e115 Nucleic Acids Research, 2005, Vol. 33, No. 13 which has been used also on commercial chips by Affymetrix. The valid oligonucleotides were deﬁned by the following conditions. They may include at most 12 A, 12 T, 10 C or 10 G nucleotides, and no window of 8 nt includes more than 6 A, 6 T, 4 C or 4 G nucleotides. Further, the oligonucleo- tides include at most 6 successive A, 6 successive T, 5 successive C or 5 successive G nucleotides. An inverse complementary oligonucleotide of an oligonucleotide can match at most six symbols from the beginning of an oligo- nucleotide. These criteria were used to avoid self-annealing, self-end annealing and to provide high enough Tm. The dis- tance threshold was four edit operations, i.e. no more than four errors were allowed. RESULTS AND DISCUSSION There is a great demand for functional oligonucleotides for a large spectrum of techniques. The oligonucleotides should be unique to allow speciﬁc and reliable binding. Genome-wide analyses are routine in many ﬁelds and therefore the probes PAGE 6 OF 16 utilized should not hybridize with any other genes or parts of genome. A method to determine, analyze and identify unique oligonucleotides from complete genomes was developed. The method was applied to the analysis of 17 complete geno- mes (Table 1). The Archaeoglobus fulgidus and Sulfolobus solfataricus represented Archae, Aquifex aeolicus and Ther- motoga maritima hyperthermophilic bacteria, Escherichia coli, Salmonella typhimurium and Buchnera sp. for Pro- bacteria gamma subdivision, Agrobacterium tumefaciens for alpha subdivision, Neisseria meningitidis for beta subdivision and Helicobacter pylori for delta/epsilon subdivision. Of the Firmicutes included were Bacillus subtilis and Clostridium acetobutylicum, and of Actinobacteria, Mycobacterium tuberculosis was included. Borrelia burgdorferi exempliﬁed Spirochete. The Eukaryotes included were Caenorhabditis elegans, a nematode, Saccharomyces cerevisiae, baker’s yeast for fungi, and Arabidopsis thaliana for plants. The genomes contained 564–25 694 genes and spanned 0.6–117 Mb. Some general properties of the genomes and oligonucleotides are in Tables 1 and 2. The organisms are Figure 5. Distribution of oligonucleotides in different sections of genes for (A) unique oligos in CDS regions and (B) valid oligos on genome. The ratio of (C) unique versus invalid oligos in coding regions and (D) valid versus invalid oligos in genome data.