High efficiency, small volume nucleic acid synthesis

Patent metadata

Field	Value
Publication number	US-10563240-B2
Application number	US-201414775648-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 14, 2013
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

Abstract

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

First claim

What is claimed is:

1. A method for assembling nucleic acid molecules, the method comprising:
(a) forming a reaction mixture by combining:
(1) two or more insert nucleic acid molecules, an acceptor nucleic acid molecule, and a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises (i) two oligonucleotides that share sequence complementarity with one terminus of a first insert nucleic acid molecule and a first terminus of the insertion site of the acceptor nucleic acid molecule, (ii) two oligonucleotides that share sequence complementarity with one terminus of a second insert nucleic acid molecule and a second terminus of the insertion site of the acceptor nucleic acid molecule, and (iii) one or more set of two oligonucleotides that each share sequence complementarity with one terminus of two different insert nucleic acid molecules, and wherein the total number of oligonucleotides is represented by the formula O=2+2I, where O is the total number of oligonucleotides and I is the number of insert nucleic acid molecules and wherein I>1,
(2) a cell extract, and
(3) a cell free protein composition comprising an exonuclease and, optionally, a single-stranded binding protein, and

(b) incubating the reaction mixture formed in (a) under conditions which allow for the introduction of the insert nucleic acid molecules into the acceptor nucleic acid molecule.

2. The method of claim 1 , wherein the cell extract is obtained from a single cellular organism selected from the group consisting of:
(a) Escherichia coli ( E. coli );
(b) Bacillus subtilis;
(c) Schizosaccharomyces pombe ; and
(d) Saccharomyces cerevisiae.

3. The method of claim 2 , wherein the Escherichia coli cells do not express redET genes.

4. The method of claim 3 , wherein the Escherichia coli cells are strain DH10B.

5. The method of claim 2 , wherein the cell extract is prepared by collecting a supernatant generated by centrifugation of a lysate of E. coli DH10B.

6. The method of claim 1 , wherein the exonuclease activity is provided by a DNA polymerase.

7. The method of claim 1 , wherein the single-stranded binding protein is encoded by T4 gene 32.

8. The method of claim 1 , wherein the number of insert nucleic acid molecules is two and the total number of oligonucleotides is six.

9. The method of claim 1 , wherein the exonuclease is T7 exonuclease.

10. The method of claim 9 , wherein the T7 exonuclease is present in a solution containing a buffer.

11. The method of claim 10 , wherein the buffer is Tris.

12. The method of claim 10 , wherein the solution containing the buffer also contains bovine serum albumin.

Assignees

• Life Technologies Corp
• Thermo Fisher Scient Geneart Gmbh

Inventors

• Peterson Todd
• Trefzer Axel
• Poehmerer Thomas
• Katzen Federico
• Liang Xiquan

Classifications

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1031 Primary

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