Nucleic Acids Research, 2005, Vol. 33, No. 13 e115 doi:10.1093/nar/gni110 Genome-wide selection of unique and valid oligonucleotides Heikki Hyyro¨ , Martti Juhola and Mauno Vihinen1,2,* Department of Computer Sciences and 1Institute of Medical Technology, FI-33014 University of Tampere, Finland and 2Research Unit, Tampere University Hospital, FI-33520 Tampere, Finland Received May 4, 2005; Revised June 1, 2005; Accepted June 22, 2005 ABSTRACT Functional genomics methods are used to investigate the huge amount of information contained in gen- omes. Numerous experimental methods rely on the use of oligo- or polynucleotides. Nucleotide strand hybridization forms the underlying principle for these methods. For all these techniques, the probes should be unique for analyzed genes. In addition to being unique for the studied genes, the probes should fulfill a large number of criteria to be usable and valid. The criteria include for example, avoid- ance of self-annealing, suitable melting temperature and nucleotide composition. We developed a method for searching unique and valid oligonucleotides or probes for genes so that there is not even a similar (approximate) occurrence in any other location of the whole genome. By using probe size 25, we ana- lyzed 17 complete genomes representing a wide range of both prokaryotic and eukaryotic organisms. More than 92% of all the genes in the investigated genomes contained valid oligonucleotides. Extens- ive statistical tests were performed to characterize the properties of unique and valid oligonucleotides. Unique and valid oligonucleotides were relatively evenly distributed in genes except for the beginning and end, which were somewhat overrepresented. The flanking regions in eukaryotes were clearly underrepresented among suitable oligonucleotides. In addition to distributions within genes, the effects on codon and amino acid usage were also studied. INTRODUCTION The complete genome of a large number of organisms includ- ing bacteria, archaea and eukaryotes have been determined along with the human genome. Currently, there are some 230 bacterial and archaeal, and 34 ﬁnished eukaryal genomes. Genomes contain overwhelming amount of information, which can be investigated with numerous experimental and computational techniques. Many experimental methods rely on the use of oligo- or polynucleotides. Nucleotide strand hybridization—preferably with unique probes—forms the underlying principle for these methods. PCR technology, the workhorse of molecular biology, utilizes oligonucleotides as primers to copy and amplify genetic material. Gene expres- sion studies such as Southern and northern blotting and more advanced SAGE and microarrays also utilize oligonucleotides. Gene function can be modulated by short oligonucleotides either by antisense technology or by RNA interference (RNAi). For all these techniques, the probes ought to be unique for analyzed genes. Oligonucleotides for an organism can be identiﬁed from complete genomes. If working with mRNA only, genes and ﬂanking regions have to be analyzed, whereas if genomic DNA is the target, the probes should be unique for the whole genome. In addition to being unique for the studied genes, the probes have to fulﬁll a large number of criteria, which vary due to the use of probes. These criteria include, for example, the avoidance of self-annealing, suitable melting temperature (Tm) and nucleotide composition. A number of methods have been developed for primer design [e.g. (1–13), http://www-genome. wi.mit.edu/genome_software/other/primer3.html; for a review see (14)]. MEDUSA shows visually the location of the primer pairs (15). Simulated annealing and Lagrangian relaxation algorithms have been used to design oligonucleotides to study microbial communities (16). Organisms can be identiﬁed with proper oligos (17). Methods for oligonucleotide selection and probe production for microarrays have also been developed (18–31). Numerous methods have been developed to predict antisense oligonucleotides (32–36) and RNAi (37–41). Probes for full gene synthesis have to be specially designed (42). Oligos can be designed also for protein interaction studies (43). When degenerate oligonucleotides are used for cloning ortho- logues and paralogues, primers have to be specially designed. The properties of genome-wide unique oligonucleotide studies have not been published, although some methods for the search of such strings have been presented (44). *To whom correspondence should be addressed. Tel: +358 3 35517735; Fax: +358 3 35517710; Email: mauno.vihinen@uta.fi The Author 2005. Published by Oxford University Press. 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