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Although the polymerase chain reaction (1,2) (PCR) can be used to produce a large amount of a specific DNA from a complex source, cloning the PCR products has not proven to be straightforward. Restriction endonuclease sites are often incorporated into the oligonucleotide primers used for amplification, so that cleavage of the product will create sticky ends that can theoretically be ligated to an equivalently cut vector (3). Unfortunately, many restriction endonucleases fail to cleave when their recognition sequences are located within a few base pairs of the end of a DNA fragment (4, 5). Ligation-independent PCR cloning schemes (6, 7, 8) involve the addition of at least 12 bases to the 5' end of the primer, which can increase the cost of synthesis substantially when done routinely. Attempts to clone PCR products as blunt-ended fragments have been very inefficient, due to the template-independent terminal transferase activity of Taq polymerase, which results in the addition of a single nucleotide at the 3' end of the fragment (9, 10). This nucleotide is almost exclusively an adenosine, due to the strong preference of the polymerase for dATP (9). Thus, cloning the products as blunt-ended fragments requires enzymatic processing to remove of the 3' overhang using an enzyme with 3' to 5' exonuclease activity (11).

Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products