Microfluidic-based Gene Synthesis

摘要

A method for synthesizing long DNA constructs from oligonucleotide precursors directly within a microfluidic device uses several oligonucleotides at once. A precursor mix containing at least two oligonucleotide precursors with at least partial base complementarity is introduced into an input of a microfluidic chip and at least one cycle of at least one gene synthesis protocol is applied to fabricate a DNA construct containing the sequence of at least two oligonucleotide precursors. A method for the synthesis of a modified DNA construct includes electroporating at least one oligonucleotide encoding for at least one point mutation and having homology with at least one DNA region of a target cell into the target cell and incorporating the oligonucleotide into the target cell DNA through the action of recombination protein beta or a recombination protein beta functional homolog.

主权项

1. A method for the synthesis of a modified DNA construct comprising the steps of:
electroporating a pool of oligonucleotides into at least one target cell, each oligonucleotide having homology with at least one DNA region of the target cell and encoding for at least one point mutation, wherein at least two of the electroporated oligonucleotides from the pool code for different point mutations; and simultaneously incorporating, in a single step, the electroporated oligonucleotides into the target cell DNA through the action of recombination protein beta or a recombination protein beta functional homolog, simultaneously bringing about a plurality of simultaneous point mutations within the target cell, wherein at least two of the simultaneous point mutations are different, wherein the steps of electroporating and incorporating are carried out entirely within a microfluidic system, the microfluidic system comprising a microfluidic device having an electroporation means and a fluidic architecture configured for implementing the steps of electroporating and incorporating.