Synthetic Genomics, Inc.

Codex Dna, Inc.

Sgi-Dna, Inc.

Lei Young

Hamilton O Smith

Daniel Glenn Gibson

J Craig Venter

Clyde A Hutchison

John Irvin Glass

Carole Lartigue

Gwynedd A Benders

John I Glass

Craig Venter

Nacyra Assad-Garcia

Clyde A Hutchison Iii

Daniel G Gibson

The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Methods for in vitro joining and combinatorial assembly of nucleic acid molecules

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5' exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

In vitro recombination method

A method to assemble any desired nucleic acid molecule by combining cassettes in vitro to form assemblies which are further combined in vivo, or by assembling large numbers of DNA fragments by recombination in a yeast culture to obtain desired DNA molecules of substantial size is described.

Assembly of large nucleic acids

A method is provided for introducing a genome into a cell or cell-like system. The introduced genome may occur in nature, be manmade with or without automation, or may be a hybrid of naturally occurring and manmade materials. The genome is obtained outside of a cell with minimal damage. Materials such as a proteins, RNAs, polycations, nucleoid condensation proteins, or gene translation systems may accompany the genome. The genome is installed into a naturally occurring cell or into a manmade cell-like system. A cell-like system or synthetic cell resulting from the practice of the provided method may be designed and used to yield gene-expression products, such as desired proteins. By enabling the synthesis of cells or cell-like systems comprising a wide variety of genomes, accompanying materials and membrane types, the provided method makes possible a broader field of experimentation and bioengineering than has been available using prior art methods.

Installation of genomes or partial genomes into cells or cell-like systems

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5' exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

A method is provided for synthesis of error-minimized nucleic acid molecules. Oligonucleotides intended to have fragments of a desired, full-length nucleotide sequence, and optionally containing other desired nucleotides, such as nucleotides for binding the oligonucleotides to a substrate, are obtained. Oligonucleotides for both strands of the desired, full-length sequence may be obtained. The oligonucleotides are amplified and assembled into a first set of molecules intended to have the desired, full-length nucleotide sequence. The first set of molecules is denatured and annealed to form a second set of molecules intended to have the desired, full-length nucleotide sequence. The second set of molecules is cut into smaller segments, for example, by mixing the molecules with endonucleases that form blunt cuts in the second set of molecules where there are sequence errors, as well as randomly along the molecules. The smaller segments are assembled into a set of molecules intended to have the desired, full-length nucleotide sequence. By promoting cutting of the molecules in this manner near the end of the nucleic acid molecule synthesis process, a set of full-length molecules can be obtained with fewer nucleotide sequence errors than can be achieved with other methods.

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Gaithersburg, MD, United States of America

Lei Young