title: Listeria monocytogenes (EGDe) macroarray, version 1
status: Public on Jul 03 2006
contact.city: Ribeirao Preto
contact.first: Ricardo
contact.middle: Z.N.
contact.last: Vêncio
contact.state: SP
contact.address: Av. Bandeirantes, 3900
contact.department: Computing and Mathematics Department
contact.country: Brazil
contact.institute: Universidade de São Paulo
organism: Listeria monocytogenes
manufacture_protocol: According to Doumith et al (2004, Infect Immun. 72:1072–1083):
  
  MATERIALS AND METHODS - Probe selection, primer design, PCR amplification, and array construction.
  
  The L. monocytogenes EGDe-specific probes spotted onto the Listeria array corresponded to the 270 genes defined previously as being specific for L. monocytogenes EGDe with respect to L. innocua CLIP 11262. As some genes were too small to allow amplification of a PCR product of optimal size, the final array contained 262 EGDe genes. The 94 probes that were specific for L. innocua CLIP 11262 relative to L. monocytogenes EGDe were also selected from the previously defined list of 149 genes specific for L. innocua CLIP 11262. For genomic regions containing several genes, only representatives were chosen for the array. In order to identify L. monocytogenes CLIP 80459-specific sequences, we used the program Cross-match (http:/bozeman.mbt.washington.edu/). With this approach, 141 sequence fragments, ranging from 33 to 3,025 bp, were identified as missing from both L. monocytogenes EGDe and L. innocua CLIP 11262. For probe design, only fragments longer than 1 kb were taken into account, allowing us finally to select 53 sequences that were specific for L. monocytogenes CLIP 80459. Primers were designed by use of a modified version of Primer 3 software (CAAT-box [18]) to amplify a specific fragment of 300 to 600 bp for each gene (melting temperatures were 55 to 65°C) (Eurogentec). Amplification reactions were performed in a 100-μl reaction volume containing 10 to 20 ng of chromosomal DNA. The concentration and size of each PCR product were verified on agarose gels. For array preparation, nylon membranes (Qfilter; Genetix) were soaked in TE solution (10 mM Tris [pH 7], 1 mM EDTA [pH 7.6]). Spot blots of PCR products and controls were performed by a Qpix robot (Genetix). Following spot deposition, membranes were fixed for 15 min in 0.5 M NaOH-1.5 M NaCl, washed briefly in distilled water, and stored wet at −20°C until use.