The Use of Microarray Technology for Cytogenetics
The use of microarray technology is revolutionizing the field of clinical cytogenetics. This new technology has transformed the cytogenetics laboratory by adapting techniques that have heretofore been the province of molecular geneticists. Intimate knowle
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1. Introduction The use of microarray technology is rapidly becoming an essential part of the diagnostic cytogenetic laboratory. DNA microarrays are solid surfaces on which nucleic acids are immobilized (spotted, lithographed, or synthesized in situ) and used as targets for hybridization. These nucleic acid molecules can be large insert clones (such as bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs)) or short (25–60 bp) oligonucleotides. In typical microarrays, they are attached to the Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_8, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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solid surface by a covalent bond through a chemical matrix (e.g., amino-silane). Most solid supports to which DNA is attached are either glass or silica, although beads are also used. The hybridization to these targets can be performed through the process of comparative genomic hybridization (CGH), or through non-CGH methods. In essence, they allow multiplex testing of many targets across the genome. The essential elements of these hybridization methods have been extensively reviewed (1–4). This chapter details the materials and methods used in oligonucleotide-based comparative genomic hybridization using microarrays (array CGH) in our laboratory. Some of the fundamental concepts of array CGH methodologies described here may apply to other platforms with minimal changes. Other methodo logies are specific to the type of array used, or to the manufacturer and the specific application for which the array is being utilized. Regardless of the platform used, it is clear that microarrays are now solidly rooted in the clinical activities of cytogenetic laboratories and will remain a part of this discipline for years to come.
2. Materials 2.1. General
1. 95% ethanol. 2. Isopropyl alcohol (isopropanol) 1 L. 3. Acetic acid, glacial, 2.5 L. 4. Tris base, 1 kg (Fisher). 5. EDTA (ethylenediamine tetra acetic acid) 1 kg.
2.2. DNA Purification
1. 5 PRIME ArchivePure DNA Blood Kit (120 ml) (Fisher). 2. Red blood cell (RBC) lysis solution, 900 ml (Fisher). 3. Cell lysis solution, 300 ml (Fisher). 4. RNase A solution, 1.5 ml (Fisher). 5. Protein precipitation solution, 100 ml (Fisher). 6. DNA hydration solution, 50 ml (Fisher). 7. Glycogen solution, 20 mg/ml, 500 ml.
2.3. Digestion of Genomic DNA (If Required)
1. DpnII Restriction Enzyme (5,000 units) (New England Biochemical, Ipswich, MA). 2. Gel loading solution, 6× concentrate, 5 ml (Sigma-Aldrich, St. Louis, MO). 3. 1 kb DNA Ladder, 1 mg (Invitrogen, Carlsbad, CA).
The Use of Microarray Technology for Cytogenetics
2.4. DNA Washing and Hybridization
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1. BioPrime Total Labeling Kit (Invitrogen). 2. Human Cot-I DNA 1 mg (Invitrogen). 3. 10× blocking reagent. 4. 2× hybridization buffer. 5. Ultrahyb hybridization buffer (Ambion, Austin, TX).
2.5. Washing
1. PBS (Invitrogen). 2. Oligo aCGH/ChIp-on-Chi
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