Method and kits for preparing multicomponent nucleic acid constructs
AssigneeVectorObjects, LLC
InventorsNone recorded
Life of this patent
Estimating the term…
Abstract
The invention provides a highly efficient, rapid, and cost effective method of linking nucleic acid components in a predetermined order to produce a nucleic acid multicomponent construct. The invention further provides nucleic acid components, each nucleic acid component comprising a double stranded nucleic acid molecule having at least one single stranded 5′ or 3′ terminal sequence, the terminal sequence having sufficient complementarity to either a terminal sequence in a separate nucleic acid component or to a sequence in a linking nucleic acid molecule so as to allow for specific annealing of complementary sequences and linkage of the components in a predetermined order. Kits containing reagents required to practice the method of the invention are also provided.
Claims38
A method of identifying a nucleic acid multicomponent construct having a desired biological activity from a library of nucleic acid constructs, comprising:
- (a) generating a combinatorial library comprising a variegated population of nucleic acid multicomponent constructs, wherein the library is generated by the following steps:
- (i) providing at least three different nucleic acid components, each comprising at least one genetic element providing a functionality and at least one single stranded 5′ or 3′ terminal sequence, wherein the terminal sequence hybridizes to either a terminal sequence in another of said nucleic acid components or to a nucleic acid adaptor molecule that is supplied in addition to the nucleic acid components;
- (ii) incubating the nucleic acid components under conditions which allow for combinatorial annealing and linkage of said at least three different nucleic acid components and nucleic acid adaptor molecules to thereby produce the library comprising a variegated population of nucleic acid multicomponent constructs, wherein each nucleic acid multicomponent construct comprises at least three different functionalities;
- (b) expressing the library in cells;
- and
- (c) identifying members of the library that, when expressed in the cells, produce a desired biological activity.
A method of identifying a nucleic acid vector having a desired biological activity from a library of nucleic acid vectors, comprising:
- (a) producing a combinatorial library comprising a variegated population of nucleic acid vectors, wherein the library is generated by the following steps:
- i) providing at least three different nucleic acid components and optionally a linking nucleic acid molecule to be assembled into the vector, each nucleic acid component comprising a double stranded nucleic acid molecule encoding a functionality and having at least one single stranded 5′ or 3′ terminal sequence, wherein the terminal sequence hybridizes to either a terminal sequence in a separate nucleic acid component or to a sequence in a linking nucleic acid molecule, wherein the nucleic acid components encode functionalities selected from an origin of replication, a selectable marker, and an insert of interest;
- (ii) incubating the nucleic acid components under conditions which allow for combinatorial annealing and linkage of said at least three different nucleic acid components to thereby produce a library comprising a variegated population of the functional vectors;
- (b) expressing the library in cells;
- and
- (c) identifying members of the library that, when expressed in the cells, encode a desired biological activity.
Dependencies are inferred from the claim text.