DNA cloning

In recent years the ISPC has adapted two Ligation Independent Cloning (LIC) strategies based on whole plasmid amplification of the vector and the insert:

  • Restriction Free (RF) cloning
  • Transfer-PCR (TPCR)

The RF (van den Ent and Lowe, Unger et al., Peleg and Unger) and TPCR (Erijman et al., Erijman, Shifman and Peleg) procedures are universal cloning methods allowing a precise insertion of a DNA fragment into any desired position within a circular plasmid. Both methods are based on synthesis of mega primers (up to several kbs) containing flanking target-vector specific sequences. The mega-primers integrate into the destination vector at predesigned positions in a linear amplification reaction.

The RF cloning is a two-stage process:

  • Synthesis and purification of the mega-primers.
  • Integration of the mega-primers into the destination vector and generation the new recombinant vector including the gene of interest.

The TPCR is a single tube cloning reaction where all reaction components are mixed in one tube. The efficiency of the reaction is highly dependent on the initial primer concentration (10-20 nM per primer).

The RF/TPCR methodologies were used successfully in a variety of applications to manipulate the target DNA sequence. The following figure illustrates some such applications.

Schematic illustration of the RF/TPCR applications 

Advantages of the RF and TPCR

  • Any vector of choice can be used
  • No need for restriction enzymes digestion of both vector and PCR product
  • Cloning can be performed at any desired position
  • No need in any pre-treatment of the recipient vector (circular vector)
  • No need in purchasing any commercial kit
  • Cheap and fast - requires only Thermostable polymerase (e.g., Phusion, Q5 from NEB)