Highly accurate de novo polynucleotide synthesis

Inventors

Peck, Bill James • NOE, Mary • Pitsch, Stefan • Weiss, Patrick A.

Assignees

Twist Bioscience Corp

Abstract

Methods, systems, compositions, and devices for the manufacturing of high-quality building blocks, such as polynucleotides, are described herein. Processes described herein provide for efficient washing of residual reagents, solvents, or byproducts from previous synthetic steps to allow for the generation of polynucleotides with low error rates. Processes described herein also provide for reduction in deletion rates during chemical nucleic acid synthesis. Further, methods and devices described herein allow for the rapid construction and assembly of large libraries of highly accurate polynucleotides.

Core Innovation

The invention relates to methods of polynucleotide synthesis that provide predetermined sequences for a plurality of polynucleotides and use a structure comprising a silicon surface. Nucleosides are coupled to polynucleotides attached to the silicon surface according to the predetermined sequences, a linkage between the nucleoside and the polynucleotide is oxidized with an oxidizing solution, and the silicon surface is washed during synthesis while repeating the coupling and linkage-formation steps.

A key feature is washing the silicon surface with a wash solvent wherein the wash solvent consists essentially of acetone. In one implementation, after washing, the process includes washing the silicon surface with a capping solution comprising at least one base, at least one O-nucleophile selected from acetic acid, formic acid, propionic acid, methoxyacetic acid, phenoxyacetic acid, and water, and tetrahydrofuran.

Another aspect uses a silicon surface comprising a plurality of discrete loci configured for polynucleotide synthesis. This implementation includes coupling at least one nucleoside to a polynucleotide attached to the silicon surface according to the predetermined sequences, oxidizing a linkage with an oxidizing solution, and washing the silicon surface with a wash solvent consisting essentially of acetone.

Claims Coverage

The patent includes two independent claims. Across these independent claims, the inventive subject matter centers on polynucleotide synthesis on a silicon surface using acetone-based washing with specified capping and oxidation steps, with one claim additionally requiring discrete loci on the silicon surface.

Taken together, the independent claims cover a repeating polynucleotide synthesis process performed on a silicon surface using a wash solvent that consists essentially of acetone, combined with oxidation of the nucleoside–polynucleotide linkage and, in one claim, a specifically defined capping solution chemistry. The other claim further requires that the silicon surface includes discrete loci configured for polynucleotide synthesis.

Stated Advantages

Documented Applications