title: Streptococcus pyogenes M1, M3, M18 GAS 2.3k array (condensed version)
status: Public on Sep 11 2007
contact.city: Atlanta
contact.first: Samantha 
contact.middle: Ann
contact.last: Roberts
contact.email: sasmit5@emory.edu
contact.state: GA
contact.address: 1510 Clifton Road
contact.department: Microbiology
contact.country: USA
contact.institute: Emory University
contact.phone: 404-727-0522
organism: Streptococcus pyogenes
description: oligonucleotide array containing unique ORFs from M1, M3 and M18 Streptococcus pyogenes.
  
  The Platform data table reflects a condensed representation of the oligo probes which were printed in triplicate. The full array layout (GPL5822_full_layout.txt) is linked as a supplementary file at the foot of this record.
manufacture_protocol: Probes were designed utilizing a proprietary algorithm to target unique non-repetitive ORFs (TIGR annotation as of 2 December, 2002) found in the sequenced genomes of serotypes M1 (SF370), M3 (MGAS315) and M18 (MGAS8232) and resulted in 2328 oligonucleotide probes (1932-M1, 165-M3, 231-M18). As the TIGR ORFs have been updated, all probes are referenced using their original Spy designation whenever possible (University of Oklahoma Sequencing Project). Oligonucleotide 70-mer probes were synthesized on a 0.2 μM scale and passed through several stringent parameters before synthesis, including a melting temperature of 76 ± 5°C, ≤ 70% cross hybridization identity to another gene within the same strain, ≤ 20 contiguous bases in common with another gene, probe location within 3' end of ORF (within 40 bp if possible), and hairpin/stem loop structures ≤ 8.
  Lyophilized oligonucleotides in 384 well plates were resuspended in sodium phosphate at a final concentration of 40 μM. Approximately 10 picolitres of each oligonucleotide probe was spotted onto slides (UltraGAPS2; Corning) using a 12 pin contact printer. Printed slides were held over steaming water for 5 s followed by drying on a heat block (70°C) for 5 s. Slides were then cross-linked using a Stratalinker (Stratagene) at 65 mJ. To reduce background, slides were post-processed in a blocking solution (0.16 M Succinic Anhydride in 1-methyl-2-pyrrolidinone followed by the addition of 0.2 M Boric Acid pH 8.0 to 0.02 M) with shaking for 15–20 min. Slides were subsequently rinsed in 95°C water for 2 min followed by a 95% ethanol wash and dried using a table top centrifuge with a microtiter plate carrier. Post-processed slides were stored in a slide storage box protected from light at ambient temperature.