Chukwuemeka Prosper Obed, Umar Haruna Isiyaku, Olukunle Oluwatoyin Folake, Oretade Oluwaseyi Matthew, Olowosoke Christopher Busayo, Akinsola Emmanuel Oluwasegun, Elabiyi Michael Omoniyi, Kurmi Usman Garba, Eigbe Joy Oseme, Oyelere Bukola Rukayat, Isunu Lucky Efe, Oretade Oyeyemi Janet
Department of Biotechnology, School of Sciences (SOS), Federal University of Technology Akure, Akure, P.M.B 704, Nigeria.
Department of Biochemistry, School of Sciences (SOS), Federal University of Technology Akure, Akure, P.M.B 704, Nigeria.
J Genet Eng Biotechnol. 2020 Nov 17;18(1):72. doi: 10.1186/s43141-020-00086-y.
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.
In this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program.
遗传物质扩增技术使得在生物系统的生物环境之外对多种生物活性进行广泛研究成为可能。最近，这种方法已扩展到扩增来自进化相关基因家族的基因群体，用于检测和评估具有多种独特潜力（如酶促降解能力）的微生物群落。可以想象，在特定位点含有不同碱基替代的引物混合物（简并引物）能够在PCR反应中扩增这些基因。然而，简并引物设计问题（DPD）是设计这类引物的一个限制因素。迄今为止，存在不同的算法来解决各种版本的DPD问题，其中许多算法只有少数能够满足设计引物的标准，即既能广泛覆盖高通量序列，又能在特异性和效率之间取得平衡。高度简并引物（HYDEN）设计软件程序主要解决DPD问题的这种变体，即“最大覆盖 - 简并引物设计（MC - DPD）”，并且其启发式方法已通过PCR中的显著成功得到证实，具有最佳效率。尽管设计简并引物有诸多优势，但文献检索表明其启发式方法应用相对较少。这被认为是由于该程序的复杂性，因为它只能通过命令行运行，从而限制了其可及性。为了解决这个问题，研究人员选择考虑手动设计简并引物或通过提供图形用户界面（GUI）可及性的软件程序进行设计。意识到这一点，我们在本研究中尝试为几乎没有或没有生物信息学背景的研究人员提供一种用户友好的方法，使用HYDEN设计简并引物。结果：通过虚拟PCR对我们设计的简并引物对进行虚拟测试，证实了效率与覆盖目标序列之间的对应关系，这与初始HYDEN输出预先定义的一致，从而验证了HYDEN有效解决MC - DPD问题的潜力。此外，设计的引物对机械地扩增了用作阳性对照的所有序列，而在阴性对照中未观察到扩增。
在本研究中，我们提供了一个交钥匙协议，以简化使用HYDEN软件程序的启发式方法设计简并引物的过程。
