De novo DNA synthesis using single molecule PCR.
The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.
Ben Yehezkel
Tuval
T
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Linshiz
Gregory
G
Buaron
Hen
H
Kaplan
Shai
S
Shabi
Uri
U
Shapiro
Ehud
E
D002138:Calibration; D003001:Cloning, Molecular; D019295:Computational Biology; D004247:DNA / Q000096:biosynthesis*; D017931:DNA Primers / Q000737:chemistry; D004272:DNA, Mitochondrial / Q000096:biosynthesis; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D013698:Templates, Genetic
D017931:DNA Primers; D004272:DNA, Mitochondrial; D009692:Nucleic Acid Heteroduplexes; D004247:DNA