Scalable method for isolation and sequence-verification of oligonucleotides from complex libraries

Patent metadata

Field	Value
Publication number	US-2016122748-A1
Application number	US-201514928928-A
Country	US
Kind code	A1
Filing date	Oct 30, 2015
Priority date	Oct 30, 2014
Publication date	May 5, 2016
Grant date	—

Abstract

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.

First claim

What is claimed is:

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.

2 . The method of claim 1 , wherein the oligonucleotides are amplified with a primer comprising an integration targeting sequence that is sufficiently homologous to a genomic target locus of the host cell, such that amplicons of the oligonucleotides integrate into the genome at the target locus in the transformed host cell by homologous recombination.

3 . The method of claim 2 , further comprising using a selectable marker that selects for clones that have undergone successful integration of an oligonucleotide at the genomic target locus.

4 . The method of claim 2 , further comprising transforming the plurality of host cells with a recombinant polynucleotide encoding a restriction enzyme operably linked to a promotor, wherein the restriction enzyme creates a double strand break at the genomic target locus that facilitates integration of an oligonucleotide at the genomic target locus.

5 . The method of claim 2 , wherein the genomic target locus is adjacent to a recombination target site capable of undergoing recombination with the recombination target site of the barcoder cell to produce a barcode-oligonucleotide fusion sequence.

6 . The method of claim 1 , wherein the oligonucleotides are amplified with a primer comprising a targeting sequence that is sufficiently homologous to a target locus on a plasmid in the host cell, such that the oligonucleotide amplicons integrate into the plasmid at the target locus in the transformed host cell.

7 . The method of claim 6 , further comprising using a selectable marker that selects for clones that have undergone successful integration of an oligonucleotide at the plasmid target locus.

8 . The method of claim 6 , further comprising transforming the plurality of host cells with a recombinant polynucleotide encoding a restriction enzyme operably linked to a promotor, wherein the restriction enzyme creates a double strand break at the target locus of the plasmid that facilitates integration of the oligonucleotide into the plasmid at the target locus.

9 . The method of claim 6 , wherein the plasmid target locus is adjacent to a recombination target site capable of undergoing recombination with the recombination target site of the barcoder cell to produce a barcode-oligonucleotide fusion sequence.

10 . The method of claim 1 , wherein the oligonucleotides are amplified with a primer comprising a recombination target site capable of undergoing recombination with the recombination target site of the barcoder cell.

11 . The method of claim 1 , wherein the unknown sequence of each oligonucleotide is flanked by a common 5′ restriction site and a common 3′ restriction site.

12 . The method of claim 11 , further comprising performing a restriction digest that selectively cleaves each oligonucleotide at the common 5′ restriction site and the common 3′ restriction site to produce a restriction fragment.

13 . The method of claim 12 , further comprising cloning the restriction fragment into a vector.

14 . The method of claim 13 , wherein the vector is a plasmid or viral vector.

15 . The method of claim 14 , wherein the vector further comprises a recombination target site capable of undergoing recombination with the recombination target site of the barcoder cell to produce a barcode-oligonucleotide fusion sequence.

16 . The method of claim 1 , wherein transforming the plurality of host cells comprises introducing vectors comprising the oligonucleotides into the host cells.

17 . The method of claim 1 , wherein the host cells are prokaryotic cells or eukaryotic cells.

18 . The method of claim 17 , wherein the host cells are yeast.

19 . The method of claim 17 , wherein the host cells are bacteria.

20 . The method of claim 1 , wherein the site-specific recombinase system is a Cre-loxP site-specific recombinase system, a Flp-FRT site-specific recombinase system, a PhiC31-att site-specific recombinase system, or a Dre-rox site-specific recombinase system.

21 . The method of claim 1 , further comprising using a selectable marker that selects for clones that have undergone successful site-specific recombination.

22 . The method of claim 1 , wherein amplifying the oligonucleotides is performed using a method selected from the group consisting of polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR).

23 . The method of claim 22 , wherein the oligonucleotides are amplified using PCR.

24 . The method of claim 1 , wherein said plating the plurality of transformed host cells in an ordered array is performed with an automated robotic device or manually.

25 . The method of claim 1 , wherein said picking a clone is performed with an automated robotic device or manually.

26 . The method of claim 1 , further comprising amplifying the sequence-verified oligonucleotide.

27 . The method of claim 1 , further comprising removing contaminating oligonucleotides that have incorrect sequences.

28 . The method of claim 1 , wherein a set of universal primers capable of hybridizing to the common priming sites are used to amplify all the oligonucleotides in parallel.

29 . The method of claim 1 , wherein a set of selective primers are used to selectively amplify a subset of the oligonucleotides.

30 . The method of claim 1 , wherein the mixture of oligonucleotides comprises a probe library or a primer library.

31 . The method of claim 30 , further comprising using the sequence-verified oligonucleotide as a primer

Assignees

• Univ Leland Stanford Junior

Inventors

• St Onge Robert P
• Schlecht Ulrich
• Levy Sasha F
• Davis Ronald W
• Horecka Joseph

Classifications

• C12N15/1031 Primary

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N15/1034

  Isolating an individual clone by screening libraries · CPC title