TipMT: Identification of PCR-based taxon-specific markers

Gabriela F Rodrigues-Luiz,Ricardo T Fujiwara,Daniella C Bartholomeu,Célia M F Gontijo,Hugo O Valdivia,Edward V Ayala,Robson S Lopes,Thiago De S Rodrigues,Mariana S Cardoso

doi:10.1186/s12859-017-1485-3

Gabriela F Rodrigues-Luiz, Ricardo T Fujiwara + Show 7 more

Open Access

https://doi.org/10.1186/s12859-017-1485-3

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Abstract

BackgroundMolecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity.ResultsHere, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species.ConclusionsThe TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/.

Highlights

Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies
Because primer specificity is a key step in a polymerase chain reaction (PCR) reaction, the pipeline identifies all potential annealing sites for the primers selected based on alignment and thermodynamics
The user provides the genomic sequences and defines the type of target sequences, orthologous genes or microsatellites, which are extracted from each taxon; in this step, the user defines the primer design constraints; 2) regions of target sequences with similarity to the other genomic sequences provided by the user are masked; cross reaction genomes correspond to templates that should not cross-react in PCR assays; 3) candidate primers for each target sequence are designed; 4) the amplification profile of each taxon is obtained based on electronic PCR and the thermodynamic

Summary

Introduction

Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. Polymerase chain reaction (PCR)-based typing methods are molecular diagnostic techniques widely used in biological and biomedical studies. Microsatellites or single sequence repeats (SSR) are tandem repeated stretches of short nucleotide motifs, usually ranging from 1 to 6 bp, ubiquitously distributed in the genomes of eukaryotic organisms. These regions are more prone to genetic variation and the differences in the length of individual SSR loci can be screened by PCR. This technique has been useful for several studies including strain typing and population genetics [4, 5]. In silico mining analysis has been used to improve marker identification [6, 7]

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