Cloning PCR Products

Abstract

Cloning PCR products into a stable vector is often desirable for subsequent analysis such as high quality DNA sequencing, hybridization studies, expression, modification or further subcloning. A wide variety of strategies have been employed and are available in commercially supplied kits (see Page 30 for a listing of manufacturers of PCR cloning kits or vectors). These approaches include simple but relatively inefficient blunt ligation schemes that rely on the 5′ to 3′ proofreading activities of some enzymes such as Pfu polymerase (Costa and Weiner 1994). These enzymes can be used to create blunt-ended PCR products or to remove the 3′ overhang generated by Taq polymerase, creating “polished ends”. A modification of this approach uses the restriction endonuclease Srf 1 to cleave the vector DNA and then supplies Srf 1 in the ligation buffer to provide a higher steady-state concentration of digested vector, which forces the reaction to proceed in the direction of ligation of the insert, as the ligated product is not digestible by Srf 1 (Simcox 1992). Other recent innovations include the addition of 5′ AATTC tails to PCR primers followed by amplification in a mix containing phosphorothioate dGTP. The PCR product is then digested with Exonuclease III, which removes bases from the 3′ end of the strand until it encounters the protected G, leaving an AATT “sticky end”, perfect for ligation into a vector’s Eco R1 site. Yet another method uses the Lambda phage recombination/integration system rather than ligation as a mechanism for cloning (reviewed in Landy 1989). In this approach, attB sites are incorporated into the PCR primers and attP sites are added to the vector. When the PCR product, vector, and Lambda phage integrase (Int) are combined, the PCR product is inserted into the vector in a site-specific manner. The same system can then be used with Lambda phage excisionase (Xis) to remove the insert for transfer to a different vector. Finally, restriction endonucleases, such as Earn 1104 I, which cuts at a defined distance from its recognition site (but not within its site) are used in PCR primers to allow specific “sticky ends” to be produced (Padgett and Sorge 1996). However, most of these approaches are either inefficient, or require the use of proprietary vectors, enzymes, or reagents and are often costly.