High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing.
The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.
Febit group, Heidelberg, Germany.
Mark
M
Matzas
Peer F
PF
Stähler
Nathalie
N
Kefer
Nicole
N
Siebelt
Valesca
V
Boisguérin
Jack T
JT
Leonard
Andreas
A
Keller
Cord F
CF
Stähler
Pamela
P
Häberle
Baback
B
Gharizadeh
Farbod
F
Babrzadeh
George M
GM
Church
D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D005813:Genes, Synthetic; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012371:Robotics
D009841:Oligonucleotides; D004247:DNA