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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Vydané v:	PloS one Ročník 20; číslo 3; s. e0306950
Hlavní autori:	Singh, Pradip Kumar, Donnenberg, Michael S.
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	United States Public Library of Science 13.03.2025 Public Library of Science (PLoS)
Predmet:	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 United States
ISSN:	1932-6203, 1932-6203
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Popis
Shrnutí:	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.
Bibliografia:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Competing Interests: NO authors have competing interests.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0306950