In the last 20 years, applications for the polymerase chain reaction (PCR) technique (Saiki et al., 1988) have been developed in almost every field in Molecular Biology. However, in a large number of situations, observing an amplification band on a gel is not enough. Sequencing, directed mutagenesis, polymorphic transcription (widely used in drug-genomics identification), obtaining labeled probes and expression of proteins for their functional study (necessary in basic science and applied mainly in biomedicine), require cloning. Cloning of PCR products has supplied the market with new kits that, although sharing some properties, offer unique features and increasingly innovative technologies.
For years, Taq DNA polymerase has been one of the most robust enzymes for the amplification of fragments by PCR (Saiki et al., 1988). For years, the market was dominated by the use of this enzyme and by those cloning kits that were adapted to the cohesive 3’A (adenine) ends that were generated with its use. However, Taq DNA polymerase produces fragments with an error rate of 7.39%. This has favored the development of DNA polymerases with proofreading activity. The enzymes with first generation proofreading activity (Pfu or Pwo) are not very robust, but the development of second-generation DNA polymerases that are much more robust and capable for the amplification of larger fragments has favored the development of cloning systems for blunt-end fragments, although these systems are not as well established in the market as T-vector Cloning Kits (Mead et al., 1991).
With the development of a new generation of proofreading polymerases with a fidelity, flexibility, and robustness superior to those of the first-generation enzymes, the lack of efficient methods for cloning of PCR products generated by these enzymes became evident, products that are characterized by their blunt ends. Even today, most of the existing kits are based on dephosphorylation of linear vectors with the consequent high background due to the recircularization of the non-dephosphorylated molecules. Some companies have solved the background problem in blunt ligations in transformation plaques using technologies that allow a positive selection of those colonies that have recombinant DNA, ie, only vectors that have incorporated the insert will give rise to colonies. Most of these cloning systems employ toxic genes in such a way that the disruption by the insert of such a toxic gene generates a viable colony. If the insert is not cloned into the vector and the vector becomes “empty”, the colony dies because of the expression of the toxic gene.
In Canvax Biotech we have developed a proprietary technology for the cloning of amplified PCR fragments with High Fidelity DNA Polymerases. pSpark® is based on the modification of the vector ends by special enzymes, with a very small background and a very high efficiency in the blunt-end cloning procedure.
In future articles in this blog, we will review and explain the existing cloning methods with emphasis on technologies aimed at cloning of fragments obtained by PCR. Based on this, we classify the cloning systems into directional and non-directional cloning systems and according to the ends of both the vector and the insert, into blunt or protruding. Each of them can be in turn analyzed according to the enzyme used for the insert-vector binding, using methods based on T4 DNA Ligase, on the activity of the CRE recombinase and/or on the Topoisomerase.
Saiki R.K.R., Gelfand D.H.D., Stoffel S.S., Scharf S.J.S., Higuchi R.R., Horn G.T.G., Mullis K.B.K., & Erlich H.A.H. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487–491.
Mead D.A.D., Pey N.K.N., Herrnstadt C.C., Marcil R.A.R., & Smith L.M.L. (1991) A universal method for the direct cloning of PCR amplified nucleic acid. Bio/Technology, 9, 657–663.