ExonSurfer: a web-tool to design primers at exon-exon junctions.

Exons Software DNA Primers / genetics Internet Humans Reverse Transcriptase Polymerase Chain Reaction / methods
Alternative splicing Exon-exon junction Gene expression Primer design RT-qPCR Web tool

Journal

BMC genomics
ISSN: 1471-2164
Titre abrégé: BMC Genomics
Pays: England
ID NLM: 100965258

Informations de publication

Date de publication:
12 Jun 2024
Historique:
received: 23 01 2024
accepted: 24 05 2024
medline: 13 6 2024
pubmed: 13 6 2024
entrez: 12 6 2024
Statut: epublish

Résumé

Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

Sections du résumé

BACKGROUND BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION CONCLUSIONS
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

Identifiants

DOI: 10.1186/s12864-024-10456-2 PMID: 38867172
pubmed: 38867172
doi: 10.1186/s12864-024-10456-2
pii: 10.1186/s12864-024-10456-2
doi:

Substances chimiques

DNA Primers 0

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

594

Informations de copyright

© 2024. The Author(s).

Références

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Auteurs

Pablo Monfort-Lanzas (P)

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.
Institute of Bioinformatics, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.

Elena Cristina Rusu (EC)

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.
SeqPlexing SL, Valencia, Spain.
Institute of Integrative Systems Biology (I2Sysbio), University of Valencia and Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain.

Lucia Parrakova (L)

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.

Cornelia A Karg (CA)

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.

Dorina-Elina Kernbichler (DE)

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.
Institute of Medical Biochemistry, Core Facility Metabolomics II, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.

Dietmar Rieder (D)

Institute of Bioinformatics, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.

Peter Lackner (P)

Department of Biosciences and Medical Biology, University of Salzburg, 5020, Salzburg, Austria.

Hubert Hackl (H)

Institute of Bioinformatics, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria.

Johanna M Gostner (JM)

Institute of Medical Biochemistry, Biochemical Immunotoxicology Group, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria. johanna.gostner@i-med.ac.at.
Institute of Medical Biochemistry, Core Facility Metabolomics II, Biocenter, Medical University of Innsbruck, Innrain 80, 6020, Innsbruck, Austria. johanna.gostner@i-med.ac.at.

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Classifications MeSH

questionsmedicales.fr › Phénomènes génétiques › Structures génétiques › Génome › Composants de génome › Gènes › Composants de gène › Exons › ExonSurfer: a web-tool to design primers at...
questionsmedicales.fr › Sciences de l'information › Méthodologies informatiques › Logiciel › ExonSurfer: a web-tool to design primers at...
questionsmedicales.fr › Actions chimiques et utilisations › Utilisations spécialisées de produits chimiques › Produits chimiques de laboratoire › Sondes moléculaires › Sondes d'acide nucléique › Sondes d'ADN › Amorces ADN › ExonSurfer: a web-tool to design primers at...
questionsmedicales.fr › Sciences de l'information › Méthodologies informatiques › Systèmes informatiques › Réseaux de communication entre ordinateurs › Internet › ExonSurfer: a web-tool to design primers at...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › ExonSurfer: a web-tool to design primers at...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Techniques d'amplification d'acides nucléiques › Réaction de polymérisation en chaîne › RT-PCR › ExonSurfer: a web-tool to design primers at...