Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments

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. (C) 2000 Elsevier Science B.V. All rights reserved.