Method and kits for preparing multicomponent nucleic acid constructs
AssigneeVectorObjects, LLC
InventorPeter D. Harney
Life of this patent
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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.
Claims17
A kit for the production of nucleic acid multicomponent constructs, comprising a package containing at least three nucleic acid components, including a first nucleic acid component selected from a category of first nucleic acid components having a common biological utility or functionality, a second nucleic acid component selected from a category of second nucleic acid components having a common biological utility or functionality, and a third nucleic acid component selected from a category of third nucleic acid components having a common biological utility or functionality and, optionally, at least one oligonucleotide bridge,
- wherein the biological utility or functionality of each of the first, second and third categories is different from the other categories,
- wherein at least two of the at least three nucleic acid components are supplied as a library of two or more different nucleic acid fragments, and
- wherein each of the first, second, and third nucleic acid components comprises a double stranded nucleic acid molecule having at least one unique non-palindromic single stranded 5′ or 3′ terminal sequence, which allows for specific annealing and linkage of the first, second, and third nucleic acid component, and optionally the at least one oligonucleotide bridge, in a predetermined order.
A kit for the production of vectors, comprising a package containing at least three nucleic acid components, including an origin of replication, a selectable marker, and an insert of interest, and, optionally, at least one oligonucleotide bridge, wherein at least two of the at least three nucleic acid components are supplied as a library of two or more different nucleic acid fragments, and wherein each of the origin of replication, the selectable marker, and the insert of interest comprises a double standard nucleic acid molecule having at least one unique non-palindromic single stranded 5′ or 3′ terminal sequence, which allows for specific annealing and linkage of the origin of replication, the selectable marker, and the insert of interest, and optionally the at least one oligonucleotide bridge, in a predetermined order.
A method of obtaining one or more species of a nucleic acid construct optimal for a particular application comprising:
- (a) providing at least three nucleic acid components, including
- (i) a first component comprising a library of two or more different nucleic acid fragments, each supplying a specific biological utility or functionality chosen from a first category of biological utilities or functionalities,
- (ii) a second component comprising a library of two or more different nucleic acid fragments, each supplying a specific biological utility or functionality chosen from a second category of biological utilities or functionalities, and
- (iii) a third component comprising one or more third nucleic acid fragments, each supplying a specific biological utility or functionality chosen from a third category of biological utilities or functionalities, wherein said nucleic acid fragments are double stranded nucleic acid molecules having at least one unique nonpalindromic single stranded 5′ or 3′ terminal sequence which facilitates specific annealing and linkage of said nucleic acid fragments in a specific order;
- and
- wherein the biological functionality or utility of each of the first, second, and third categories is different from that of the other categories,
- (b) contacting said first, second and third nucleic acid components so that said nucleic acid fragments become specifically annealed and linked so as to produce a library of at least 4 different vector sequences from which one or more species optimal for a particular application may be obtained, and
- (c) isolating one or more species optimal for a particular application.
A method for generating a library of vectors, comprising
- (i) providing at least 3 different types of nucleic acid components, each representing a functionality to be included in a vector, wherein
- (a) at least two of said nucleic acid components types are provided as a library of two or more different nucleic acids, and
- (b) said nucleic acid components are double-stranded nucleic acid molecules having at least one unique non-palindromic single stranded 5′ or 3′ terminal sequence which facilitates specific annealing and linkage of said components;
- and
- (ii) annealing and ligating said nucleic acid components to generate a library of at least 4 different vector sequences.
Dependencies are inferred from the claim text.