The P. furiosus full-genome microarray represented 2065 protein encoding genes. DNA fragments for spotting were produced by PCR. The array was constructed by designing oligonucleotide primers to span the most unique portions of each gene, averaging 350 nucleotides in length. A custom Perl program was written to select primers with the most desirable thermodynamic conditions possible for each gene using the Whitehead Institute's Primer3 program (version 3.09) as its core analysis engine. The polymerase chain reaction was performed in 100 µl reactions with 1µM of each primer, 2mM MgCl2, 1.0 mM dNTPs, 1.75 U AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) 1x AmpliTaq buffer, and 20ng genomic DNA for 30 cycles (94°C 30 sec, 50°C 1 min, 72°C 2 min). All PCR products were checked by agarose gel electrophoresis and failures were repeated or new primers designed. Each PCR amplicon was spotted on poly-L-lysine coated glass slides twice, each time by a different print pin (print plates were rotated 180 degrees for second spotting) using a 16-pins ArrayMaker Version 1.0 (http://cmgm.stanford.edu/pbrown/mguide/index.html).
Less...
More...