Use of Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes

Although the polymerase chain reaction (PCR) (1 ,2 ) is invaluable for the cloning and manipulation of existing DNA sequences, PCR also makes it possible to create new DNA fragments consisting of a nucleic acid sequence that is specified entirely by the investigator. In this chapter we describe a simple two-step PCR method for the rapid construction of synthetic genes (3 ). This method is based on early observations by Mullis et al. (4 ) in which multiple overlapping oligonucleotides could be used to generate synthetic DNA through several sequential rounds of Klenow-based PCR amplification. The method described in this chapter utilizes the thermostabile Taq polymerase and allows for the generation of synthetic genes in as little as 1 d. This method has proven useful in studies in which synthetic genes were constructed for the HIV-2 Rev protein (3 ,5 ) and the Wilms’ tumor locus zinc finger protein (6 ). Furthermore, this method has been successfully employed in extensive mutagenesis of the HIV-1 rev response element (7 ).