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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Bibliographic Details
Published in:PloS one Vol. 20; no. 3; p. e0306950
Main Authors: Singh, Pradip Kumar, Donnenberg, Michael S.
Format: Journal Article
Language:English
Published: United States Public Library of Science 13.03.2025
Public Library of Science (PLoS)
Subjects:
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
Online Access:Get full text
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Summary: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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Competing Interests: NO authors have competing interests.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0306950