Gene and library synthesis without amplification: polymerase step reaction (PSR).
Current gene synthesis methods often incorporate a PCR amplification step in order to yield final material sufficient for resolution from multiple off-products. These amplification steps can cause stochastic sampling effects that propagate errors in gene synthesis or decrease variability when applied to the construction of randomized libraries. We have developed a simple DNA polymerase-based gene synthesis reaction, polymerase step reaction (PSR), that assembles DNA oligonucleotides in a unidirectional fashion without the need for amplification. We demonstrate that PSR is efficient, with little off-product production, no detectable error propagation, and maximized variability in the synthesis of a phage display library.
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO.
Zhuo-Bin
ZB
Lee
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO.
Christopher
C
Firnhaber
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO.
Jesse
J
Clarke
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO.
Brian S
BS
DeDecker
D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction; D061905:Single-Domain Antibodies / Q000235:genetics
D017931:DNA Primers; D004277:DNA, Single-Stranded; D061905:Single-Domain Antibodies; D004247:DNA; D004259:DNA-Directed DNA Polymerase
DNA polymerase; gene assembly; gene synthesis; randomized library construction; stochastic sampling effects