A novel method for conserved sequence extraction with prospective mutation prediction for SARS-CoV-2 PCR primer design.
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ABSTRACT:
SUBMITTER: Portakal SH
PROVIDER: S-EPMC8015351 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).
| S-EPMC168931 | biostudies-literature
Project description:We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.
| S-EPMC2715258 | biostudies-literature
Project description:UnlabelledDesigned degenerate primers unlike conventional primers are superior in matching and amplification of large number of genes, from related gene families. DPPrimer tool was designed to predict primers for PCR amplification of homologous gene from related or diverse plant species. The key features of this tool include platform independence and user friendliness in primer design. Embedded features such as search for functional domains, similarity score selection and phylogebetic tree further enhance the user friendliness of DPPrimer tool. Performance of DPPrimer tool was evaluated by successful PCR amplification of ADP-glucose phosphorylase genes from wheat, barley and rice.AvailabilityDPPrimer is freely accessible at http://202.141.12.147/DGEN_tool/index.html.
| S-EPMC3842581 | biostudies-literature
Project description:UnlabelledBackgroundAlthough Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3'end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge.ResultsIn this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3rd nucleotide from the 3' end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species.ConclusionThe method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.
| S-EPMC3495711 | biostudies-literature
Project description:Microsatellites or SSRs (simple sequence repeats) are ubiquitous short tandem duplications occurring in eukaryotic organisms. These sequences are among the best marker technologies applied in plant genetics and breeding. The abundant genomic, BAC, and EST sequences available in databases allow the survey regarding presence and location of SSR loci. Additional information concerning primer sequences is also the target of plant geneticists and breeders. In this paper, we describe a utility that integrates SSR searches, frequency of occurrence of motifs and arrangements, primer design, and PCR simulation against other databases. This simulation allows the performance of global alignments and identity and homology searches between different amplified sequences, that is, amplicons. In order to validate the tool functions, SSR discovery searches were performed in a database containing 28 469 nonredundant rice cDNA sequences.
| S-EPMC2486402 | biostudies-literature
Project description: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.
| S-EPMC1538803 | biostudies-literature
Project description:As the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive. To extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently. A total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5'-/3'-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR. Out of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations. In addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks.
| S-EPMC9160177 | biostudies-literature
Project description:UNLABELLED: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.
| S-EPMC3039988 | biostudies-literature
Project description:BackgroundThe accurate tracking of Cryptosporidium in faecal, water and/or soil samples in water catchment areas is central to developing strategies to manage the potential risk of cryptosporidiosis transmission to humans. Various PCR assays are used for this purpose. Although some assays achieve specific amplification from Cryptosporidium DNA in animal faecal samples, some do not. Indeed, we have observed non-specificity of some oligonucleotide primers in the small subunit of nuclear ribosomal RNA gene (SSU), which has presented an obstacle to the identification and classification of Cryptosporidium species and genotypes (taxa) from faecal samples.ResultsUsing a novel bioinformatic approach, we explored all available Cryptosporidium genome sequences for new and diagnostically-informative, multi-copy regions to specifically design oligonucleotide primers in the large subunit of nuclear ribosomal RNA gene (LSU) as a basis for an effective nested PCR-based sequencing method for the identification and/or classification of Cryptosporidium taxa.ConclusionThis newly established PCR, which has high analytical specificity and sensitivity, is now in routine use in our laboratory, together with other assays developed by various colleagues. Although the present bioinformatic workflow used here was for the specific design of primers in nuclear DNA of Cryptosporidium, this approach should be broadly applicable to many other microorganisms.
| S-EPMC5654123 | biostudies-literature
Project description:Globally more than 100 million SNPs in populations. These variations approximately 4-5 million SNPs in a people genome, occur almost every 1000 nucleotides on average and present either unique or in many in individuals. They can act as genetic signs, associated with illness and respond to chemicals and drugs. SNPs occurrence within or near a gene play important role in disease throughout affecting gene task. Frequently many protocols have been used to study single nucleotide polymorphism (SNP) among human variants genome. Restriction fragment length polymorphism (RFLP), Amplification refractory mutation system PCR(ARMS-PCR), sequencing and SNaPshot assays considered familial methods. The potential risk of contamination after PCR is common due to further other steps. In this direction, a high resolution melting (HRM) real-time PCR method is an alternative, reducing the post-PCR transferring steps. uVariants is clarified as appropriate website for designing primers used for SNP recognition by easy and inexpensive protocol called HRM. The researchers can focus on the interest of reference SNP ID number, or "rs" ID to avoid loss time. In this article description how to uses uVariants website for primer design used in HRM technique. Aims:To describe uVariants and uDesign software, application and usefulness of HRM technique primer design in the genotyping SNPs among people and public health. Accessibility and requirements:uVariants and uDesign are freely accessible at: https://www.dna.utah.edu/variants/;https://www.dna.utah.edu/udesign/app.php respectively.The network server supports the browsers: Chrome, Firefox, Torch, CoolNovo, 360 Browser, Internet Explorer, Opera, and Safari.
| S-EPMC6458491 | biostudies-other