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

Prosper Obed Chukwuemeka,Haruna Isiyaku Umar,Oyeyemi Janet Oretade,Lucky Efe Isunu,Usman Garba Kurmi,Oluwaseyi Matthew Oretade,Bukola Rukayat Oyelere,Michael Omoniyi Elabiyi,Christopher Busayo Olowosoke,Joy Oseme Eigbe,Emmanuel Oluwasegun Akinsola,Oluwatoyin Folake Olukunle

doi:10.1186/s43141-020-00086-y

Prosper Obed Chukwuemeka, Haruna Isiyaku Umar + Show 10 more

Open Access

https://doi.org/10.1186/s43141-020-00086-y

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Abstract

BackgroundThe 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 ResultsVirtual 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. ConclusionIn this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program.

Highlights

The techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems
All the selected bacterial strains used in this study to design the highly degenerate primer pair possess the C12O genes, the failure of primer catAf and catAr to bind the residual gene sequence might conceivably result from lower homology between the sequences of the gene and that of the C12O primer pair
The current study addresses the accessibility constraint of designing highly degenerate primers through programs that are extensively run by command-line which have been thought to be the main problem decreasing the number of degenerate primers more recently reported since many of these reliable degenerate primer design programs often lacks Graphical User Interface (GUI) to simplify use in spite holding a good profile to design degenerate primers

Summary

Introduction

The techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems. 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, limiting its accessibility. Since DNA polymerases do not possess de novo activity and often requires a free 3’ –OH end to facilitate DNA elongation, the role of primers in PCR studies is of great consideration and cannot be overruled Owing to this striking point, various paradigms to design primers have been proposed over time and several commercial software programs have been developed to design different types of primers that would fit the primer design constraints. Free web servers to effectively determine the properties of these oligonucleotide strings have been made available reducing time implications, ambiguity, and errors of humans that might occur from the manual design of primers, resulting in improved primer design accuracy [4]

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