Molecular cloning
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Advances in Molecular Cloning: Techniques and Applications
Introduction to Molecular Cloning
Molecular cloning is a fundamental technology in molecular biology that allows for the propagation of DNA segments in bacterial cells. This technique has been pivotal in driving advancements in biotechnology, clinical microbiology, and synthetic biology1 2 5. The process involves the recombination of DNA fragments with a vector, which is then introduced into a host cell for amplification and expression2.
Nimble Cloning: A Versatile and Efficient System
One of the recent advancements in molecular cloning is the development of Nimble Cloning. This method utilizes the restriction enzyme SfiI and T5 exonuclease to linearize vectors and generate 3′-overhangs, making it suitable for both PCR products and plasmids. Nimble Cloning is highly efficient and versatile, supporting gene expression in both prokaryotic and eukaryotic systems. It also allows for the reuse of DNA fragments and is applicable for single or multiple fragment cloning, as well as multi-site cloning1.
In Vivo DNA Assembly: Simplifying Cloning Procedures
In vivo DNA assembly is re-emerging as a powerful tool for molecular cloning, leveraging a RecA-independent recombination pathway present in common laboratory E. coli strains. This method simplifies cloning procedures by eliminating the need for in vitro DNA assembly or specialized bacterial strains. It offers seamless, directional, and sequence-independent cloning, making it an attractive option for the molecular biology community4 8 9.
IVA Cloning: A Single-Tube Universal System
IVA (In Vivo Assembly) cloning exploits the RecA-independent pathway to perform all molecular cloning procedures in a single-tube, single-step PCR. This system is efficient, seamless, and reduces the time required for complex cloning procedures, such as multi-fragment assembly and library construction, to less than two hours9.
Hot Fusion: Efficient Multi-Fragment Cloning
Hot Fusion is another innovative method that allows for the cloning of multiple DNA fragments without the use of ligase. This technique relies on shared homology regions at the junctions and is carried out in a one-step, single-tube reaction. Hot Fusion is highly efficient, with cloning efficiencies of 90-95%, and is particularly useful for applications requiring the cloning of DNA fragments with inverted repeats10.
Applications in Clinical Microbiology
Molecular cloning has become an essential tool in clinical microbiology due to its simplicity, cost-effectiveness, and reliability. It is used for the production of recombinant antigens for screening infectious agents, development of recombinant vaccines, and creation of diagnostic probes. Additionally, cloning techniques have facilitated the production of antimicrobial peptides and recombinant cytokines, which are used as therapeutic agents5.
PCR-Based Cloning: A Comprehensive Protocol
Polymerase chain reaction (PCR) is a critical tool in molecular cloning, enabling the amplification of specific DNA sequences. Recent advancements in PCR reagents, cloning kits, and software have streamlined the cloning process. A comprehensive protocol for PCR-based cloning includes steps for primer design, PCR amplification, sequencing, and gene expression analysis, providing a roadmap for researchers in cellular engineering6.
Conclusion
Molecular cloning continues to evolve with the development of new techniques such as Nimble Cloning, in vivo DNA assembly, IVA cloning, and Hot Fusion. These advancements have simplified cloning procedures, increased efficiency, and expanded the applications of molecular cloning in various fields, including clinical microbiology and synthetic biology. As these methods become more accessible, they will undoubtedly drive further innovations in molecular biology research.
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Most relevant research papers on this topic
Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning
Nimble Cloning is a novel, efficient, and versatile method for standardized molecular cloning, suitable for gene expression in both prokaryotic and eukaryotic expression systems.
Highly CitedMolecular cloning of DNA
Molecular cloning techniques enable the propagation of single DNA segments in bacterial cells, potentially enabling useful functions from other organisms.
Molecular cloning of a new transforming gene from a chemically transformed human cell line
The new transforming gene identified in this study is unrelated to known oncogenes and may serve as a potential therapeutic target for osteosarcoma.
Highly CitedIn vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning
In vivo DNA assembly in E. coli offers a powerful tool for molecular cloning, simplifying procedures and potentially revolutionizing molecular biology research.
Advances and applications of molecular cloning in clinical microbiology
Molecular cloning has revolutionized clinical microbiology by producing recombinant antigens, vaccines, diagnostic probes, and new antibiotics for treating various infectious agents.
Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering
This paper provides a comprehensive PCR-cloning protocol with important tips for researchers focusing on functional aspects of cellular engineering.
A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.
The mini-vector pANY1 enables convenient molecular biological manipulations by meeting the requirements of various cloning strategies and avoiding false positives.
Rapid cloning by homologous recombination in vivo.
This novel cloning method, using the natural intracellular enzyme machinery, allows for rapid production of recombinant plasmid molecules in vivo.
Highly CitedIVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly
IVA cloning simplifies molecular cloning procedures, reducing them to a single-tube, single-step PCR in 2 hours, offering significant advantages over alternative methods.
Highly CitedHot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase
Hot Fusion is an efficient method for cloning multiple DNA fragments and inverted repeats without ligase, simplifying construct design and achieving 90-95% cloning efficiency.
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