![]() Accordingly, the resulting single -stranded DNA templates are enzymatically extended by the activity of a thermostable polymerase (called DNA polymerase) into new double-stranded DNA from free nucleotides contained within a reaction mixture. Subsequently, short single-stranded nucleotide strings called “primers” bind to complementary regions within the melted DNA molecule in a process called hybridization. To execute PCR, the genetic material is first denatured in a melting step involving thermal elevation of double-stranded DNA molecules to a temperature nearly at the boiling point of ordinary water, thus, converting double-stranded DNA molecules within a reaction mixture into single strands. The technique generally involves three steps all of which are temperature-dependent: denaturation, annealing (primer hybridization), and extension (primer elongation). Polymerase chain reaction is an in vitro technique used to make genetic materials (DNA) in several orders of magnitude by amplifying DNA segment of known sequences or a portion of DNA that lies between two known sequences to exponential copies. PCR is not a technique limited to just a single field of study and has been exploited in several interdisciplinary researches spanning across specialties such as molecular biology, medicine, biotechnology, agriculture, engineering, biochemistry, microbiology, genetics, and a good number of fascinating applications in newer scope of biological or life sciences. ![]() The advent of polymerase chain reaction (PCR) has revolutionize the understanding of genetic materials (DNA and RNA) with a wide range applicability in many biological studies including amplification, gene expression, cloning, mutation detection, mutagenesis, and a large list of genome typing experiments that are of pertinence in the metagenomics era. ![]() ![]() In this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program. Additionally, the designed primer-pair mechanistically amplified all sequences used as a positive control with no amplification observed in the negative controls. 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. 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 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). This has been thought to result from the complexity of the program since it is run only by command-line, hence limiting its accessibility. In spite of the premium presented for designing degenerate primers, literature search has indicated relatively little use of its heuristics. 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. 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. However, the degenerate primer design problem (DPD) is a constraint to designing this kind of primer. Conceivably, primer mixtures containing substitutions of different bases at specific sites (degenerate primers) have enabled the amplification of these genes in PCR reaction. 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). The techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems.
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