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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Cold Spring Harbor Laboratory
03.07.2024
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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. |
| Author | Donnenberg, Michael S. Singh, Pradip Kumar |
| 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. email: michael.donnenberg@vcuhealth.org |
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| Cites_doi | 10.1385/1-59259-192-2:365 10.3389/fbioe.2021.692797 10.1093/bioinformatics/btp163 10.1002/0471142727.mb0320s110 10.1128/jb.178.9.2613-2628.1996 10.1007/978-1-4842-4470-8_7 10.1186/1472-6750-11-92 10.1371/journal.pone.0273873 10.1145/360262.360268 10.1128/ecosalplus.esp-0003-2023 10.1038/nprot.2010.129 |
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| Copyright | 2024, Posted by Cold Spring Harbor Laboratory |
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| Notes | Competing Interest Statement: The authors have declared no competing interest. |
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| References | Wang, Zheng, Lu (2024.06.28.601205v1.5) 2021; 9 Bisong (2024.06.28.601205v1.7) 2019 Jiang, Meng, Lu, Chen, Luo, Chen (2024.06.28.601205v1.4) 2022; 17 Chapman, Chang (2024.06.28.601205v1.8) 2000; 20 Li, Wen, Shen, Lu, Huang, Chang (2024.06.28.601205v1.6) 2011; 11 Lessard (2024.06.28.601205v1.1) 2013 Hoffmann, Gaietta, Zürn, Adams, Terrillon, Ellisman (2024.06.28.601205v1.14) 2010; 5 2024.06.28.601205v1.11 2024.06.28.601205v1.2 Rozen, Skaletsky (2024.06.28.601205v1.10) 2000; 132 Little, Singh, Zhao, Dunn, Matz, Donnenberg (2024.06.28.601205v1.12) 2024 Park, Throop, LaBaer (2024.06.28.601205v1.3) 2015; 110 Cock, Antao, Chang, Chapman, Cox, Dalke (2024.06.28.601205v1.9) 2009; 25 Sohel, Puente, Ramer, Bieber, Wu, Schoolnik (2024.06.28.601205v1.13) 1996; 178 |
| References_xml | – volume: 132 start-page: 365 year: 2000 end-page: 386 ident: 2024.06.28.601205v1.10 article-title: Primer3 on the WWW for general users and for biologist programmers publication-title: Methods Mol Biol doi: 10.1385/1-59259-192-2:365 – volume: 9 start-page: 692797 year: 2021 ident: 2024.06.28.601205v1.5 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 – start-page: 85 year: 2013 end-page: 98 ident: 2024.06.28.601205v1.1 article-title: Molecular Cloning publication-title: Methods in Enzymology – volume: 25 start-page: 1422 year: 2009 end-page: 1423 ident: 2024.06.28.601205v1.9 article-title: Biopython: freely available Python tools for computational molecular biology and bioinformatics publication-title: Bioinformatics doi: 10.1093/bioinformatics/btp163 – ident: 2024.06.28.601205v1.2 article-title: Foundations of Molecular Cloning - Past, Present and Future publication-title: NEB – volume: 110 start-page: 3.20.1 year: 2015 end-page: 3.20.23 ident: 2024.06.28.601205v1.3 article-title: Site-specific recombinational cloning using gateway and in-fusion cloning schemes publication-title: Curr Protoc Mol Biol doi: 10.1002/0471142727.mb0320s110 – volume: 178 start-page: 2613 year: 1996 end-page: 2628 ident: 2024.06.28.601205v1.13 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 – start-page: 59 year: 2019 end-page: 64 ident: 2024.06.28.601205v1.7 publication-title: Building machine learning and deep learning models on google cloud platform: A comprehensive guide for beginners doi: 10.1007/978-1-4842-4470-8_7 – volume: 11 start-page: 92 year: 2011 ident: 2024.06.28.601205v1.6 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 – ident: 2024.06.28.601205v1.11 publication-title: tkinter — Python interface to Tcl/Tk — Python 3.12.2 documentation – volume: 17 start-page: e0273873 year: 2022 ident: 2024.06.28.601205v1.4 article-title: High-throughput FastCloning technology: A low-cost method for parallel cloning publication-title: PLoS ONE doi: 10.1371/journal.pone.0273873 – volume: 20 start-page: 15 year: 2000 end-page: 19 ident: 2024.06.28.601205v1.8 article-title: Biopython publication-title: SIGBIO Newsl doi: 10.1145/360262.360268 – start-page: eesp00032023 year: 2024 ident: 2024.06.28.601205v1.12 article-title: Type IV pili of Enterobacteriaceae species publication-title: Ecosal Plus doi: 10.1128/ecosalplus.esp-0003-2023 – volume: 5 start-page: 1666 year: 2010 end-page: 1677 ident: 2024.06.28.601205v1.14 article-title: Fluorescent labeling of tetracysteine-tagged proteins in intact cells publication-title: Nat Protoc doi: 10.1038/nprot.2010.129 |
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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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