METHOD OF ON-CHIP NUCLEIC ACID MOLECULE SYNTHESIS

摘要

A method of synthesizing a nucleic acid molecule, such as a gene, on a substrate or microchip is described. In particular, a method for synthesizing, amplifying, and assembling DNA oligonucleotides into a nucleic acid molecule or gene product, on a single substrate or microchip is described. Also described are a method of correcting a sequence error in a synthesized nucleic acid molecule, as well as a method for synthesizing and screening a library of codon variants to identify a nucleic acid molecule with an optimized level of protein expression.

主权项

1. A method of synthesizing a nucleic acid molecule having a target sequence, comprising:
(1) obtaining a substrate having a chamber comprising a plurality of immobilized oligonucleotides for the synthesis of the target sequence, (2) adding to the chamber a reaction mixture comprising dNTPs, a primer, a strand-displacing polymerase, a nicking endonuclease, a heat-stable DNA polymerase, and a buffer; (3) amplifying the plurality of oligonucleotides to obtain free amplified oligonucleotides by a nicking strand displacement amplification reaction in the chamber containing the reaction mixture; and (4) assembling the free amplified oligonucleotides by a polymerase cycling assembly reaction to obtain the nucleic acid molecule; wherein step (4) is conducted in the chamber without the need for a buffer change after step (3).