Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides

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 DNA 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 DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 β-lactamase-encoding 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 amplified and cloned in a vector containing the tetracycline-resistance gene (Tc R ) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the Tc R colonies were Ap R , making this approach a general method for the rapid and cost-effective synthesis 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 α-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli , yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Gene Elsevier

Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides

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Publisher
Elsevier
Copyright
Copyright © 1995 Elsevier Ltd
ISSN
0378-1119
eISSN
1879-0038
D.O.I.
10.1016/0378-1119(95)00511-4
Publisher site
See Article on Publisher Site

Abstract

Here, we describe assembly PCR as a method for the synthesis of long DNA 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 DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 β-lactamase-encoding 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 amplified and cloned in a vector containing the tetracycline-resistance gene (Tc R ) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the Tc R colonies were Ap R , making this approach a general method for the rapid and cost-effective synthesis 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 α-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli , yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.

Journal

GeneElsevier

Published: Oct 16, 1995

References

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