Construction of Synthetic Genes by Polymerase Chain Reaction

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 thermostable
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
).