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.     

EMILiO: a fast algorithm for genome-scale strain design

Systems-level design of cell metabolism is becoming increasingly important for renewable production of fuels, chemicals, and drugs. Computational models are improving in the accuracy and scope of predictions, but are also growing in complexity. Consequently, efficient and scalable algorithms are increasingly important for strain design. Previous algorithms helped to consolidate the utility of computational modeling in this field. To meet intensifying demands for high-performance strains, both the number and variety of genetic manipulations involved in strain construction are increasing. Existing algorithms have experienced combinatorial increases in computational complexity when applied toward the design of such complex strains. Here, we present EMILiO, a new algorithm that increases the scope of strain design to include reactions with individually optimized fluxes. Unlike existing approaches that would experience an explosion in complexity to solve this problem, we efficiently generated numerous alternate strain designs producing succinate, l-glutamate and l-serine. This was enabled by successive linear programming, a technique new to the area of computational strain design.     

QuickLIMS: facilitating the data management for DNA-microarray fabrication

SUMMARY: QuickLIMS is a Microsoft Access-based laboratory information and management system, capable of processing all information for microarray production. The program's operational flow is protocol-based, dynamically adapting to changes of the process. It interacts with the laboratory robot and with other database systems over the network, and it represents a complete solution for the management of the entire manufacturing process. AVAILABILITY AND SUPPLEMENTARY INFORMATION: http://www.dkfz-heidelberg.de/kompl_genome/Other/QuickLims/index.html     

MPprimer: a program for reliable multiplex PCR primer design

Background  

Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility.     

PrimerIdent: A web based tool for conserved primer design

Conserved primers across multiple species and simultaneously specific for a certain isozyme can be rare and difficult to find. PrimerIdent was developed aiming to automate this primer design and selection process in a given nucleotide sequence alignment, providing an intuitive, easy to interpret graphical result, which offers a list of all possible primers that meet the user criteria, with a colour-code identity to each sequence in the alignment. The software here presented is a simple and intuitive web based tool that is suitable for distinguishing very similar nucleotide sequences, such as isozymes-coding sequences, to enable the conserved primer design across multiple species, necessary for approaches that rely on knowing if a primer is suitable for a certain set of pre-aligned sequences, to design a specific primer to a certain sequence variation, or a combination thereof. This extremely useful software can, therefore, be used as a tool for the specific amplification of individual members of multigenic families across related species and also to evaluate the differential expression of isogenes for a given species. AVAILABILITY: http://primerident.up.pt.     

UniPrime: a workflow-based platform for improved universal primer design

UniPrime is an open-source software (http://uniprime.batlab.eu), which automatically designs large sets of universal primers by simply inputting a gene ID reference. UniPrime automatically retrieves and aligns homologous sequences from GenBank, identifies regions of conservation within the alignment and generates suitable primers that can amplify variable genomic regions. UniPrime differs from previous automatic primer design programs in that all steps of primer design are automated, saved and are phylogenetically limited. We have experimentally verified the efficiency and success of this program by amplifying and sequencing four diverse genes (AOF2, EFEMP1, LRP6 and OAZ1) across multiple Orders of mammals. UniPrime is an experimentally validated, fully automated program that generates successful cross-species primers that take into account the biological aspects of the PCR.     

Efficient primer design algorithms

MOTIVATION: Primer design involves various parameters such as string-based alignment scores, melting temperature, primer length and GC content. This entails a design approach from multicriteria decision making. Values of some of the criteria are easy to compute while others require intense calculations. RESULTS: The reference point method was found to be tractable for trading-off between deviations from ideal values of all the criteria. Some criteria computations are based on dynamic programs with value iteration whose run time can be bounded by a low-degree polynomial. For designing standard PCR primers, the scheme offers in a relative gain in computing speed of up to 50: 1 over ad-hoc computational methods. Single PCR primer pairs have been used as model systems in order to simplify the quantization of the computational acceleration factors. The program has been structured so as to facilitate the analysis of large numbers of primer pairs with minor modifications. The scheme significantly increases primer design throughput which in turn facilitates the use of oligonucleotides in a wide range of applications including: multiplex PCR and other nucleic acid-based amplification systems, as well as in zip code targeting, oligonucleotide microarrays and nucleic acid-based nanoengineering.     

DNA-microarray analysis of brain cancer: molecular classification for therapy

Primary brain tumours are among the most lethal of all cancers, largely as a result of their lack of responsiveness to current therapy. Numerous new therapies hold great promise for the treatment of patients with brain cancer, but the main challenge is to determine which treatment is most likely to benefit an individual patient. DNA-microarray-based technologies, which allow simultaneous analysis of expression of thousands of genes, have already begun to uncover previously unrecognized patient subsets that differ in their survival. Here, we review the progress made so far in using DNA microarrays to optimize brain cancer therapy.     

D-MaPs - DNA-microarray projects: Web-based software for multi-platform microarray analysis

The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix). Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus) submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner). In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core.     

CloneAssistant 1.0: a stand-alone software for automated cloning primer design

"CloneAssistant 1.0" is a stand-alone software compatible with the current Windows operating systems, which can automatically design cloning primers with full consideration of the sequence information of vectors and genes, cloning strategies, the principles of primer design, reading frames, position effects, and enzymatic reaction conditions for users. Five internal XML (extensible markup language) databases [restriction enzymes, plasmids, universal buffers, PCR (polymerase chain reaction) protection bases, and an MCS (multiple cloning site) double digest interference database] were established to serve as the basic support for "CloneAssistant 1.0". The primer pairs designed are sorted according to the difficulty of the follow-up experiments. Once a primer pair is selected by the user, detailed experimental guidance for this primer pair will be provided. In addition, "CloneAssistant 1.0" can be used for restriction map analysis, ORF (open reading frame) finding, sequence alignment and complementary analysis, translation, restriction enzyme and universal buffer queries, and isocaudamer analysis. "CloneAssistant 1.0" makes gene clone design much easier, and it can be freely downloaded from http://bis.zju.edu.cn/clone.     

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences.     

Primer Prim'r: A web based server for automated primer design

The Northeast Structural Genomics Consortium (NESG) is one of nine NIH-funded pilot projects created to develop technologies needed for structural studies of proteins on a genome-wide scale. One of the most challenging aspects of this emerging field is the production of protein samples amenable to structural determination. To do this efficiently, all steps in the protein production pipeline must be automated. Here we describe the Primer program (linked from http://www-nmr.cabm.rutgers.edu/bioinformatics, www-nmr.cabm.rutgers.edu/bioinformatics, a web-based primer design program freely available to the scientific community, which was created to automate this time consuming and laborious task. This program has the ability to simultaneously calculate plasmid specific primer sets for multiple open reading frame (ORF) targets, including 96-well and greater formats. Primer includes a library of commonly used plasmid systems and possesses the ability to upload user-defined plasmid systems. In addition to calculating gene-specific annealing regions for each target, the program also adds appropriate restriction endonuclease recognition or viral recombination sites while preserving a reading frame with plasmid based fusions. Primer has several useful features such as sorting calculated primer sets by target size, facilitating interpretation of PCR amplifications by agarose gel electrophoresis, as well as supplying the molecular biologist with many important characteristics of each target such as the expected size of the PCR amplified DNA fragment and internal restriction sites. The NESG has cloned over 1500 genes using oligonucleotide primers designed by Primer.     

Web-based primer design for single nucleotide polymorphism analysis

The detection of single nucleotide polymorphisms by PCR is necessary for many types of genetic analysis, from mapping genomes to tracking specific mutations. This technique is most commonly used when polymorphisms alter restriction endonuclease recognition sites. Here we describe a web-based program, dCAPS Finder 2.0, that facilitates the design of mismatched PCR primers to create or remove a restriction endonuclease recognition site relative to the polymorphism being analyzed.     

Olga--oligonucleotide primer design program for the Atari ST

A program to facilitate the design of oligonucleotide primnershas been devised. Olga is written in draft ANSI standard ‘C’and makes use of the implementation of Digital Research GEM(Graphics Environment Manager) on the Atari ST Olga is specificallysuited to the polymnerase chain reaction (PCR) allowing si analysisof two primer sequences. The advantage of Olga is that it providesin one program analyses for direct repeats, secondary structuresand primer dimerization as well as several useful ‘finishing’tools for workers engaged in PCR optimization and oligonucleotidesyntheses.     

Homolog-specific PCR primer design for profiling splice variants

To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org.     

Specific primer design for the polymerase chain reaction

The design of primers has a major impact on the success of PCR in relation to the specificity and yield of the amplified product. Here, we introduce the applications of PCR as well as the definition and characteristics for PCR primer design. Recent primer design tools based on Primer3, along with several computational intelligence-based primer design methods which have been applied in primer design, are also reviewed. In addition, characteristics of population-based methods used in primer design are discussed in detail.     

Methylation-specific PCR: four steps in primer design

Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design.     

The degenerate primer design problem

A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur when studying a family of genes that is known only in part, or is known in a related species. We prove that various simplified versions of the problem are hard, show the polynomiality of some restricted cases, and develop approximation algorithms for one variant. Based on these algorithms, we implemented a program called HYDEN for designing highly-degenerate primers for a set of genomic sequences. We report on the success of the program in an experimental scheme for identifying all human olfactory receptor (OR) genes. In that project, HYDEN was used to design primers with degeneracies up to 10(10) that amplified with high specificity many novel genes of that family, tripling the number of OR genes known at the time.