title: Gene Assembly from Chip-Synthesized Oligonucleotides.
abstract: De novosynthesis of long double-stranded DNA constructs has a myriad of applications in biology and biological engineering. However, its widespread adoption has been hindered by high costs. Cost can be significantly reduced by using oligonucleotides synthesized on high-density DNA chips. However, most methods for using off-chip DNA for gene synthesis have failed to scale due to the high error rates, low yields, and high chemical complexity of the chip-synthesized oligonucleotides. We have recently demonstrated that some commercial DNA chip manufacturers have improved error rates, and that the issues of chemical complexity and low yields can be solved by using barcoded primers to accurately and efficiently amplify subpools of oligonucleotides. This article includes protocols for computationally designing the DNA chip, amplifying the oligonucleotide subpools, and assembling 500-800 basepair (bp) constructs.
authors: Harvard School of Engineering and Applied Sciences, Cambridge, Massachusetts.
authors: Nikolai
authors: N
authors: Eroshenko
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts ; Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts.
authors: Sriram
authors: S
authors: Kosuri
authors: Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts ; Harvard Biophysics Program, Cambridge, Massachusetts.
authors: Adam H
authors: AH
authors: Marblestone
authors: Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts.
authors: Nicholas
authors: N
authors: Conway
authors: Department of Genetics, Harvard Medical School, Boston, Massachusetts ; Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts.
authors: George M
authors: GM
authors: Church
keywords: gene synthesis; nucleic acids; oligonucleotide; synthetic biology