Molecular Biology
Cloning Methods
Cloning Methods
The generation of recombinant DNA molecules is an essential tool in modern molecular biology. Conventional DNA cloning strategies that have been used for several decades typically involve the use of type II restriction enzymes to generate appropriate DNA fragments with blunt or sticky ends and the ligation of the DNA fragments to generate plasmid or other type DNA constructs [1]. However, these procedures depend on the presence of appropriate restriction sites and often leave unwanted sequences at the junction sites. To avoid such limitations and complicated procedures, various seamless DNA cloning methods (Gibson assembly, SLIC) have been developed over the last decade.
The generation of recombinant DNA molecules is an essential tool in modern molecular biology. Conventional DNA cloning strategies that have been used for several decades typically involve the use of type II restriction enzymes to generate appropriate DNA fragments with blunt or sticky ends and the ligation of the DNA fragments to generate plasmid or other type DNA constructs [1]. However, these procedures depend on the presence of appropriate restriction sites and often leave unwanted sequences at the junction sites. To avoid such limitations and complicated procedures, various seamless DNA cloning methods (Gibson assembly, SLIC) have been developed over the last decade.
The Auer group has developed a modified seamless ligation method based on the Gibson Assembly, showing that highly efficient insert-plasmid assembly can be achieved, using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, significantly reducing the cost [2].
The Auer group has developed a modified seamless ligation method based on the Gibson Assembly, showing that highly efficient insert-plasmid assembly can be achieved, using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, significantly reducing the cost [2].
The Auer group is also using a second seamless ligation method called SLiCE (Seamless Ligation Cloning Extract). The method is based on the DNA recombination characteristics of E. coli laboratory strain cell lysates. Although the ligation efficiency is lower than that observed for other seamless ligation methods, multiple DNA fragments have been ligated simultaneously. In addition, by using standard laboratory chemicals the SLiCE- method becomes an ultra-low cost homemade seamless DNA cloning method [3-4].
The Auer group is also using a second seamless ligation method called SLiCE (Seamless Ligation Cloning Extract). The method is based on the DNA recombination characteristics of E. coli laboratory strain cell lysates. Although the ligation efficiency is lower than that observed for other seamless ligation methods, multiple DNA fragments have been ligated simultaneously. In addition, by using standard laboratory chemicals the SLiCE- method becomes an ultra-low cost homemade seamless DNA cloning method [3-4].
1. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual, Fourth Edition edn. Cold Spring Harbor: NY Cold Spring Harbor Laboratory Press; 2012.
2. Benoit RM, Ostermeier C, Geiser M, Li JSZ , Widmer H, Auer M (2016) Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. PLOS One 11(4) e0153158.
3. Zhang Y, Werling U & Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Research doi: 10.1093/nar/gkr1288.
4. Okegawa Y, Motohashi K (2015) A simple and ultra-lowcost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit. Biochemistry and Biophysics Reports 4: 148-151.