Microfluidic-based gene synthesis
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.
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, wherein the pool of oligonucleotides comprises at least two different types of oligonucleotides such that each different type of oligonucleotide in the pool has homology with a different DNA region of the target cell than the DNA regions with which the other types of oligonucleotides have homology and each different type of oligonucleotide encodes for a different point mutation than the point mutations encoded for by the other types of oligonucleotides; 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 of different types,
wherein the steps of electroporating and incorporating are carried out entirely within a microfluidic chip, the microfluidic chip comprising a microfluidic device having a fluidic architecture configured for implementing the steps of electroporating and incorporating.
2. The method of claim 1 , wherein the step of electroporating is repeated a plurality of times using a plurality of oligonucleotide pools.
3. The method of claim 1 , comprising steps for hierarchical assembly of the oligonucleotides in the pool, comprising:
synthesizing small oligonucleotide segments in parallel in separate microfluidic chambers;
introducing the synthesized small oligonucleotide segments into a subsequent chamber; and
assembling the oligonucleotides,
wherein the microfluidic chambers and the subsequent chamber are contained on one or more microfluidic chips.
4. The method of claim 1 , further comprising the step of applying at least one form of error correction.
5. The method of claim 4 , wherein the error correction comprises at least one cycle of hybridization by selection and is carried out within the microfluidic system.
6. A method for the synthesis of a modified DNA construct, comprising the steps of:
introducing, into at least one target cell, at least one polynucleotide construct having homology with a plurality of DNA regions of the target cell and encoding for a plurality of different point mutations;
generating, using the polynucleotide construct, a plurality of oligonucleotides within the target cell, wherein the plurality of oligonucleotides comprises at least two different types of oligonucleotides such that each different type of oligonucleotide within the target cell has homology with a different DNA region of the target cell than the DNA regions with which the other types of oligonucleotides have homology and each different type of oligonucleotide encodes for a different point mutation than the point mutations encoded for by the other types of oligonucleotides; and
simultaneously incorporating, in a single step, the 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 introducing, generating, and incorporating are carried out entirely within a single microfluidic chip, the microfluidic chip comprising a microfluidic device having a fluidic architecture configured for implementing the steps of introducing, generating, and incorporating.
7. The method of claim 6 , wherein the step of introducing is carried out through electroporation.
8. The method of claim 6 , further comprising the step of applying at least one form of error correction.
9. The method of claim 8 , wherein the error correction comprises at least one cycle of hybridization by selection and is carried out within the microfluidic system.