Cloning Polymerase Chain Reaction (PCR) Products: TA Cloning

Abstract

The nontemplate-dependent terminal transferase activity inherent in nonproofreading DNA polymerases such as Taq provides a highly efficient method to clone PCR products. The enzyme adds a single, unpaired residue—preferentially an adenosyl residue—to each 3′ end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3′-thymidyl residue at each end. The two chief advantages of TA cloning are speed and lack of reliance on restriction enzymes. The major disadvantage is an inability to clone directionally. For this reason, it is important to pick and analyze several transformed clones when a particular orientation of the amplified fragment is required.