Abstract A novel DNA assembly method, chain reaction cloning (CRC), is described. CRC enables the ordered assembly of multiple DNA fragments in a single step. The power of the technique was demonstrated by the directed in vitro assembly of a plasmid comprised of six DNA fragments from a pool of 12 available fragments. The odds of obtaining the correct plasmid clone in a single step, using conventional techniques, is less than 1 in 191 000 000. Using CRC, the desired plasmid was recovered at a frequency of one in two. Ligation is no longer the rate limiting step in cloning, and limitless possibilities exist for the reconstruction of complex genomes.