Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products.

The ligation-independent cloning of PCR products (LIC-PCR) is a versatile and highly efficient cloning procedure resulting in recombinant clones only. Recombinants are generated between PCR products and a PCR-amplified vector through defined complementary single-stranded (ss) ends artificially generated with T4 DNA polymerase. This procedure does not require restriction enzymes, alkaline phosphatase, or DNA ligase. The primers used for amplification contain an additional 12-nucleotide sequence at their 5' ends that is complementary in the vector- and insert-specific primers. The (3'-->5') exonuclease activity of T4 DNA polymerase is used in combination with a predetermined dNTP (dGTP for the inserts and dCTP for the vector) to specifically remove 12 nucleotides from each 3' end of the PCR fragments. Because of the complementarity of the ends that are generated, circularization can occur between vector and insert. The recombinant molecules do not require in vitro ligation for efficient bacterial transformation. To make this technique widely applicable, we have simplified the handling of the PCR fragments prior to LIC. The PCR products do not need further purification following the T4 DNA polymerase treatment. Incubation of vector and insert PCR fragments for as little as 5 min is sufficient for a high yield of recombinants. Comparison of the transformation efficiencies using different-length LIC tails revealed that using 12-nucleotide cohesive ends produced four times more transformants than were obtained with the LIC with 10-nucleotide cohesive ends. When the LIC tails were 8 nucleotides long, no transformants were obtained. PCR fragment purification, T4 DNA polymerase treatment, and LIC is complete in < 1 hr.