title: UR_TWT_version 1.0
status: Public on Dec 02 2005
contact.city: Marseille
contact.first: nicolas
contact.last: crapoulet
contact.email: nicolas.crapoulet@medecine.univ-mrs.fr
contact.address: 27 BD J. Moulin
contact.country: France
contact.institute: Unité des Rickettsies (CNRS-UMR 6020)
contact.phone: 33 (0)4 91 32 43 75
organism: Tropheryma whipplei
manufacture_protocol: T. whipplei whole-genome microarray construction. 1,608 PCR primers were designed in order to amplify by PCR 804 annoted ORFs representing about 99.5% of T. whipplei genome {Raoult, 2003 26 /id}. The 0.5% remaining genes can not be targeted because their sequences share repeated regions impairing the design of specific probes.
  The following criteria were used to identify optimal forward and reverse primers to generate PCR products specific to each of the selected ORFs: (i) primers were highly specific for T. whipplei genome; (ii) length of amplicons were roughly 260 bp; (iii) amplicons were highly specific for their corresponding genes; (iv) each primer contained 20 to 25 bases and annealing temperature of primers was ~60°C; (v) each primer contained an additional sequence in 5’-area specific for the forward and the reverse primer. These universal sequences added were used for a second round of amplification. The sequences of primers used are listed in Table S1.
  ORF-specific fragments were amplified using T. whipplei DNA as template and under following conditions: 40 cycles including 20 sec. denaturation at 95°C, 30 sec. annealing at 60°C, and 30 sec. extension at 72°C, along with an initial 5 min. denaturation at 95°C and a final extension reaction at 72°C for 5 min. Following amplification, PCR products were visualized by ethidium bromide staining after migration in a 2% agarose gel. Purified amplicons (QIAquick 96-well purification kit ; QIAGEN, Courtaboeuf, France) were sequenced (3100 Genetic Analyzer, Applied Biosystems, Courtaboeuf, France) to check for the identity of amplified samples and processed in a second PCR run by using universal primers.
  Amplified DNA fragments resuspended in 50% DMSO solution were printed in quadruplicate onto Nexterion® Slide A+ (SCHOTT AG, Mainz, Germany) by using a SDDC-2 (Bio-Rad, Marnes-la-Coquette, France). A set of 23 artificial genes that serve as analytical controls to validate, filter, and normalize microarray data contained in Universal ScoreCard™ kit (AMERSHAM BIOSCIENCES, Orsay, France) was also printed onto glass slides. The spotted slides were crosslinked by using a UV Stratalinker (Stratagene, La Jolla, CA, USA) each at a total energy of 250 mJ, and then were stored in a dry environment at room temperature.