Twin PCRs: a simple and efficient method for directional cloning of PCR products

A simple and efficient method was developed for directional cloning of PCR products without any restriction enzyme digestion of the amplified sequence. Two pairs of primers were designed in which parts of two restriction enzyme recognition sequences were integrated, and the primers were used for two parallel PCRs. The PCR products were mixed, heat denatured and re-annealed to generate hybridized DNA fragments bearing sticky ends compatible with restriction enzymes. This method is particularly useful when it is necessary to use a restriction enzyme but there is an additional internal restriction site within the amplified sequence, or when there are problems caused by end sensitivity of restriction enzymes.