Simplified gene synthesis: a one-step approach to PCR-based gene construction.
PCR-based gene synthesis conventionally requires two steps: first, all overlapping oligonucleotides are assembled by self-priming; then an additional pair of primers is used to amplify the full-length gene product. Here we propose a simplified method of gene synthesis which combines these two steps into one. We have found that the efficiency of this one-step method, which we term "Simplified Gene Synthesis", is affected by multiple parameters of the PCR reactions. In particular, the choice of polymerase is critical for successful one-step assembly. Other important factors include the concentration of assembly oligonucleotides and amplification primers. Moreover, we offer a general method to estimate, given a known mutation rate, how many clones should be sequenced in order to be confident of obtaining at least one correct gene product. Having determined the accuracy of gene products synthesized under optimal conditions with Simplified Gene Synthesis, we show that our estimation works well. Overall, the simplified gene synthesis provides an easier and more efficient approach to gene synthesis, providing a further step towards the future goal of generalized automation for this process.
Wu
Gang
G
Applied Molecular Biology Laboratory, Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA. wug1@umbc.edu
Wolf
Julie B
JB
Ibrahim
Ameer F
AF
Vadasz
Stephanie
S
Gunasinghe
Muditha
M
Freeland
Stephen J
SJ
D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics*; D005796:Genes; D005818:Genetic Engineering / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004247:DNA