Microarray Comparative Genomic Hybridization for the Analysis of Bacterial Population Genetics and Evolution - Bacterial Pathogenesis: Methods and Protocols - page 45

Studies on the evolution and pathogenesis of an increasing number of
bacterial pathogens including Escherichia coli O157:H7 (2) , Mycobacterium
spp. (3) , Helicobacter pylori (4) , Yersinia spp. (5) , and Staphylococcus aureus
(6,7,8,9) have been performed with microarrays, resulting in a greatly enhanced
understanding of their biology.
Several different approaches to microarray construction have been developed,
including oligonucleotide- or DNA-based arrays on glass slides or nylon
membranes (10) . Here, we describe a robust CGH protocol based on the use of a
PCR-generated microarray (6) . Detailed descriptions of genomic DNA template
preparation, labeling, microarray hybridization, and data analysis are provided.
2. Materials
2.1. Genomic DNA Template Preparation (S. aureus)
1. Edge Biosystems DNA extraction kit (Edge Biosystems, Gaithersburg, MD). All
components are kept at 4 °C; spheroplast buffer is stored at -20 °C.
2. 100% isopropanol (molecular grade). Chill before use.
3. 70% ethanol in sterile deionized water. Chill before use.
4. Lysostaphin (AMBI products, Lawrence, NY) lyophilized powder is resuspended
in nuclease-free water (Stock; 5 mg/mL). Store at -20 °C.
5. Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA) or equivalent.
6. Hin P1I restriction endonuclease (New England Biolabs, Ipswich, MA). Store
at -20 °C.
2.2. Labeling of DNA
1. 400 ng of genomic DNA templates (test and reference strains; see Note 2 ).
2. Prime-a-gene labeling kit (Promega, Madison, WI):
a. Bovine serum albumin (BSA, 10 mg/mL); nuclease free.
b. Unlabeled dNTPs (1.5 m M ). Store at -20 °C .
c. DNA polymerase I large fragment (Klenow). Store at -20 °C.
d. Labeling buffer (5×) including 26.0 A 260 U/mL random hexadeoxyribonu-
cleotides.
3. TE Buffer: 10 m M Tris-HCl, 1 m M EDTA, pH 7. Store at room temperature.
4. 0.2 M EDTA (pH 8.0). Store at room temperature.
5. Cyanine 3-dCTP (Cy3); Cyanine 5-dCTP (Cy5) (NEN-Dupont, Boston, MA).
Store at -20 °C; light sensitive.
6. Sephadex®G-50 spin columns (Roche Diagnostics, Basel, Switzerland) (optional).
7. Centri-spin 20 label clean up kit (Princeton Separations, Adelphia, NJ). Store at
room temperature.
8. Agilent 2100 Bioanalyser (Agilent Technologies) or equivalent to determine
genomic DNA labeling efficiency.