In silico design and validation of a highly degenerate primer pair: a systematic approach
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 dete...
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| Veröffentlicht in: | Journal of Genetic Engineering and Biotechnology 2020-11, Vol.18 (1), p.72-17, Article 72 |
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| creator | 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 |
| description | 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 |
| doi_str_mv | 10.1186/s43141-020-00086-y |
| format | Article |
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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.</description><identifier>ISSN: 1687-157X</identifier><identifier>EISSN: 2090-5920</identifier><identifier>DOI: 10.1186/s43141-020-00086-y</identifier><identifier>PMID: 33205353</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Accessibility ; Accessible ; Algorithms ; Amplification ; Bioinformatics ; Biomedical Engineering and Bioengineering ; C12O ; Cloning ; Computer graphics ; Coverage ; Degenerate primers ; Degradability ; Design ; DNA polymerase ; Efficiency ; Engineering ; Enzymes ; Experiments ; Gene expression ; Genes ; Genetic engineering ; Genetic materials ; Genomes ; Graphical user interface ; Methods ; Microorganisms ; Mutagenesis ; Mutation ; Polymerase chain reaction ; Primers ; Problem solving ; Proteins ; Software ; Success</subject><ispartof>Journal of Genetic Engineering and Biotechnology, 2020-11, Vol.18 (1), p.72-17, Article 72</ispartof><rights>The Author(s) 2020</rights><rights>COPYRIGHT 2020 Springer</rights><rights>The Author(s) 2020. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c556t-29a8fda3810083b73e3d27936882f395e8a8cdbdf649b740841eb9839a04a4943</citedby><cites>FETCH-LOGICAL-c556t-29a8fda3810083b73e3d27936882f395e8a8cdbdf649b740841eb9839a04a4943</cites><orcidid>0000-0001-8381-2802 ; 0000-0001-9427-9804</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671957/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671957/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27922,27923,53789,53791</link.rule.ids></links><search><creatorcontrib>Chukwuemeka, Prosper Obed</creatorcontrib><creatorcontrib>Umar, Haruna Isiyaku</creatorcontrib><creatorcontrib>Olukunle, Oluwatoyin Folake</creatorcontrib><creatorcontrib>Oretade, Oluwaseyi Matthew</creatorcontrib><creatorcontrib>Olowosoke, Christopher Busayo</creatorcontrib><creatorcontrib>Akinsola, Emmanuel Oluwasegun</creatorcontrib><creatorcontrib>Elabiyi, Michael Omoniyi</creatorcontrib><creatorcontrib>Kurmi, Usman Garba</creatorcontrib><creatorcontrib>Eigbe, Joy Oseme</creatorcontrib><creatorcontrib>Oyelere, Bukola Rukayat</creatorcontrib><creatorcontrib>Isunu, Lucky Efe</creatorcontrib><creatorcontrib>Oretade, Oyeyemi Janet</creatorcontrib><title>In silico design and validation of a highly degenerate primer pair: a systematic approach</title><title>Journal of Genetic Engineering and Biotechnology</title><addtitle>J Genet Eng Biotechnol</addtitle><description>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.</description><subject>Accessibility</subject><subject>Accessible</subject><subject>Algorithms</subject><subject>Amplification</subject><subject>Bioinformatics</subject><subject>Biomedical Engineering and Bioengineering</subject><subject>C12O</subject><subject>Cloning</subject><subject>Computer graphics</subject><subject>Coverage</subject><subject>Degenerate primers</subject><subject>Degradability</subject><subject>Design</subject><subject>DNA polymerase</subject><subject>Efficiency</subject><subject>Engineering</subject><subject>Enzymes</subject><subject>Experiments</subject><subject>Gene expression</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Genetic materials</subject><subject>Genomes</subject><subject>Graphical user interface</subject><subject>Methods</subject><subject>Microorganisms</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Polymerase chain reaction</subject><subject>Primers</subject><subject>Problem solving</subject><subject>Proteins</subject><subject>Software</subject><subject>Success</subject><issn>1687-157X</issn><issn>2090-5920</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNp9kk1r3DAQhk1paZY0f6AnQ89O9WFbUg-FEPqxEOilhfYkxpLs1eKVXMkb8L_vOA4NuQQdBKP3fXhnNEXxnpJrSmX7Mdec1rQijFSEENlWy6tix4giVaMYeV3saCtFRRvx-6K4yvmIItLUkjb0bXHBOSMNb_iu-LMPZfajN7G0LvshlBBseQ-jtzD7GMrYl1Ae_HAYF1QMLrgEsyun5E8ulRP49AkFecmzO6HDlDBNKYI5vCve9DBmd_V4Xxa_vn75efu9uvvxbX97c1eZpmnniimQvQUuKXbBO8Edt0wo3krJeq4aJ0Ea29m-rVUnaiJr6joluQJSQ61qflnsN66NcNRrLkiLjuD1QyGmQUPCYKPTYIQ0XEniVpoSnTW85jgvrBpqBLI-b6zp3J2cNS7MCcZn0OcvwR_0EO-1aAVVzQr48AhI8e_Z5Vkf4zkF7F8zwdQ681ai6npTDYCpfOgjwgwe6074E8H1Hus3rUCDkJyhgW0Gk2LOyfX_I1Gi13XQ2zpoXAf9sA56QRPfTBnFYXDpKcsLrn_dTLZQ</recordid><startdate>20201117</startdate><enddate>20201117</enddate><creator>Chukwuemeka, Prosper Obed</creator><creator>Umar, Haruna Isiyaku</creator><creator>Olukunle, Oluwatoyin Folake</creator><creator>Oretade, Oluwaseyi Matthew</creator><creator>Olowosoke, Christopher Busayo</creator><creator>Akinsola, Emmanuel Oluwasegun</creator><creator>Elabiyi, Michael Omoniyi</creator><creator>Kurmi, Usman Garba</creator><creator>Eigbe, Joy Oseme</creator><creator>Oyelere, Bukola Rukayat</creator><creator>Isunu, Lucky Efe</creator><creator>Oretade, Oyeyemi Janet</creator><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><general>Elsevier</general><scope>C6C</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>L6V</scope><scope>LK8</scope><scope>M7P</scope><scope>M7S</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-8381-2802</orcidid><orcidid>https://orcid.org/0000-0001-9427-9804</orcidid></search><sort><creationdate>20201117</creationdate><title>In silico design and validation of a highly degenerate primer pair: a systematic approach</title><author>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</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c556t-29a8fda3810083b73e3d27936882f395e8a8cdbdf649b740841eb9839a04a4943</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Accessibility</topic><topic>Accessible</topic><topic>Algorithms</topic><topic>Amplification</topic><topic>Bioinformatics</topic><topic>Biomedical Engineering and Bioengineering</topic><topic>C12O</topic><topic>Cloning</topic><topic>Computer graphics</topic><topic>Coverage</topic><topic>Degenerate primers</topic><topic>Degradability</topic><topic>Design</topic><topic>DNA polymerase</topic><topic>Efficiency</topic><topic>Engineering</topic><topic>Enzymes</topic><topic>Experiments</topic><topic>Gene expression</topic><topic>Genes</topic><topic>Genetic engineering</topic><topic>Genetic materials</topic><topic>Genomes</topic><topic>Graphical user interface</topic><topic>Methods</topic><topic>Microorganisms</topic><topic>Mutagenesis</topic><topic>Mutation</topic><topic>Polymerase chain reaction</topic><topic>Primers</topic><topic>Problem solving</topic><topic>Proteins</topic><topic>Software</topic><topic>Success</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chukwuemeka, Prosper Obed</creatorcontrib><creatorcontrib>Umar, Haruna Isiyaku</creatorcontrib><creatorcontrib>Olukunle, Oluwatoyin Folake</creatorcontrib><creatorcontrib>Oretade, Oluwaseyi Matthew</creatorcontrib><creatorcontrib>Olowosoke, Christopher Busayo</creatorcontrib><creatorcontrib>Akinsola, Emmanuel Oluwasegun</creatorcontrib><creatorcontrib>Elabiyi, Michael Omoniyi</creatorcontrib><creatorcontrib>Kurmi, Usman Garba</creatorcontrib><creatorcontrib>Eigbe, Joy Oseme</creatorcontrib><creatorcontrib>Oyelere, Bukola Rukayat</creatorcontrib><creatorcontrib>Isunu, Lucky Efe</creatorcontrib><creatorcontrib>Oretade, Oyeyemi Janet</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>CrossRef</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>Journal of Genetic Engineering and Biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chukwuemeka, Prosper Obed</au><au>Umar, Haruna Isiyaku</au><au>Olukunle, Oluwatoyin Folake</au><au>Oretade, Oluwaseyi Matthew</au><au>Olowosoke, Christopher Busayo</au><au>Akinsola, Emmanuel Oluwasegun</au><au>Elabiyi, Michael Omoniyi</au><au>Kurmi, Usman Garba</au><au>Eigbe, Joy Oseme</au><au>Oyelere, Bukola Rukayat</au><au>Isunu, Lucky Efe</au><au>Oretade, Oyeyemi Janet</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>In silico design and validation of a highly degenerate primer pair: a systematic approach</atitle><jtitle>Journal of Genetic Engineering and Biotechnology</jtitle><stitle>J Genet Eng Biotechnol</stitle><date>2020-11-17</date><risdate>2020</risdate><volume>18</volume><issue>1</issue><spage>72</spage><epage>17</epage><pages>72-17</pages><artnum>72</artnum><issn>1687-157X</issn><eissn>2090-5920</eissn><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.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>33205353</pmid><doi>10.1186/s43141-020-00086-y</doi><tpages>17</tpages><orcidid>https://orcid.org/0000-0001-8381-2802</orcidid><orcidid>https://orcid.org/0000-0001-9427-9804</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Journal of Genetic Engineering and Biotechnology, 2020-11, Vol.18 (1), p.72-17, Article 72 |
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| source | DOAJ Directory of Open Access Journals; Springer Nature OA Free Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Alma/SFX Local Collection |
| subjects | Accessibility Accessible Algorithms Amplification Bioinformatics Biomedical Engineering and Bioengineering C12O Cloning Computer graphics Coverage Degenerate primers Degradability Design DNA polymerase Efficiency Engineering Enzymes Experiments Gene expression Genes Genetic engineering Genetic materials Genomes Graphical user interface Methods Microorganisms Mutagenesis Mutation Polymerase chain reaction Primers Problem solving Proteins Software Success |
| title | In silico design and validation of a highly degenerate primer pair: a systematic approach |
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