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

Patent metadata

Field	Value
Publication number	US-10787661-B2
Application number	US-201514928928-A
Country	US
Kind code	B2
Filing date	Oct 30, 2015
Priority date	Oct 30, 2014
Publication date	Sep 29, 2020
Grant date	Sep 29, 2020

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 sequence verification of an 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 common priming sites for amplification;
b) amplifying one or more oligonucleotides;
c) transforming a plurality of host cells with the amplified oligonucleotides, wherein each host cell comprises a target locus, wherein each amplified oligonucleotide is integrated at a site within the target locus of a host cell, said target locus comprising a first recombination target site for a site-specific recombinase, wherein the first recombination site is adjacent to the site where the oligonucleotide is integrated at the target locus;
d) plating the plurality of transformed host cells in a first 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 first ordered array, wherein each colony of clones has a position in the first ordered array and comprises a different oligonucleotide integrated at the target locus, and further wherein the position of each oligonucleotide in the first ordered array is not known;
f) providing a plurality of barcoder cells in a second ordered array, wherein each position of the first ordered array corresponds to a position in the second ordered array, and further wherein each barcoder cell comprises a nucleic acid comprising a second recombination target site for the site-specific recombinase adjacent to a barcode sequence that identifies a position of the barcoder cell in the second ordered array;
g) introducing the oligonucleotide integrated at the target locus of a clone from each position in the first ordered array into a barcoder cell from each corresponding position in the second ordered array, thereby forming a plurality of oligonucleotide-barcoder cells;
h) introducing the site-specific recombinase into each oligonucleotide-barcoder cell, wherein the site-specific recombinase catalyzes site-specific recombination between the first recombination target site and the second recombination target site, thereby translocating the oligonucleotide to a position adjacent to the barcode sequence to generate a nucleic acid comprising a barcode-oligonucleotide fusion sequence in each oligonucleotide-barcoder cell;
i) combining the barcode-oligonucleotide fusion sequences from each of the plurality of oligonucleotide-barcoder cells; and
j) sequencing the combined barcode-oligonucleotide fusion sequences to identify and verify each oligonucleotide sequence, wherein the barcode in each barcode-oligonucleotide fusion sequence identifies the position in the first ordered array of a colony comprising the adjacent sequence-verified oligonucleotide.

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

6. The method of claim 5 , further comprising using a selectable marker that selects for clones that have undergone successful integration of an oligonucleotide at the plasmid target locus.

7. The method of claim 5 , 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 is expressed in the plurality of host cells and creates a double strand break at the target locus of the plasmid that facilitates integration of the oligonucleotide at the target locus into the plasmid.

8. 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.

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

10. The method of claim 9 , 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.

11. The method of claim 10 , further comprising cloning the restriction fragment into a vector.

12. The method of claim 11 , wherein the vector is a plasmid or viral vector.

13. The method of claim 12 , wherein the vector further comprises the first recombination target site.

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

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

16. The method of claim 15 , wherein the host cells are yeast.

17. The method of claim 15 , wherein the host cells are bacteria.

18. 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.

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

20. 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).

21. The method of claim 20 , wherein the oligonucleotides are amplified using PCR.

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

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

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

25. The method of claim 23 , wherein a set of universal primers capable of hybri

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

• C12N15/1034

  Isolating an individual clone by screening libraries · 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

• C12Q2565/514

  characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array · CPC title