Revolutionizing Molecular cloning: Introducing FastCloneAssist, a Streamlined Python tool for optimizing primer design in restriction & ligation-independent PCR cloning
FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired ve...
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| Published in: | PloS one Vol. 20; no. 3; p. e0306950 |
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| Main Authors: | , |
| Format: | Journal Article |
| Language: | English |
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United States
Public Library of Science
13.03.2025
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| ISSN: | 1932-6203, 1932-6203 |
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| Abstract | FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease.
However, efficient primer design remains a critical hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, we present FastCloneAssist, a user-friendly Python program that automates FastCloning primer design with minimal user input. Users simply provide vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist provides best primer pairs after calculating optimal primer parameters for efficient PCR amplification and seamless DNA integration using established bioinformatics libraries. This open-source, freely available tool simplifies and accelerates cloning, making this powerful technique accessible to researchers of all levels and expediting scientific discovery. |
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| AbstractList | FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease.
However, efficient primer design remains a critical hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, we present FastCloneAssist, a user-friendly Python program that automates FastCloning primer design with minimal user input. Users simply provide vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist provides best primer pairs after calculating optimal primer parameters for efficient PCR amplification and seamless DNA integration using established bioinformatics libraries. This open-source, freely available tool simplifies and accelerates cloning, making this powerful technique accessible to researchers of all levels and expediting scientific discovery. FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease. However, efficient primer design remains a critical hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, we present FastCloneAssist, a user-friendly Python program that automates FastCloning primer design with minimal user input. Users simply provide vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist provides best primer pairs after calculating optimal primer parameters for efficient PCR amplification and seamless DNA integration using established bioinformatics libraries. This open-source, freely available tool simplifies and accelerates cloning, making this powerful technique accessible to researchers of all levels and expediting scientific discovery. FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease. FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease. However, efficient primer design remains a critical hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, we present FastCloneAssist, a user-friendly Python program that automates FastCloning primer design with minimal user input. Users simply provide vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist provides best primer pairs after calculating optimal primer parameters for efficient PCR amplification and seamless DNA integration using established bioinformatics libraries. This open-source, freely available tool simplifies and accelerates cloning, making this powerful technique accessible to researchers of all levels and expediting scientific discovery.FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease. However, efficient primer design remains a critical hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, we present FastCloneAssist, a user-friendly Python program that automates FastCloning primer design with minimal user input. Users simply provide vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist provides best primer pairs after calculating optimal primer parameters for efficient PCR amplification and seamless DNA integration using established bioinformatics libraries. This open-source, freely available tool simplifies and accelerates cloning, making this powerful technique accessible to researchers of all levels and expediting scientific discovery. |
| Audience | Academic |
| Author | Donnenberg, Michael S. Singh, Pradip Kumar |
| AuthorAffiliation | Virginia Commonwealth University, Richmond, Virginia, United States of America Nuclear Science and Technology Research Institute, IRAN, ISLAMIC REPUBLIC OF |
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| Author_xml | – sequence: 1 givenname: Pradip Kumar orcidid: 0000-0003-0551-6877 surname: Singh fullname: Singh, Pradip Kumar – sequence: 2 givenname: Michael S. orcidid: 0000-0002-8818-3184 surname: Donnenberg fullname: Donnenberg, Michael S. |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/40080516$$D View this record in MEDLINE/PubMed |
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| Cites_doi | 10.1186/1472-6750-11-92 10.1145/360262.360268 10.1016/B978-0-12-418687-3.00007-0 10.1093/bioinformatics/btp163 10.1002/0471142727.mb0320s110 10.1007/978-1-4842-4470-8_7 10.1128/ecosalplus.esp-0003-2023 10.1371/journal.pone.0273873 10.3389/fbioe.2021.692797 10.1038/nprot.2010.129 10.1128/jb.178.9.2613-2628.1996 |
| ContentType | Journal Article |
| Copyright | Copyright: © 2025 Singh, Donnenberg. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. COPYRIGHT 2025 Public Library of Science 2025 Singh, Donnenberg. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. 2025 Singh, Donnenberg 2025 Singh, Donnenberg 2025 Singh, Donnenberg. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. |
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| References | PJA Cock (pone.0306950.ref009) 2009; 25 I Sohel (pone.0306950.ref013) 1996; 178 J Park (pone.0306950.ref003) 2015; 110 J Lessard (pone.0306950.ref001) 2013 W Wang (pone.0306950.ref005) 2021; 9 C Hoffmann (pone.0306950.ref014) 2010; 5 E Bisong (pone.0306950.ref007) 2019 H Jiang (pone.0306950.ref004) 2022; 17 Foundations of Molecular Cloning - Past, Present and Future | NEB (pone.0306950.ref002) pone.0306950.ref011 C Li (pone.0306950.ref006) 2011; 11 B Chapman (pone.0306950.ref008) 2000; 20 S Rozen (pone.0306950.ref010) 2000; 132 JI Little (pone.0306950.ref012) 2024; 12 |
| References_xml | – volume: 11 start-page: 92 year: 2011 ident: pone.0306950.ref006 article-title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method publication-title: BMC Biotechnol doi: 10.1186/1472-6750-11-92 – volume: 20 start-page: 15 issue: 2 year: 2000 ident: pone.0306950.ref008 article-title: Biopython publication-title: SIGBIO Newsl doi: 10.1145/360262.360268 – start-page: 85 year: 2013 ident: pone.0306950.ref001 article-title: Molecular cloning publication-title: Methods in Enzymology doi: 10.1016/B978-0-12-418687-3.00007-0 – ident: pone.0306950.ref011 – volume: 25 start-page: 1422 issue: 11 year: 2009 ident: pone.0306950.ref009 article-title: Biopython: freely available Python tools for computational molecular biology and bioinformatics publication-title: Bioinformatics doi: 10.1093/bioinformatics/btp163 – volume: 110 start-page: 3.20.1-3.20.23 year: 2015 ident: pone.0306950.ref003 article-title: Site-specific recombinational cloning using gateway and in-fusion cloning schemes publication-title: Curr Protoc Mol Biol doi: 10.1002/0471142727.mb0320s110 – start-page: 59 volume-title: Building machine learning and deep learning models on google cloud platform: A comprehensive guide for beginners year: 2019 ident: pone.0306950.ref007 article-title: Google Colaboratory doi: 10.1007/978-1-4842-4470-8_7 – volume: 12 start-page: eesp00032023 issue: 1 year: 2024 ident: pone.0306950.ref012 article-title: Type IV pili of Enterobacteriaceae species publication-title: EcoSal Plus doi: 10.1128/ecosalplus.esp-0003-2023 – volume: 132 start-page: 365 year: 2000 ident: pone.0306950.ref010 article-title: Primer3 on the WWW for general users and for biologist programmers publication-title: Methods Mol Biol – volume: 17 start-page: e0273873 issue: 9 year: 2022 ident: pone.0306950.ref004 article-title: High-throughput FastCloning technology: A low-cost method for parallel cloning publication-title: PLoS One doi: 10.1371/journal.pone.0273873 – volume: 9 start-page: 692797 year: 2021 ident: pone.0306950.ref005 article-title: Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters publication-title: Front Bioeng Biotechnol doi: 10.3389/fbioe.2021.692797 – ident: pone.0306950.ref002 – volume: 5 start-page: 1666 issue: 10 year: 2010 ident: pone.0306950.ref014 article-title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells publication-title: Nat Protoc doi: 10.1038/nprot.2010.129 – volume: 178 start-page: 2613 issue: 9 year: 1996 ident: pone.0306950.ref013 article-title: Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis publication-title: J Bacteriol doi: 10.1128/jb.178.9.2613-2628.1996 |
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| SubjectTerms | Applications software Automation Bioinformatics Biology and Life Sciences Boxes Cloning Cloning, Molecular - methods Cloud computing Computer and Information Sciences Deoxyribonucleic acid Design Design optimization DNA DNA Primers - genetics Engineering and Technology Genetic Vectors - genetics Melt temperature Melting Methods Physical Sciences Polymerase chain reaction Polymerase Chain Reaction - methods Python (Programming language) Research and Analysis Methods Software |
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| Title | Revolutionizing Molecular cloning: Introducing FastCloneAssist, a Streamlined Python tool for optimizing primer design in restriction & ligation-independent PCR cloning |
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| Volume | 20 |
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