: 4) Reference DNA Labeling Biotinylated and digoxigenin conjugated deoxynucleotides – fluorochrome conjugated antibodies ( avidin–fluorescein isothiocyanate (FITC) and sheep antidigoxigenin – tetramethyl rhodamine isothiocyanate (TRITC), respectively Deoxynucleotides that have been directly conjugated with fluorochromes – weaker signal Tumor and reference DNA are in the same range of lengths – 500 – 1500bp 1% agarose gel 5) Blocking: 5) Blocking Unlabelled Cot-1 DNA -- placental DNA from 50 to 100 bp High number at all centromeres , telomeres, and some specific regions Removal of repetitive sequences from the probe 6) Hybridisation: 6) Hybridisation Tumor and reference labeled DNA (the probes) are mixed with 100 times the same amount of human Cot-1 DNA Hybridization mix containing 50% formamide (which decreases the melting temperature of DNA) and 10% dextran sulphate (which increases the effective probe concentration), in 2 % saline sodium citrate (SSC), pH 7.0 The probes in a water bath at 80°C for 10 minutes Hybridization at 40°C for two to four days Washed and counterstained with DAPI -- an anti fade solution 7) Detection : 7) Detection Visualization of Fluorescence Mercury arc lamp -- without chromatic variation Fluorescence microscope: DAPI (blue; for chromosome identification), TRITC (red; normal reference DNA), and FITC (green; tumor DNA) Narrow band pass filters are required for excitation and emission ×63 or ×100 magnification 7) Detection : 7) Detection Recording the Image Hybridization signals should be strong and homogeneous Background should be minimal Minimal overlapping of chromosomes Capturing can be done both automatically or manually Image Processing (1) Background subtraction (2) Segmentation and removal of non-chromosome objects (3) Normalization of the FITC:TRITC ratio for the whole metaphase (4) Interactive karyotyping (5) Scaling of chromosomes to a standard length Advantages: Advantages Fast screening technique CGH results, more specific molecular biological techniques such as FISH, loss of heterozygosity analysis, and sequencing Limitations: Limitations Relatively time consuming Cannot detect structural chromosomal aberrations without copy number changes, such as balanced chromosomal translocations, inversions, or ring chromosomes Less sensitive than PCR based methods in detecting deletions CGH results are not linearly distributed Applications: Applications Screening of tumors for genetic aberrations Searching for genes involved in the carcinogenesis of particular subsets of cancers Analyzing tumors in experimental models to obtain an insight into tumor progression Diagnosis

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