Cloning PCR Products Using T-Vectors

Abstract

The polymerase chain reaction (PCR) has revolutionized the way that molecular biologists approach the manipulation of nucleic acids through its ability to amplify specific DNA sequences (1–3). It has numerous applications (4), many of which require the cloning of amplified DNA products into vectors for further analysis. This is commonly achieved by so-called “sticky end” cloning, in which restriction endonuclease recognition sites are incorporated into the 5′ ends of the PCR primers (5). Following amplification, the DNA fragment is purified, digested with the appropriate enzyme(s), and then ligated into an identically restricted vector. Obtaining efficient cleavage at the extreme ends of linear PCR products can be difficult (6), and moreover, their use can result in the restriction of sites that lie within the amplified DNA fragment. The one particular advantage to this method is that the PCR product can be force-cloned using designed restriction sites, so, for example, a DNA fragment, such as a leader signal sequence, can be fused in-frame to a structural gene for expression studies.