A T-Linker Strategy for Modification and Directional Cloning of PCR Products

The propensity of Taq polymerase to add 3′-A overhangs (1 ,2 ) to polymerase chain reaction (PCR)-amplified DNA has made possible a simple method for cloning PCR products into a T-vector (Invitrogen, San Diego, CA) (3 –5 ). Here, we present a related strategy that uses T-linkers to add sequences, such as restriction sites, to the ends of PCR products (see Note 1 ). A single-base T overhang at the end of a synthetic double-stranded oligonucleotide linker allows ligation of the linker to the unpolished ends of a PCR product. This avoids the expense of adding the “extra” sequences to sequence-specific primers.