Protocol

Streamlined Gene Assembly PCR

This protocol was adapted from "Streamlined Gene Assembly PCR," Chapter 33, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

To construct genes with artificial, designed sequences, the temperature-cycling steps of the PCR process can be used to assemble whole genes and plasmids from identically sized pieces as small as 40 nucleotides. The original protocol for this process entailed two sequential PCR-like reactions. The first cycling protocol of 55 cycles (called "assembly") contained no PCR primers per se; instead, the 40-mers all primed on each other, building up the product gene by extension of 20 bp at each extension step. In the second cycling protocol, real PCR primers were supplied, and 1-2 µL from the first PCR served as the template for an additional 23 cycles (for a total of 78 cycles). Finally, the PCR product was digested by restriction enzymes and gel-purified for cloning. The procedure presented here is a streamlined version of the original methodology, requiring only one round of 20-25 cycles to obtain a single or major product band, as detected by agarose gel analysis. This can be followed directly by cloning without gel-purification of the target DNA.