Methods for assembling genetic constructs
Plasmid assembly plays a crucial role in a wide range of molecular biology research and applied tasks involving recombinant DNA molecules. Modern technologies make it possible to assemble plasmids with high accuracy and speed and at minimal cost. Below we review the main modern methods of plasmid assembly, along with their advantages and disadvantages.
Restriction enzyme–based ligation
A classic method that involves digestion of the plasmid and the insert (e.g., a gene) with restriction enzymes, followed by ligation with DNA ligase. This approach was foundational to the development of genetic engineering and remains popular due to its reliability and simplicity. However, it has several limitations, such as the need for specific restriction sites and relatively high labor intensity [Green, M. R., & Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual. 4th edition, 448].
Gibson assembly
One of the most popular modern methods for plasmid assembly, developed by Daniel Gibson [doi.org/10.1038/nmeth.1318]. The method enables simultaneous joining of multiple DNA fragments in a single tube. The principle is based on the use of exonucleases, DNA polymerases, and DNA ligases, which facilitate the joining of fragments to one another. Key advantages include high accuracy, the ability to assemble large constructs, and no requirement for specific restriction sites (Fig. 1).
Restriction enzyme–based ligation
A classic method that involves digestion of the plasmid and the insert (e.g., a gene) with restriction enzymes, followed by ligation with DNA ligase. This approach was foundational to the development of genetic engineering and remains popular due to its reliability and simplicity. However, it has several limitations, such as the need for specific restriction sites and relatively high labor intensity [Green, M. R., & Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual. 4th edition, 448].
Gibson assembly
One of the most popular modern methods for plasmid assembly, developed by Daniel Gibson [doi.org/10.1038/nmeth.1318]. The method enables simultaneous joining of multiple DNA fragments in a single tube. The principle is based on the use of exonucleases, DNA polymerases, and DNA ligases, which facilitate the joining of fragments to one another. Key advantages include high accuracy, the ability to assemble large constructs, and no requirement for specific restriction sites (Fig. 1).
Figure 1. Schematic of Gibson assembly for genetic constructs [doi.org/10.1038/nmeth.1318].
Golden Gate assembly
This method is based on the use of type IIS restriction enzymes. A key feature of the method is the generation of matching cohesive (sticky) ends in pairs, which enables simultaneous ligation. Golden Gate is widely used in modern cloning [doi.org/10.1371/journal.pone.0003647].
Site-specific recombination–based methods
These approaches rely on bacterial recombination sites (attP, attB, attL, attR). The system enables insertion of DNA fragments into a vector via recombination using multiple variants of phage recombinases [doi.org/10.1101/gr.143000].
There is also a range of other, more exotic approaches, such as methods using PCR, bacterial extracts, etc. [doi.org/10.1093/nar/gkr1288, doi.org/10.1371/journal.pone.0006441].
DUOX BIOTECHNOLOGIES uses all modern approaches for assembling genetic constructs, selecting the most optimal method for each task. This makes it possible to obtain the desired molecules within short timeframes. To order genetic constructs, please contact our specialists.
This method is based on the use of type IIS restriction enzymes. A key feature of the method is the generation of matching cohesive (sticky) ends in pairs, which enables simultaneous ligation. Golden Gate is widely used in modern cloning [doi.org/10.1371/journal.pone.0003647].
Site-specific recombination–based methods
These approaches rely on bacterial recombination sites (attP, attB, attL, attR). The system enables insertion of DNA fragments into a vector via recombination using multiple variants of phage recombinases [doi.org/10.1101/gr.143000].
There is also a range of other, more exotic approaches, such as methods using PCR, bacterial extracts, etc. [doi.org/10.1093/nar/gkr1288, doi.org/10.1371/journal.pone.0006441].
DUOX BIOTECHNOLOGIES uses all modern approaches for assembling genetic constructs, selecting the most optimal method for each task. This makes it possible to obtain the desired molecules within short timeframes. To order genetic constructs, please contact our specialists.