SINGLE-STEP ASSEMBLY OF A GENE AND ENTIRE PLASMID FROM LARGE NUMBERS OF OLIGODEOXYRIBONUCLEOTIDES

Here, we describe assembly PCR as a method for the synthesis of long D NA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DN A polymerase to build increasingly longer DNA fragments during the ass embly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-e ncoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplifed and cloned in a vector containing the tetracycline-resist ance gene (Tc-R) as the sole selectable marker. Without relying on amp icillin (Ap) selection, 76% of the Tc-R colonies were Ap(R), making th is approach a general method for the rapid and cost-effective synthesi s of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alph a-fragment of the lacZ gene and the pUC origin of replication. Digesti on with a unique restriction enzyme, followed by ligation and transfor mation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.