PCR Assembly of Synthetic Promoters

In this chapter, we describe a two-step assembly PCR method to construct synthetic promoters. Essentially, this method takes advantage of specific annealing between complimentary DNA sequences to build random TFBS combinations within the assembled PCR products. A DNA polymerase is then employed to fill in the unpaired nucleotides in the generated sequences and also to amplify the assembled PCR products. We have used this method to generate synthetic promoters whereby the orientation of the TFBS can be controlled, the spacing between TFBS can be predetermined, and also the full diversity of the consensus TFBS can be covered through the use of degenerate oligonucleotides .