SCALABLE METHOD FOR ISOLATION AND SEQUENCE-VERIFICATION OF OLIGONUCLEOTIDES FROM COMPLEX LIBRARIES

摘要

A novel method for preparing sequence-verified oligonucleotides is disclosed. In particular, the invention relates to a simple, affordable, and scalable method that combines high-throughput mating of yeast clones, a unique selectable system for combining DNA sequences in yeast, and next-generation sequencing. This method allows sequence-verified oligonucleotides to be readily isolated from complex libraries.

主权项

1. A method for isolating a sequence-verified oligonucleotide from a composition comprising a mixture of oligonucleotides, the method comprising:
a) providing the composition comprising the mixture of oligonucleotides, wherein each oligonucleotide comprises an unknown sequence and common priming sites for amplification; b) amplifying one or more oligonucleotides; c) transforming a plurality of host cells with the amplified oligonucleotides; d) plating the plurality of transformed host cells in an ordered array on media suitable for growth of the host cells; e) culturing the plurality of transformed host cells under conditions whereby each host cell produces a colony of clones in the ordered array; f) introducing an oligonucleotide from a colony in the ordered array into a barcoder cell, wherein the barcoder cell comprises a nucleic acid comprising a recombination target site for a site-specific recombinase and a barcode sequence that identifies the colony in the ordered array to which the oligonucleotide corresponds; g) translocating the oligonucleotide to a position adjacent to the barcode sequence of the barcoder cell using a site-specific recombinase system, wherein site-specific recombination with the recombination target site of the barcoder cell generates a nucleic acid comprising a barcode-oligonucleotide fusion sequence; h) sequencing the barcode-oligonucleotide fusion sequence to identify a colony in the ordered array comprising a sequence-verified oligonucleotide; i) picking a clone comprising the sequence-verified oligonucleotide from the colony in the ordered array identified by the barcode; and j) isolating the sequence-verified oligonucleotide from the clone.