title: Complementary DNA arrays produced with PCR fragments of 6116 ORFs of Saccharomyces cerevisiae
status: Public on Jun 21 2007
contact.city: Heidelberg
contact.first: Ana
contact.last: Kitanovic
contact.email: Ana.Kitanovic@urz.uni-heidelberg.de
contact.address: Im Neuenheimer Feld 364
contact.department: Biology
contact.country: Germany
contact.institute: Institute for Pharmacy and Molecular Biotechnology
contact.phone: ++49 6221 546460
organism: Saccharomyces cerevisiae
manufacture_protocol: Open reading frame PCR-amplification:
  A set of 12,232 PCR primers was commercially
  obtained (Research Genetics, USA). The DNA
  was transferred into microtitre dishes and simultaneously
  diluted to a concentration of 4 ìM using
  a Biomek-2000 robot (Beckman, USA). The oligomer
  sequences were designed to bind close to
  either end of the open reading frames (ORFs).
  They were about 45 nucleotides in length, of which
  two common tag-sequences accounted for 20 and
  22 nucleotides at the 5*-end, respectively [forward
  primers: d(GGAATTCCAGCTGACCACCATG);
  reverse primers: d(GATCCCCGGGAATTGCC
  ATG)]. PCR was done in microtitre dishes using
  PTC-200 cyclers (MJ Research, USA). For the
  amplification of the most 3*-located region of genes,
  a second primer set was designed according to the
  specifications of the above set but binding about
  500 bp upstream of the respective 3* end. The oligonucleotides were obtained from Interactiva
  (Germany).
  First-round PCR was done on a template of
  25 ng genomic DNA in a total volume of 50 ìl
  of 10 mM-Tris–HCl, pH 8·8, 50 mM-KCl, 1·5 mMMgCl2,
  0·2 mM of each nucleotide triphosphate,
  a mixture of 1 unit of Taq-polymerase and 0·25
  unit Pfu-polymerase (Stratagene, USA) and both
  primers at a concentration of 0·4 ìM each. Initial
  denaturation was at 95)C for 3 min, followed by 35
  cycles of 1 min at 95)C, 45 s at 50)C and 3·5 min at
  72)C; post-treatment at 72)C was for 10 min.
  For reactions that failed to yield product under
  the above conditions, the Expand= High Fidelity
  System (Boehringer Mannheim, Germany) was
  used in a second amplification reaction on 25 ng
  genomic template DNA under the conditions recommended
  by the manufacturer. After 2 min denaturation
  at 94)C, there were 10 cycles of 15 s at
  94)C, 30 s at 50)C and 4 min at 68)C, followed by
  another 26 cycles during which the period at 68)C
  was extended by 20 s per cycle. Again, there was a
  final incubation at 72)C to fill-in ends.
  Array production:
  Initially, the PCR-products were transferred to
  positively charged filters (Pall Biodyne B; DuPont,
  USA) using 96- or 384-pin gadgets mounted to a
  BioGrid spotting device (BioRobotics, UK). Flatbottomed
  pins of 0·7 mm diameter transferred
  about 60 nl of liquid per spotting event, while only
  15 nl was spotted with pins of 0·4 mm. PCRproducts
  were placed repeatedly onto the same
  position in order to accumulate DNA. During
  production, filters were kept humid by being
  placed onto Whatman 3MM paper soaked in
  denaturing solution (1·5 M-NaCl, 0·5 M-NaOH).
  For the production of higher-density grids, a
  special pin tool (BioRobotics, UK) was used that
  produced arrays of densities of up to 750 spots
  per square centimetre. More recently, DNA was
  placed onto the surface by a device equipped with
  an eight-channel array of piezo printing elements
  (GeSim, Germany). By this technique, samples
  could be sprayed in multiples of 15 picolitres onto
  solid support at a grid density of up to 10,000 per
  centimetre square.