Methods for generating pools of variants of a DNA template

Patent metadata

Field	Value
Publication number	US-11274295-B2
Application number	US-201314420881-A
Country	US
Kind code	B2
Filing date	Aug 9, 2013
Priority date	Aug 10, 2012
Publication date	Mar 15, 2022
Grant date	Mar 15, 2022

Abstract

The invention provides methods for generating pools of variants of DNA templates, and methods of using pools of variants to identify sequences involved in conferring sensitivity or resistance to environmental factors.

First claim

What is claimed is:

1. A method of generating a mixture of variants of a template DNA molecule, said method comprising:
a) providing a plurality of oligonucleotides synthesized on one or more solid supports comprising one or more sets of oligonucleotides, each set corresponding to one of multiple (“n”) 100 to 500 nucleotide regions of said template DNA molecule, wherein the oligonucleotides in each set of oligonucleotides comprise:
i) 5′ and 3′ ends identical to the 5′ and 3′ ends of the region, and
ii) a central variable region comprising at least one sequence variation as compared to the sequence of said region of said template DNA molecule and the other oligonucleotides in the set;
b) amplifying a set of oligonucleotides on the one or more solid supports corresponding to a first region of said template DNA molecule from the plurality of oligonucleotides using polymerase chain reaction (PCR);
c) providing a plurality of linearized plasmid vectors comprising the template DNA molecule, but lacking said first region of said template DNA molecule, wherein said plurality of linearized plasmid vectors is generated by polymerase chain reaction using primers having 5′ ends that are facing each other on the vector and 3′ ends oriented such that extension amplifies the entire sequence of the vector except for the first region, wherein the ends of said linearized plasmid vectors are configured for joining with the ends of the amplified set of oligonucleotides to complete the template DNA molecule; and
d) joining said amplified set of oligonucleotides to said plurality of linearized plasmid vectors, thereby generating a mixture of circular plasmids comprising a mixture of variants of said first region of said template DNA molecule.

2. The method of claim 1 , wherein said template DNA molecule encodes a polypeptide, a non-coding RNA, an untranslated regulatory sequence, a promoter sequence, and/or an enhancer sequence.

3. The method of claim 1 , wherein said variation is a substitution or deletion of at least one nucleotide.

4. The method of claim 1 , further comprising repeating steps b) to d) for a second region of said template DNA molecule; and
e) mixing said mixture of circular plasmids comprising variants of said first region of said template DNA molecule with said mixture of circular plasmids comprising variants of said second region of said template DNA molecule to generate a mixture of variants of said first and second regions of said template DNA molecule.

5. The method of claim 1 , wherein each of the one or more sets of oligonucleotides of step (a) is a set of oligonucleotides that encodes at least one variation of every amino acid encoded by the central variable region of the set of oligonucleotides.

6. The method of claim 1 , wherein each of the one or more sets of oligonucleotides of step (a) is a set of oligonucleotides that comprises at least one variation of every nucleotide of the central variable region of the set of oligonucleotides, and/or is a set of oligonucleotides that each have variation in more than one nucleotide of the central variable region.

7. The method of claim 1 , wherein said method further comprises repeating steps b) to d) for each of said “n” regions to generate a mixture of circular plasmids, wherein each mixture of circular plasmids has variants of one of said “n” regions of said template molecule; and e) mixing each mixture of circular plasmids to generate a mixture of circular plasmids having variants of said “n” regions of said template DNA molecule.

8. The method of claim 5 , wherein said at least one variation of every amino acid is at least one naturally occurring variation of every amino acid.

9. The method of claim 1 , further comprising:
e) transforming said mixture of circular plasmids generated by said joining into a host cell;
f) selecting for recombinant clones containing said plasmids; and
g) isolating said plasmids containing said variants.

10. A method of generating a mixture of variants of a template DNA molecule, said method comprising:
a) providing a plurality of oligonucleotides synthesized on one or more solid supports comprising two or more sets of oligonucleotides, each set corresponding to one of multiple (“n”) 100 to 500 nucleotide consecutive regions of said template DNA molecule, wherein the oligonucleotides in each set of oligonucleotides comprise:
i) 5′ and 3′ ends identical to the 5′ and 3′ ends of the region, and
ii) a central variable region comprising at least one sequence variation as compared to the sequence of said region of said template DNA molecule and the other oligonucleotides in the set;
b) amplifying two or more sets of oligonucleotides on the one or more solid supports corresponding to consecutive multiple regions of said template DNA molecule from the plurality of oligonucleotides using polymerase chain reaction (PCR);
c) providing a plurality of linearized plasmid vectors comprising the template DNA molecule, but lacking said consecutive multiple regions of said template DNA molecule, wherein said plurality of linearized plasmid vectors is generated by polymerase chain reaction using primers having 5′ ends that are facing each other on the vector and 3′ ends oriented such that extension amplifies the entire sequence of the vector except for the consecutive multiple regions, wherein the ends of said linearized vectors are configured for joining with the ends of the amplified sets of oligonucleotides corresponding to the two outermost of the consecutive multiple regions of said template DNA molecule to complete the template DNA molecule upon joining of all of said amplified plurality of oligonucleotides corresponding to consecutive multiple regions; and
d) joining said amplified sets of oligonucleotides to each other and said plurality of linearized plasmid vectors, thereby generating circular plasmids comprising a mixture of variants of said multiple regions of said template DNA molecule.

11. A method of identifying a variant nucleic acid molecule which, when introduced into a cell, selectively increases or decreases the sensitivity of the cell to an environmental factor, said method comprising:
a) introducing a mixture of variants of a template DNA molecule into both a first population of cells and a second population of cells, wherein said mixture of variants of a template DNA molecule are generated by the methods of claim 1 ,
b) incubating said first population of cells in the presence of a first environmental factor;
c) incubating said second population of cells in the absence of said first environmental factor;
d) isolating cells exhibiting a phenotype associated with increased or decreased sensitivity to said first environmental factor; and
e) determining which variants of said template DNA molecule are enriched or depleted in cells isolated from said first population of cells as compared to said second population of cells; thereby identifying a variant nucleic acid that selectively increases or decreases the sensitivity of said cells to said first environmental factor.

12. The method of claim 11 , wherein said second population of cells are grown in the presence of a second environmental factor, thereby identifying a variant that selectively increases or decreases the sensitivity of said cells to said first environmental factor relative to said second environmental factor.

13. The method of claim 11 , wherein said isolating of cells comprises collection of surviving cells, separation of cells having a morphological characteristic, optionally wherein said morphological characteristic is cell size, expression of a differentiation marker, cell adhesion, or cell me

Assignees

• Broad Inst Inc

Inventors

• Mikkelsen Tarjei
• Melnikov Alexandre

Classifications

• C12N15/1079

  Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086) · 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

• C12N15/1031

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

• C12N15/1027 Primary

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title