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.