Accurate multiplex gene synthesis from programmable DNA microchips

Abstract

Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

Protein Interfacial Pocket Engineering via Coupled Computational Filtering and Biological Focusing Criterion

“…Preliminary empirical validation was carried out using a cell survival assay. R.PvuII Putative Engineered IP mutant was synthesized de novo using a similar protocol as described in 41 and the correct synthetic sequence was selected and confirmed by standard DNA sequencing technology. This synthetic gene was sub-cloned into the pET-21a expression vector (Novagen), which was then transformed into E. coli BL21 cells.…”

Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes

“…It perhaps shows that the starting oligonucleotides that we used for PCA had an error rate of less than 1:6000. High-throughput gene synthesis on microchips using improved oligonucleotide synthesis method was recently reported (Tian et al, 2004;Engels, 2005). Tian et al (2004) developed a stringent hybridization method to remove error-containing oligonucleotides and using such oligos, a complete operon of 14.6 kb for 21 genes was assembled.…”

“…High-throughput gene synthesis on microchips using improved oligonucleotide synthesis method was recently reported (Tian et al, 2004;Engels, 2005). Tian et al (2004) developed a stringent hybridization method to remove error-containing oligonucleotides and using such oligos, a complete operon of 14.6 kb for 21 genes was assembled. The error rate for the oligonucleotides was estimated to be 1:7300 bp, confirming that error rate of synthetic oligonucleotides is one of the limiting step.…”

In situ Synthesis of DNA Microarray on Functionalized Cyclic Olefin Copolymer Substrate

“…In addition to on-chip assays, in situ synthesized oligonucleotide arrays can also be used to generate pools of mixed oligos for off-chip use as oligo libraries or gene building blocks (6)(7)(8)(9).…”