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PubMed2025

AutoPVPrimer: A comprehensive AI-Enhanced pipeline for efficient plant virus primer design and assessment.

Plant viruses pose a significant threat to global agriculture and require efficient tools for their timely detection. We present AutoPVPrimer, an innovative pipeline that integrates artificial intelligence (AI) and machine learning to accelerate the development of plant virus primers. The pipeline uses Biopython to automatically retrieve different genomic sequences from the NCBI database to increase the robustness of the subsequent primer design. The design_primers_with_tuning module uses a random forest classifier that optimizes parameters and provides flexibility for different experimental conditions. Quality control measures, including the evaluation of poly-X content and melting temperature, increase primer reliability. Unique to AutoPVPrimer is the visualize_primer_dimer module, which supports the visual evaluation of primer dimers-a feature missing in other tools. Primer specificity is validated via primer BLAST, which contributes to the overall efficiency of the pipeline. AutoPVPrimer has been successfully applied to the tomato mosaic virus, proving its adaptability and efficiency. The modular design allows customization by the user and extends the applicability to different plant viruses and experimental scenarios. The pipeline represents a significant advance in primer design and provides researchers with an effective tool to accelerate molecular biology experiments. Future developments aim to extend compatibility and incorporate user feedback to consolidate AutoPVPrimer as an innovative contribution to the bioinformatics toolbox and a promising resource for the advancement of plant virology research.

DNA Primers↗
PubMed2001

Set of novel tools for PCR primer design.

We have developed a new package of computer programs and algorithms for different PCR applications, including allele-specific PCR, multiplex PCR, and long PCR. The package is included in the upcoming VectorNTI suite software and attempts to incorporate most of the current knowledge about PCR primer design. A wide range of primer characteristics is available for user manipulation to provide improved efficiency and increased flexibility of primer design. To accelerate the primer calculations, we have optimized algorithms using recent advances in computer science such as dynamic trees and lazy evaluation. Proper structural organization of input parameters provides further program acceleration. New Vector NTI primer design software allows calculations of primer pairs for long PCR amplification of 120-kb genomic DNA in 5 min under most stringent input parameters and clustering 435 primer pairs for multiplex PCR within 30 min on a standard Pentium III PC. Our program allows the user to take advantage of molecule annotation by applying different kinds of filtering features during PCR primer design.

Algorithms↗
PubMed2006

Recent developments in primer design for DNA polymorphism and mRNA profiling in higher plants.

Primer design is a critical step in the application of PCR-based technologies in gene expression and genetic diversity analysis. As more plant genomes have been sequenced in recent years, the emphasis of primer design strategy has shifted to genome-wide and high-throughput direction. This paper summarizes recent advances in primer design for profiling of DNA polymorphism and mRNA in higher plants, as well as new primer systems developed for animals that can be adapted for plants.

Journal Article↗
PubMed2006

Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA.

BACKGROUND: The amplification of variable regions of immunoglobulins has become a major challenge in the cloning of antibody genes, whether from hybridoma cell lines or splenic B cells. Using conventional protocols, the heavy-chain variable region genes often are not amplified successfully from the hybridoma cell lines. RESULTS: A novel method was developed to design the degenerated primer of immunoglobulin cDNA and to amplify cDNA ends rapidly. Polymerase chain reaction protocols were performed to recognize the VH gene from the hybridoma cell line. The most highly conserved region in the middle of the VH regions of the Ig cDNA was identified, and a degenerated 5'primer was designed, using our algorithms. The VH gene was amplified by both the 3'RACE and 5'RACE. The VH sequence of CSA cells was 399 bp. CONCLUSION: The new protocol rescued the amplifications of the VH gene that had failed under conventional protocols. In addition, there was a notable increase in amplification specificity. Moreover, the algorithm improved the primer design efficiency and was shown to be useful both for building VH and VL gene libraries and for the cloning of unknown genes in gene families.

Cell Line↗
PubMed2005

Applying instructional design theories to bioinformatics education in microarray analysis and primer design workshops.

The need to support bioinformatics training has been widely recognized by scientists, industry, and government institutions. However, the discussion of instructional methods for teaching bioinformatics is only beginning. Here we report on a systematic attempt to design two bioinformatics workshops for graduate biology students on the basis of Gagne's Conditions of Learning instructional design theory. This theory, although first published in the early 1970s, is still fundamental in instructional design and instructional technology. First, top-level as well as prerequisite learning objectives for a microarray analysis workshop and a primer design workshop were defined. Then a hierarchy of objectives for each workshop was created. Hands-on tutorials were designed to meet these objectives. Finally, events of learning proposed by Gagne's theory were incorporated into the hands-on tutorials. The resultant manuals were tested on a small number of trainees, revised, and applied in 1-day bioinformatics workshops. Based on this experience and on observations made during the workshops, we conclude that Gagne's Conditions of Learning instructional design theory provides a useful framework for developing bioinformatics training, but may not be optimal as a method for teaching it.

Computational Biology↗
PubMed2002

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.

Algorithms↗
PubMed2004

SNPbox: a modular software package for large-scale primer design.

UNLABELLED: We developed a modular software package SNPbox that automates and standardizes the generation of PCR primers and is used in the strategy for constructing single nucleotide polymorphisms (SNPs) maps. In this strategy, the focus of primer design can be either on the validation of annotated public SNPs or on the SNP discovery in exon regions or extended genomic regions, both by resequencing. SNPbox relies on Primer3 for the primer design and combines this program with other publicly available software tools such as BLAST, Spidey and RepeatMasker, and newly developed algorithms. Primer conditions were chosen such that PCR amplifications are uniform for each PCR amplicon facilitating the use of high-throughput genetic platforms. SNPbox can also be used for the design of primer sets for mutation analysis, STR marker genotyping and microarray oligo design. Of the 2500 primer sets designed by SNPbox, 95% successfully amplified genomic DNA under uniform PCR conditions. AVAILABILITY: The software is available from the authors upon request. SUPPLEMENTARY INFORMATION: SNPbox_supplement.

Algorithms↗
PubMed2003

DePIE: Designing Primers for Protein Interaction Experiments.

Several primer prediction and analysis programs have been developed for diverse applications. However, none of these existing programs can be directly used for the design of primers in protein interaction experiments, since proteins may have transmembrane domains (TMDs) and/or a signal peptide that must be excluded from experiments. Furthermore, it is frequently the case that a short restriction sequences must be added to each primer in order to clone PCR products into a given destination vectors for expression. DePIE, a web-based primer design tool, was developed to address these deficiencies. The program takes as input NCBI protein accession numbers and returns primer information including nucleotide sequences, thermodynamic melting temperature of the nucleotide sequences and the target positions. DePIE is implemented in JAVA, PERL and PHP and has proven to be very efficient in designing primers for our interaction experiments. DePIE services can be accessed at the web site: http://biocore.unl.edu/primer/primerPI.html.

DNA Primers↗
PubMed2005

Oligonucleotide properties determination and primer designing: a critical examination of predictions.

MOTIVATION: Precise prediction of melting temperature (T(m)), secondary structures and design of oligonucleotides determine the efficiency and success of experimentation in molecular biology. Availability of a plethora of software and the users unawareness about their limitations compromises the accuracy and reliability of the predictions. RESULTS: Comparative analysis of 56 modules was done for T(m) prediction using a large set of oligonucleotide sequences spanning the whole range of GC-content and length. Allawi module of the calculator 'MELTING', Nearest Neighbor (NN) of oligo calculator (McLab), NN of T(m) Calculation for Oligos (Biomath Calculator, Promega) and HYTHER provided the most precise T(m) predictions. A model has also been proposed to calculate the optimum annealing temperature integrating the already reported formulations. Secondary structure predictions of oligonucleotides reveal a large number of structures in contrast to the experimental observations. Of the 11 primer designing tools evaluated, Primer 3 and WebPrimer performed the best for the AT-rich templates, Exon Primer for AT = GC templates, and Primer Design Assistant, Primer3 and Primer Quest for GC-rich templates. This study provides optimal choice for application to the user, increasing the success of a variety of experimentations, especially those that have high-throughput and complex assay designs. CONTACT: db@igib.res.in SUPPLEMENTARY INFORMATION: The details of the oligonucleotides and of the different modules of T(m) prediction considered for the study are provided as Supplementary Information, available at Bioinformatics online.

Computer Simulation↗
PubMed2004

Primer Prim'er: 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.

Computer-Aided Design↗
PubMed2001

GST-PRIME: a genome-wide primer design software for the generation of gene sequence tags.

The availability of sequenced genomes has generated a need for experimental approaches that allow the simultaneous analysis of large, or even complete, sets of genes. To facilitate such analyses, we have developed GST-PRIME, a software package for retrieving and assembling gene sequences, even from complex genomes, using the NCBI public database, and then designing sets of primer pairs for use in gene amplification. Primers were designed by the program for the direct amplification of gene sequence tags (GSTs) from either genomic DNA or cDNA. Test runs of GST-PRIME on 2000 randomly selected Arabidopsis and Drosophila genes demonstrate that 93 and 88% of resulting GSTs, respectively, fulfilled imposed length criteria. GST-PRIME primer pairs were tested on a set of 1900 Arabidopsis genes coding for chloroplast-targeted proteins: 95% of the primer pairs used in PCRs with genomic DNA generated the correct amplicons. GST-PRIME can thus be reliably used for large-scale or specific amplification of intron-containing genes of multicellular eukaryotes.

Arabidopsis Proteins↗
PubMed2003

Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences.

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (approximately 0.4-0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (approximately 0.4-0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4-0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Hepatitis C virus whole genome position weight matrix and robust primer design.

BACKGROUND: The high degree of sequence heterogeneity found in Hepatitis C virus (HCV) isolates, makes robust nucleic acid-based assays difficult to generate. Polymerase chain reaction based techniques, require efficient and specific sequence recognition. Generation of robust primers capable of recognizing a wide range of isolates is a difficult task. RESULTS: A position weight matrix (PWM) and a consensus sequence were built for each region of HCV and subsequently assembled into a whole genome consensus sequence and PWM. For each of the 10 regions, the number of occurrences of each base at a given position was compiled. These counts were converted to frequencies that were used to calculate log odds scores. Using over 100 complete and 14,000 partial HCV genomes from GenBank, a consensus HCV genome sequence was generated along with a PWM reflecting heterogeneity at each position. The PWM was used to identify the most conserved regions for primer design. CONCLUSIONS: This approach allows rapid identification of conserved regions for robust primer design and is broadly applicable to sets of genomes with all levels of genetic heterogeneity.

Conserved Sequence↗
PubMed2006

Simplified primer design for PCR-based gene targeting and microarray primer database: two web tools for fission yeast.

PCR-based gene targeting is a popular method for manipulating yeast genes in their normal chromosomal locations. The manual design of primers, however, can be cumbersome and error-prone. We have developed a straightforward web-based tool that applies user-specified inputs to automate and simplify the task of primer selection for deletion, tagging and/or regulated expression of genes in Schizosaccharomyces pombe. This tool, named PPPP (for Pombe PCR Primer Programs), is available at http://www.sanger.ac.uk/PostGenomics/S_pombe/software/. We also present a searchable Microarray Primer Database to retrieve the sequences and accompanying information for primers and PCR products used to build our in-house Sz. pombe microarrays. This database contains information on both coding and intergenic regions to provide context for the microarray data, and it should be useful also for other applications, such as quantitative PCR. The database can be accessed at http://www.sanger.ac.uk/PostGenomics/S_pombe/microarray/.

DNA Primers↗
PubMed2005

The degenerate primer design problem: theory and applications.

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 several applications, one of which is 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.

Algorithms↗
PubMed2006

MutScreener: primer design tool for PCR-direct sequencing.

In searching for susceptibility genes, both positional cloning and candidate gene strategies have been helpful. Mutation screening is one of the many technologies that have been implemented in order to identify mutations or polymorphisms in candidate genes or genomic regions. Since human genome sequence is available, PCR-direct sequencing is one of the major methods for mutation screening or resequencing. Unfortunately, assay design can be laborious if multiple genes or large regions need to be investigated. To solve this conundrum a web-based application, MutScreener, has been developed. MutScreener assists in the analysis of human gene structure and design of PCR/sequencing primer. This application supports batch assay design based on either existing public gene annotation or custom gene annotation. The optional universal tagged primers can support high throughput resequencing processes. MutScreener is available for public use at http://bioinfo.bsd.uchicago.edu/MutScreener.html.

DNA Primers↗
PubMed2005

BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes.

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.

Algorithms↗
PubMed2005

ExPrimer: to design primers from exon--exon junctions.

SUMMARY: ExPrimer is a web-based computer program to design primers mainly from a specified exon-exon junction (E-E-jn) of a gene of interest. The tool suggests the optimum primer-pair(s) of which the right (reverse) primer represents a particular E-E-jn of the mRNA. The 'product length' decides the location of the left primer. The results also include all other primer pairs considered and their 'scores'. ExPrimer can use the NCBI BLASTn program for sequence specificity of primers. The tool is useful in many areas of molecular biology research that involve hybridization of short sequences with mRNA or cDNA. AVAILABILITY: http://exprimer.ibab.ac.in/exprimer_html/exprimer.html

Algorithms↗