ROSO: optimizing oligonucleotide probes for microarrays

ROSO is software to design optimal oligonucleotide probe sets for microarrays. Selected probes show no significant cross-hybridization, no stable secondary structures and their Tm are chosen to minimize the Tm variability of the probe set. AVAILABILITY: The program is available on the internet. Sources are freely available, for non-profit use, on request to the authors. Supplementary information: http://pbil.univ-lyon1.fr/roso     

PROBFIND: a computer program for selecting oligonucleotide probes from peptide sequences.

Synthetic oligonucleotides have proven to be extremely useful probes for screening cDNA and genomic libraries. Selection of the appropriate probe can be more easily and accurately achieved with the use of the computer program PROBFIND. The user enters the amino acid sequence from a file or from the keyboard, selects the minimum length allowed for the probe and the maximum allowable degeneracy. The computer prints a list of the sequences of potential probes which meet these minimum specifications and the location of the corresponding sequence in the protein to the screen and to a file. The user may modify the specifications for length and degeneracy at any time during the output of data, which allows for rapid selection of the desired probe. The program is interactive, accepts any file format with only a single modification of the file, is written in BASIC, and requires less than 6 kbytes of memory. This makes the program easy to use and adaptable even to unsophisticated microcomputers.     

MProbe: computer aided probe design for oligonucleotide microarrays

The present work describes a complete probe design software system for oligonucleotide microarrays based on Kane's research on probe sensitivity and specificity (Kane's rule). Combining Kane's rule and traditional criteria for probe design we constructed MProbe, the software system for oligonucleotide microarrays using Java. The general criteria for probe design are: (1) probes may have different lengths that range from 20 to 100 bases; (2) they should have a similar melting temperature (Tm) or GC content; (3) they should not contain stable secondary structures; and (4) they abide by Kane's rule.     

DNAPROBE, a computer program which generates oligonucleotide probes from protein alignments.

We describe a program to assist in designing oligonucleotide probes on the basis of protein alignments and the codon usage of the target organism. If necessary, the input sequences can be weighted to neutralise the effect of closely similar sequences or to bias the output in favour of a particular taxon.     

BIGPROBE: a computer program that predicts the sequence of long oligonucleotide probes with high reliability

We have written a computer program, BIGPROBE, which facilitates the design of long nucleic acid probes from the partial or complete amino acid sequence of a protein. BIGPROBE relies upon information on codon usage, intercodon dinucleotide frequency, and potential probe self-complementarity. We have examined the accuracy with which the program predicts coding sequences using sample human and rat genes and probe lengths of 30-60 nucleotides. Rat probe sequences selected by BIGPROBE using either codon usage or dinucleotide frequency data alone averaged 86-92% homology with the known exons of the corresponding gene sequences. Predictive accuracy with rat gene probes could be improved to 89-94%, depending upon probe length, by applying codon usage and dinucleotide frequency data in combination. Similar accuracy was achieved for human genes.     

Fabrication of DNA microarrays using unmodified oligonucleotide probes

Microarrays printed on glass slides are often constructed by covalently linking oligonucleotide probes to a derivatized surface. These procedures typically require relatively expensive amine- or thiol-modified oligonucleotide probes that add considerable expense to larger arrays. We describe a system by which unmodified oligonucleotide probes are bound to either nonderivatized or epoxy-silane-derivatized glass slides. Biotinylated PCR products are heat denatured, hybridized to the arrays, and detected using an enzymatic amplification system. Unmodified probes appear to detach from the slide surface at high pH (> 10.0), suggesting that hydrogen bonding plays a significant role in probe attachment. Regardless of surface preparation, high temperature (up to 65 degrees C) and low ionic strength (deionized water) do not disturb probe attachment; hence, the fabrication method described here is suitable for a wide range of hybridization stringencies and conditions. We illustrate kinetics of room temperature hybridizations for probes attached to nonderivatized slides, and we demonstrate that unmodified probes produce hybridization signals equal to amine-modified, covalently bound probes. Our method provides a cost-effective alternative to conventional attachment strategies that is particularly suitable for genotyping PCR products with nucleic acid microarrays.     

A computer program for the design of optimal synthetic oligonucleotide probes for protein coding genes

A computer program has been written in FORTRAN 77 to locateon a protein sequence a region with optimum length and limiteddegeneracy in order to design artificial oligonucleotide probesfor use in molecular cloning. In addition the program checksfor regions of homology between this probe and any other basesequence found in nucleotide sequence data banks. There areoptions in the program to eliminate rare codons or to make preferentialchoices of bases in order to minimize the degeneracy of probes. Received on February 17, 1987; accepted on June 26, 1987     

Specific and nonspecific hybridization of oligonucleotide probes on microarrays

Gene expression analysis by means of microarrays is based on the sequence-specific binding of RNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving sequences other than the intended target is problematic because it adds a chemical background to the signal, which is not related to the expression degree of the target gene. The article presents a molecular signature of specific and nonspecific hybridization with potential consequences for gene expression analysis. We analyzed the signal intensities of perfect match (PM) and mismatch (MM) probes of GeneChip microarrays to specify the effect of specific and nonspecific hybridization. We found that these events give rise to different relations between the PM and MM intensities as function of the middle base of the PM, namely a triplet-like (C > G approximately T > A > 0) and a duplet-like (C approximately T > 0 > G approximately A) pattern of the PM-MM log-intensity difference upon binding of specific and nonspecific RNA fragments, respectively. The systematic behavior of the intensity difference can be rationalized on the level of basepairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Nonspecific binding is characterized by the reversal of the central Watson-Crick (WC) pairing for each PM/MM probe pair, whereas specific binding refers to the combination of a WC and a self-complementary (SC) pairing in PM and MM probes, respectively. The Gibbs free energy contribution of WC pairs to duplex stability is asymmetric for purines and pyrimidines of the PM and decreases according to C > G approximately T > A. SC pairings on the average only weakly contribute to duplex stability. The intensity of complementary MM introduces a systematic source of variation which decreases the precision of expression measures based on the MM intensities.     

The application of oligonucleotide probes and microarrays for the identification of freshwater diatoms

Diatoms are major contributors to global carbon fixation and constitute a significant portion of biofilms found in lotic ecosystems. Despite their widespread abundance and the fact that extensive studies have been performed on morphological features of frustules, molecular tools for the identification of diatoms are not commonly available. This study focuses on the development of oligonucleotide probes for the detection of diatom species relevant to water quality assessment. The selected panel of diatoms covers all the species found in water of varying quality from the rivers of central-East Apennine (Italy). Small subunit rRNA-targeted probes were applied to a microarray platform as well as to a new technique termed Primer–Probe, with the aim of obtaining a molecular tool suitable for accurate identification of both single and mixed species diatom populations. The Primer–Probe technique together with dot-blot assays proved to be ideal for the preliminary screening of a large set of DNA oligonucleotides designed by ARB software. It was shown that microarrays, as a promising technology for rapid and simultaneous detection of a wide range of species-specific genetic markers, can be adapted to monitor changes within a diatom community. It is suggested that microarrays will provide a molecular basis for microbial identification to support standard microscopy techniques used by ecologists and environmental scientists for monitoring water quality.     

OligoArray: genome-scale oligonucleotide design for microarrays

SUMMARY: OligoArray is a program that computes gene specific and secondary structure free oligonucleotides for genome-scale oligonucleotide microarray construction or other applications. AVAILABILITY: The program code is distributed under the GNU General Public License and is freely available for non-profit use via request from the authors.     

High-resolution genotyping via whole genome hybridizations to microarrays containing long oligonucleotide probes

To date, microarray-based genotyping of large, complex plant genomes has been complicated by the need to perform genome complexity reduction to obtain sufficiently strong hybridization signals. Genome complexity reduction techniques are, however, tedious and can introduce unwanted variables into genotyping assays. Here, we report a microarray-based genotyping technology for complex genomes (such as the 2.3 GB maize genome) that does not require genome complexity reduction prior to hybridization. Approximately 200,000 long oligonucleotide probes were identified as being polymorphic between the inbred parents of a mapping population and used to genotype two recombinant inbred lines. While multiple hybridization replicates provided ～97% accuracy, even a single replicate provided ～95% accuracy. Genotyping accuracy was further increased to >99% by utilizing information from adjacent probes. This microarray-based method provides a simple, high-density genotyping approach for large, complex genomes.     

Design and synthesis of versatile ganglioside probes for carbohydrate microarrays

A series of ganglioside GM1-, GM2-, and GM3-type probes, in which the ceramide portion is replaced with a glucose residue, were systematically synthesized based on a convergent synthetic method.     

Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays

Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis.     

PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database

We describe PRIMROSE, a computer program for identifying 16S rRNA probes and PCR primers for use as phylogenetic and ecological tools in the identification and enumeration of bacteria. PRIMROSE is designed to use data from the Ribosomal Database Project (RDP) to find potentially useful oligonucleotides with up to two degenerate positions. The taxonomic range of these, and other existing oligonucleotides, can then be explored, allowing for the rapid identification of suitable oligonucleotides. PRIMROSE includes features to allow user-defined sequence databases to be used. An in silico trial of the program using the RDP database identified oligonucleotides that described their target taxa with a degree of accuracy far greater than that of equivalent currently used oligonucleotides. We identify oligonucleotides for subdivisions of the Proteobacteria and for the Cytophaga–Flexibacter–Bacteroides (CFB) division. These oligonucleotides describe up to 94.7% of their target taxon with fewer than 50 non-target hits, and the authors recommend that they be investigated further. A comparison with PROBE DESIGN within the ARB software package shows that PRIMROSE is capable of identifying oligonucleotides with a higher specificity. PRIMROSE has an intuitive graphical user interface and runs on the Microsoft Windows 95/NT/2000 operating systems. It is open source and is freely available from the authors.     

Selection of optimal oligonucleotide probes for microarrays using multiple criteria, global alignment and parameter estimation

The oligonucleotide specificity for microarray hybridization can be predicted by its sequence identity to non-targets, continuous stretch to non-targets, and/or binding free energy to non-targets. Most currently available programs only use one or two of these criteria, which may choose ‘false’ specific oligonucleotides or miss ‘true’ optimal probes in a considerable proportion. We have developed a software tool, called CommOligo using new algorithms and all three criteria for selection of optimal oligonucleotide probes. A series of filters, including sequence identity, free energy, continuous stretch, GC content, self-annealing, distance to the 3′-untranslated region (3′-UTR) and melting temperature (T_m), are used to check each possible oligonucleotide. A sequence identity is calculated based on gapped global alignments. A traversal algorithm is used to generate alignments for free energy calculation. The optimal T_m interval is determined based on probe candidates that have passed all other filters. Final probes are picked using a combination of user-configurable piece-wise linear functions and an iterative process. The thresholds for identity, stretch and free energy filters are automatically determined from experimental data by an accessory software tool, CommOligo_PE (CommOligo Parameter Estimator). The program was used to design probes for both whole-genome and highly homologous sequence data. CommOligo and CommOligo_PE are freely available to academic users upon request.     

Design of long oligonucleotide probes for functional gene detection in a microbial community

MOTIVATION: Analysis of the functions of microorganisms and their dynamics in the environment is essential for understanding microbial ecology. For analysis of highly similar sequences of a functional gene family using microarrays, the previous long oligonucleotide probe design strategies have not been useful in generating probes. RESULTS: We developed a Hierarchical Probe Design (HPD) program that designs both sequence-specific probes and hierarchical cluster-specific probes from sequences of a conserved functional gene based on the clustering tree of the genes, specifically for analyses of functional gene diversity in environmental samples. HPD was tested on datasets for the nirS and pmoA genes. Our results showed that HPD generated more sequence-specific probes than several popular oligonucleotide design programs. With a combination of sequence-specific and cluster-specific probes, HPD generated a probe set covering all the sequences of each test set. AVAILABILITY: http://brcapp.kribb.re.kr/HPD/     

Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 5'-end-labeled oligonucleotide probes and nick-translated plasmid probes

Terminal deoxynucleotidyl transferase was used to add labeled dAMP residues to the 3' end of oligonucleotide probes that hybridize to the 5' end of the neomycin phosphotransferase II gene. Southern hybridization conditions were described in which the sensitivity per unit of exposure time was about 30-fold greater for the tailed probe as compared to the 5'-end-labeled probe. The tailed oligonucleotide probe had the sensitivity per unit of exposure time comparable to that of a nick-translated probe of high specific activity: in 3 h of autoradiographic exposure both easily detected an amount of target equivalent to a single-copy gene in 10 micrograms of human DNA. The thermal dissociation profiles of 5'-end-labeled and tailed oligonucleotide probes were virtually identical and the tailed oligonucleotide probe was as allele specific as the 5'-end-labeled oligonucleotide probe. The useful lifetime of a 32P-tailed probe was about 1-2 weeks. Finally, by adding 50 35S-labeled nucleotides to the 3' end, we prepared a stable oligonucleotide probe with a sensitivity per unit of exposure time comparable to that of the unstable 5'-32P-labeled oligonucleotide probe.     

A computer program for selection of oligonucleotide primers for polymerase chain reactions.

We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest.