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Results for “primer design”

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At least 19 recordsLinked to original sources
PubMed1995

SSCP primer design based on single-strand DNA structure predicted by a DNA folding program.

To predict alterations in single-strand DNA mobility in non-denaturing electrophoretic gels, Zuker's RNA folding program was modified. Energy files utilized by the LRNA RNA folding algorithm were modified to emulate folding of single-strand DNA. Energy files were modified to disallow G-T base pairing. Stacking energies were corrected for DNA thermodynamics. Constraints on loop nucleotide sequences were removed. The LRNA RNA folding algorithm using the DNA fold energy files was applied to predict folding of PCR generated single-strand DNA molecules from polymorphic human ALDH2 and TPH alleles. The DNA-Fold version 1.0 program was used to design primers to create and abolish SSCP mobility shifts. Primers were made that add a 5' tag sequence or alter complementarity to an internal sequence. Differences in DNA secondary structure were assessed by SSCP analysis and compared to single-strand DNA secondary structure predictions. Results demonstrate that alterations in single-strand DNA conformation may be predicted using DNA-Fold 1.0.

Aldehyde Dehydrogenase↗
PubMed2026

Primer design through submodular function estimation.

MOTIVATION: Multiplex PCR-based enrichment is widely used in viral genome sequencing and pathogen surveillance. However, designing large sets of primers that maximize genome coverage while minimizing primer-primer interactions remains a major computational challenge. Existing methods such as SADDLE and Olivar use heuristics to optimize a Badness score for primer dimers but lack theoretical guarantees on solution quality. RESULTS: We introduce PRISM, a new framework that formulates multiplex primer design as a constrained submodular maximization problem. Our method defines an objective that balances genome coverage and dimer risk, and applies a local search algorithm with a constant-factor approximation guarantee. Evaluations on viral genome datasets demonstrate that PRISM consistently achieves lower Badness scores compared to PrimalScheme, Olivar, and primerJinn. These results highlight the scalability and theoretical rigor of submodular optimization in primer design. AVAILABILITY: PRISM is open-source and available at https://github.com/yhhan19/PRISM-new. The experimental data, scripts, and results used in this paper are archived on Figshare at https://doi.org/10.6084/m9.figshare.32806499.

Algorithms↗
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↗
PubMed1995

Primer design for automated DNA sequencing utilizing T7 DNA polymerase and internal labeling with fluorescein-15-dATP.

We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.

Binding Sites↗
PubMed1990

Olga--oligonucleotide primer design program for the Atari ST.

A program to facilitate the design of oligonucleotide primers has 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 specifically suited to the polymerase chain reaction (PCR) allowing simultaneous analysis of two primer sequences. The advantage of Olga is that it provides in one program analyses for direct repeats, secondary structures and primer dimerization as well as several useful 'finishing' tools for workers engaged in PCR optimization and oligonucleotide syntheses.

Drug Design↗
PubMed1992

Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus.

A PCR assay for the specific detection and identification of viral sequences that correlate with established serotypes of foot-and-mouth disease virus (FMDV) has been developed. A new analysis based on homology profiles among reported sequences was used for primer design. RNA replicase (3D) gene regions that showed high homology among FMDVs, and low homology to other picornaviruses, were used for PCR amplification. Specific and highly sensitive detection was achieved for RNA of FMDV types C, A, and O, either purified or extracted from vesicular fluids of infected animals, under reaction conditions permissive for the detection of variants present in the virus population. Similarly, serotype-specific primers were designed to amplify the carboxy-terminal end of VP1 gene of FMDV types either C, A, or O. The results of PCR amplification of 15 different FMDV RNAs using type-specific primers are in agreement with the serological typing of the corresponding viruses and show that the primer-selection procedure developed for FMDV constitutes a reliable method of viral diagnosis.

Animals↗
PubMed2025

Designated primers targeted canine TP53 gene hotspot regions.

BACKGROUND: Tumor protein 53 gene (TP53) is a critical factor that controls different cell activities such as cell cycle, DNA repair mechanism, autophagy, apoptosis, and metabolism. The TP53 gene is the most commonly mutated gene, especially in the 4-8 exons region. This mutation enhances the development of many abnormalities, such as the initiation of different types of cancer. AIM: The main objective of this study was to design and evaluate the efficacy of three different primer sets that targeted the TP53 gene at the hotspot regions. METHODS: To do that, twelve blood samples were collected from dogs belonging to the German Shepherd breed/K9 aged between 8-12 years. Then, the DNA extraction and polymerase chain reaction (PCR) took place by using the three primer sets, which were designed using SnapGene. The primer sets, namely, first primer, the second and the third targeted exons 5-9 located in the canine TP53 gene. In the following step, all the PCR products were sent for Sanger sequencing and then phylogenetic analysis. RESULTS: Our findings indicated that the first primer set consistently showed higher amplification signal efficiency and reduced dimer formation compared with the second and third primer sets, respectively, with a 60ºC annealing temperature. In addition, all the sequenced samples aligned with the reference canine TP53 gene in the phylogenetic tree. CONCLUSION: This study offered the best TP53 primer design that targeted the hotspot regions of the canine TP53 gene for researchers who are interested in targeting such regions in this gene.

Animals↗
PubMed1995

Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers.

Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.

Base Sequence↗
PubMed1991

Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR.

To facilitate the rapid cloning and sequencing of rearranged murine heavy-chain variable regions, we have designed a set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes. RNA was extracted from the mouse hybridoma cells secreting monoclonal antibodies: IOR-T3 (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), CB-Fib.1 (anti-human fibrin) and CB-Hep.2 (anti-hepatitis B surface antigen). First-strand cDNA was synthesized and amplified using PCR. The primers successfully amplified correct size fragments from cDNA prepared from all hybridomas. These methods will facilitate the cloning and sequencing of mouse immunoglobulin variable regions.

Animals↗
PubMedDate not supplied

Primers designed for amplification of Echinococcus multilocularis DNA amplify the DNA of Encephalitozoon-like spores in the polymerase chain reaction.

Microsporidian spores were developed from cells which were grown in vitro from a human liver lesion which was due to larval Echinococcus multilocularis. The microsporidian spores developed in the same fashion as an Encephalitozoon cuniculi. The Encephalitozoon-like spores were completely separated on Percoll gradients. The separated spores contained DNA capable of amplification by two different primer sets designed for the polymerase chain reaction (PCR) of E. multilocularis DNA. However, the cell DNA from which microsporidium developed was thoroughly insensitive to the PCR using the E. multilocularis primer sets. The results strongly suggested that Encephalitozoon should be taken into consideration, when DNA isolated from larval E. multilocularis is analyzed.

Animals↗
PubMed2026

Primer design for the voltage-gated sodium channel, the pyrethroids' target site, in the triatomine vector Triatoma infestans (Hemiptera: Reduviidae).

Resistance to pyrethroid insecticides has become more frequent in triatomines, associated with point mutations in their target site, the voltage-gated sodium channel (VGSC). These mutations have been reported in Argentina, Bolivia and Mexico, by means of nested PCR. In Chile, the repeated intradomiciliary presence of the vector Triatoma infestans after spraying may be related to resistance; however, target-site mutations have yet to be evaluated. Using a partial sequence of the VGSC as a search query for the T. infestans genome, the complete VGSC gene sequence was obtained. Ten primer pairs were generated with Primer-BLAST and tested in silico by BLAST against the T. infestans genome and against the NCBI nucleotide database (nr) to discard those with low specificity. Conventional PCRs were performed with T. infestans' DNA and the remaining primers, selecting one pair based on sensitivity and the absence of nonspecific bands. The 993 bp sequenced product allows for the evaluation of the three point mutations reported in the VGSC of triatomines. Primer validation was performed with L925I mutation-positive samples from Argentina. Chilean samples from five localities were also amplified and Sanger sequenced in search of mutations. The L925I mutation was detected only in the Argentinean controls. None of the variants evaluated were found in the Chilean samples. These primers will allow rapid evaluation of point mutations in the VGSC, which may be associated with reduced affinity of pyrethroids for their target site; thereby facilitating insecticide resistance surveillance.

Animals↗
PubMed1990

Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells.

In this article we show how the polymerase chain reaction (PCR) and primers designed for conserved sequences of leader (L), framework one (FR1) and constant (CONST) regions of immunoglobulin light and heavy chain genes can be used for the cloning and sequencing of rearranged antibody variable regions from mouse hybridoma cells. RNA was extracted from the mouse hybridoma cells secreting MAbs: IOR-T3a (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), and CB-Fib.1 (anti-human fibrin). First strand cDNA was synthesized and amplified using PCR. The newly designed primers are superior to others reported recently in the literature. Isolated PCR DNA fragments of C6 and IOR-T3a were sequenced after asymmetric amplification, or M13 cloning. The FR1/CONST primer combinations selectively amplified mouse lights chain of groups kappa II, V, and VI, and heavy chains of groups IIa and IIc. The L/CONST primers for light chains amplified light chains from all four hybridomas. These methods greatly facilitate structural and functional studies of antibodies by reducing the efforts required to clone and sequence their variable regions.

Amino Acid Sequence↗
PubMed1995

Determination of gene structure by intron trapping using polymerase chain reaction: application to the human plasma prekallikrein gene.

We have devised a method to determine gene structure that utilizes the known gene structure of homologous proteins and the polymerase chain reaction (PCR). Because homologous proteins have evolved from a common ancestral gene, it is possible to design primers corresponding to the adjacent exon sequences in the protein of interest. These primers could then be used in a PCR using normal genomic DNA as template. The resultant PCR product is then subcloned and the nuceotide sequence of the insert is determined. This information provides the exon-intron junction sequences and the partial sequences of the intron. We have applied this method to trap introns A, H, and J of human plasma prekallikrein gene and determined their exon-intron junction sequences. The primers were designed based on the known cDNA sequence of human plasma prekallikrein and gene structures of rat plasma prekallikrein and human coagulation factor XI (which is 50% identical in the primary sequence). The intron-exon junctions are at identical sites to those of the rat plasma prekallikrein gene. The intron sequences thus obtained will be useful in designing primers for exon trapping and sequence analysis of plasma prekallikrein gene from patients with defective plasma prekallikrein. This technique will also allow the determination of the entire gene structure.

Amino Acid Sequence↗
PubMed1991

The use of the polymerase chain reaction for the detection of human papillomavirus type 13.

Human papillomavirus type 13 (HPV-13) is associated with oral focal epithelial hyperplasia (FEH). The purpose of this study was to establish conditions for the application of polymerase chain reaction (PCR) to the specific detection and amplification of HPV-13 DNA. To design primers for HPV-13 a part of the HPV-13 genome was sequenced first: the smallest BamHI fragment (597 bp) of HPV-13 was subcloned and sequenced. The sequence was found to be part of a large open reading frame and had significant homology with the L1 gene of other HPVs. HPV-13 specific primers were designed to amplify a 240 bp fragment from the L1 gene by PCR. Conditions for PCR were standardized for this set of primers.

Base Sequence↗