DNA Restriction-Free Overlapping Sequence Cloning Techniques: A Modern Toolkit for Synthetic Biology and Genetic Engineering
Abstract
Background: Traditional restriction enzyme–based cloning techniques are limited by dependence on specific recognition sites, reduced flexibility, and introduction of unwanted sequences. To overcome these barriers, restriction-free overlapping sequence cloning methods have emerged as powerful alternatives. These strategies exploit homologous sequence overlaps and enzymatic reactions to enable scarless, seamless, and efficient DNA assembly. Over the past two decades, approaches such as Gibson Assembly, Circular Polymerase Extension Cloning (CPEC), In Vivo Cloning, Polymerase Incomplete Primer Extension (PIPE), Sequence and Ligation Independent Cloning (SLIC), and Overlap Extension PCR Cloning (OE-PCR) have reshaped synthetic biology and genetic engineering. Methods: A comprehensive literature search was performed for studies published between 2000 and 2025 in peer-reviewed journals. Inclusion criteria encompassed experimental studies, reviews, and mechanistic reports focusing on restriction-free, overlap-based cloning methods. Exclusion criteria removed articles not in English, lacking full-text availability, providing insufficient methodological details, or relying exclusively on computational predictions. Non–peer-reviewed publications, editorials, conference abstracts, and unrelated molecular biology studies were also excluded. Results: Analysis of selected literature demonstrated that overlap-based cloning strategies provide higher versatility and fidelity compared with conventional methods. Gibson Assembly and In Vivo Cloning showed the highest adoption due to efficiency and user-friendliness, while CPEC, PIPE, SLIC, and OE-PCR offered cost-effective and flexible alternatives for specific applications. These methods enable efficient construction of genetic circuits, gene synthesis, metabolic pathway engineering, and therapeutic constructs. Comparative assessments revealed trade-offs in error rates, fragment size limits, scalability, and cost across different techniques. Integration into high-throughput and automated systems has further enhanced their applicability in synthetic biology. Conclusion: Restriction-free overlapping sequence cloning has become a cornerstone of modern molecular biology, providing a robust toolkit for seamless DNA assembly. Its adoption facilitates rapid progress in synthetic biology, functional genomics, and therapeutic engineering. Future directions include integration with CRISPR-based editing, cell-free systems, and emerging DNA synthesis technologies, which together promise to expand the scope and efficiency of genetic engineering.
Keywords: Restriction-free cloning, Gibson Assembly, Circular Polymerase Extension Cloning, In Vivo Cloning, Polymerase Incomplete Primer Extension, Sequence and Ligation Independent Cloning, Overlap Extension PCR Cloning
https://doi.org/10.5281/zenodo.16977920
