Current gene synthesis methods often incorporate a PCR-amplifying step in order to yield sufficient final product that is detectable and resolvable from multiple off-products. This amplification step can cause stochastic sampling effects that propagate errors during the synthesis and lower the variability when applied towards the construction of randomized libraries. We present the method for polymerase step reaction (PSR), a simple DNA polymerase-based gene synthesis reaction that assembles DNA oligonucleotides in a unidirectional fashion without the need for a PCR-type amplification (Lee et al., BioTechniques 59:163–166, 2015). The PSR method is simple and efficient with little off-product production, undetected stochastic sampling effects, and maximized variability when used to synthesize phage display libraries.
Gene synthesis Gene assembly Randomized library construction DNA polymerase Stochastic sampling effect Thermal cycling reaction Variable library and single-stranded DNA
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This work was supported in part by a grant from the National Institute of Health.
LeProust EM, Peck BJ, Spirin K et al (2010) Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res 38:2522–2540CrossRefPubMedPubMedCentralGoogle Scholar
Xiong A-S, Peng R-H, Zhuang J et al (2008) Non-polymerase-cycling-assembly-based chemical gene synthesis: strategies, methods, and progress. Biotechnol Adv 26:121–134CrossRefPubMedGoogle Scholar
Van den Brulle J, Fischer M, Langmann T et al (2008) A novel solid phase technology for high-throughput gene synthesis. Biotechniques 45:340–343CrossRefPubMedGoogle Scholar
Zhou L, Myers AN, Vandersteen JG et al (2004) Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin Chem 50:1328–1335CrossRefPubMedGoogle Scholar
Rydzanicz R, Zhao XS, Johnson PE (2005) Assembly PCR oligo maker: a tool for designing oligodeoxynucleotides for constructing long DNA molecules for RNA production. Nucleic Acids Res 33:W521–W525CrossRefPubMedPubMedCentralGoogle Scholar