ArticleJournal, genetic engineering & biotechnology2020
In silico design and validation of a highly degenerate primer pair: a systematic approach.
Article in Journal, genetic engineering & biotechnology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed, 12 citations in OpenAlex.
- In silico PCR-RFLP identification of forensically important Calliphoridae.Forensic science, medicine, and pathology · 2026Article
- Review
- Development of a highly degenerate primer-based molecular tool for detecting and classifying the four major classes of polyhydroxyalkanoate synthase (phaC) genes in bacteria.Microbial cell factories · 2025Article
- Designated primers targeted canineOpen veterinary journal · 2025Article
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Authors and funding
12 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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