Agilent Technologies, Inc.

Other

Derek Lee Lindstrom

Jeffrey Robert Sampson

Gusti Zeiner

Robert A Ach

Brian Jon Peter

Methods for making a synthetic nucleic acid which comprise:

Pairing code directed assembly

This disclosure provides, among other things, a method of combining nucleic acid fragments, comprising: (a) providing two double-stranded DNA molecules with a common sequence, wherein the common sequence is at the end of each molecule; (b) nicking one strand in the common sequence of both molecules at a respective nicked site; (c) moderately denaturing both molecules to remove a single-stranded fragment from the nicked site to one end of each molecule, wherein the single-stranded fragment includes the common sequence in part or in whole, resulting in an overhanging sequence in each molecule, and the overhanging sequences in both molecules are complementary to each other; (d) allowing the overhanging sequences of both molecules to anneal to each other, and ligating the molecules. Alternative ways for performing the method are also provided.

DNA assembly using an RNA-programmable nickase

Provided herein is a method comprising: producing a first complex by annealing: a first nucleic acid comprising, in order, a first unique sequence, a first central sequence and a second unique sequence; and a second nucleic acid comprising, in order, said second unique sequence, a second central sequence and a third unique sequence; wherein the first, second and third unique sequences do not hybridize with each other; subjecting said first complex to multiple rounds of primer extension to extend the first and second nucleic acids using each other as a template, thereby producing a first product molecule that contains, in order, the first unique sequence, the first central sequence, the second unique sequence, the second central sequence and the third unique sequence; and circularizing said first product molecule by intramolecularly ligating the ends of said product molecule together. Kits and compositions relating to the method are also provided.

A method for fragmenting a genome is provided. In certain embodiments, the method comprises: (a) combining a genomic sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and (b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA. Also provided is a composition comprising at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in a genome. Kits for performing the method are also provided. In addition, other methods, compositions and kits for manipulating nucleic acids are also provided.

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Cupertino, CA, United States of America

Derek Lee Lindstrom