Primer Design, Evaluation of Primer Universality, and Estimation of Identification Power of Amplicon Sequences In Silico

2019 ◽  
pp. 21-36
Author(s):  
Akifumi S. Tanabe ◽  
Satoshi Nagai ◽  
Yuki Hongo ◽  
Motoshige Yasuike ◽  
Yoji Nakamura ◽  
...  
Keyword(s):  
In Silico ◽  
Primer Design ◽  
Design Evaluation

scholarly journals PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers

2016 ◽  
Author(s):  
Vasco Elbrecht ◽  
Florian Leese
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Dna Barcode ◽  
R Package ◽  
Primer Design ◽  
Reference Sequence ◽  
Gene Marker ◽  
Sequence Alignments ◽  
Variable Binding ◽  
Dna Metabarcoding

1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that serve as a backbone for primer design is currently available. 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in another available software for that purpose, ecoPCR. 3) We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. 4) With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU based reference alignments generated with PrimerMiner can be used for manual primer design, or processed with bioinformatic tools for primer development.

scholarly journals Methylation-specific PCR: four steps in primer design

2014 ◽  
Vol 9 (12) ◽  
pp. 1127-1139 ◽  
Author(s):  
Radoslav Davidović ◽  
Ana Božović ◽  
Vesna Mandušić ◽  
Milena Krajnović
Keyword(s):  
In Silico ◽  
Single Gene ◽  
In Silico Analysis ◽  
Promoter Sequence ◽  
Primer Design ◽  
Gene Methylation ◽  
Methylation Specific Pcr ◽  
Specific Pcr ◽  
Proper Design ◽  
Silico Analysis

AbstractMethylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design.

In-Silico Automated Allele-Specific Primer Design for Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
George Alexandrou ◽  
Jesus Rodriguez-Manzano ◽  
Kenny Malpartida-Cardenas ◽  
Pantelis Georgiou ◽  
Chris Toumazou ◽  
...  
Keyword(s):  
In Silico ◽  
Primer Design ◽  
Isothermal Amplification ◽  
Specific Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Allele Specific

scholarly journals In silico design and validation of a highly degenerate primer pair: a systematic approach

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Prosper Obed Chukwuemeka ◽  
Haruna Isiyaku Umar ◽  
Oluwatoyin Folake Olukunle ◽  
Oluwaseyi Matthew Oretade ◽  
Christopher Busayo Olowosoke ◽  
...  
Keyword(s):  
In Silico ◽  
Biological Activities ◽  
Positive Control ◽  
Primer Design ◽  
Extensive Study ◽  
Degenerate Primer ◽  
Degenerate Primers ◽  
Software Program ◽  
Optimal Efficiency ◽  
Enzymatic Degradability

Abstract Background The techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems. More recently, this approach has been extended to amplify population of genes, from evolutionarily related gene family for detection and evaluation of microbial consortial with several unique potentialities (e.g., enzymatic degradability). Conceivably, primer mixtures containing substitutions of different bases at specific sites (degenerate primers) have enabled the amplification of these genes in PCR reaction. However, the degenerate primer design problem (DPD) is a constraint to designing this kind of primer. To date, different algorithms now exist to solve various versions of DPD problem, many of which, only few addresses and satisfy the criteria to design primers that can extensively cover high through-put sequences while striking the balance between specificity and efficiency. The highly degenerate primer (HYDEN) design software program primarily addresses this variant of DPD problem termed “maximum coverage-degenerate primer design (MC-DPD)” and its heuristics have been substantiated for optimal efficiency from significant successes in PCR. In spite of the premium presented for designing degenerate primers, literature search has indicated relatively little use of its heuristics. This has been thought to result from the complexity of the program since it is run only by command-line, hence limiting its accessibility. To solve this problem, researchers have optionally considered the manual design of degenerate primers or design through software programs that provides accessibility through a graphical user interface (GUI). Realizing this, we have attempted in this study to provide a user-friendly approach for researchers with little or no background in bioinformatics to design degenerate primers using HYDEN Results Virtual Tests of our designed degenerate primer pair through in silico PCR substantiated the correspondence between efficiency and coverage with the target sequences as pre-defined by the initial HYDEN output, thereby validating the potentials of HYDEN to effectively solve the MC-DPD problem. Additionally, the designed primer-pair mechanistically amplified all sequences used as a positive control with no amplification observed in the negative controls. Conclusion In this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program.

scholarly journals Lessons and Considerations for the Creation of Universal Primers Targeting Non-Conserved, Horizontally Mobile Genes

2020 ◽  
Author(s):  
Damon C. Brown ◽  
Raymond J. Turner
Keyword(s):  
In Silico ◽  
Primer Design ◽  
Detection Methods ◽  
Universal Primers ◽  
Sequence Alignments ◽  
Universal Primer ◽  
Conserved Genes ◽  
Primer Sets ◽  
Conserved Core ◽  
Core Genes

Effective and accurate primer design is an increasingly important skill as the use of PCR-based diagnostics in clinical and environmental settings is on the rise. While universal primer sets have been successfully designed for highly conserved core genes such as 16S rRNA and characteristic genes such as dsrAB and dnaJ, primer sets for mobile, accessory genes such as multidrug resistance efflux pumps (MDREP) have not been explored. Here, we describe an approach to create universal primer sets for select MDREP genes chosen from five superfamilies (SMR, MFS, MATE, ABC and RND) identified in a model community of six members (Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica). Using sequence alignments and in silico PCR analyses, a new approach for creating universal primers sets targeting mobile, non-conserved genes has been developed and compared to more traditional approaches used for highly conserved genes. A discussion of the potential shortfalls of the primer sets designed this way are described. The approach described here can be adapted to any unique gene set and aid in creating a wider, more robust library of primer sets to detect less conserved genes and improve the field of PCR-based screening research. IMPORTANCE Increasing use of molecular detection methods, specifically PCR and qPCR, requires utmost confidence in the results while minimizing false positives and negatives due to poor primer designs. Frequently, these detection methods are focused on conserved, core genes which limits their applications. These screening methods are being used in various industries for specific genetic targets or key organisms such as viral or infectious strains, or characteristic genes indicating the presence of key metabolic processes. The significance of this work is to improve primer design approaches to broaden the scope of detectable genes. The use of the techniques explored here will improve detection of non-conserved genes through unique primer design approaches. Additionally, the approaches here highlight additional, important information which can be gleaned during the in silico phase of primer design which will improve our gene annotations based on percent identities.

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