Directional assembly of large viral genomes and chromosomes
AssigneeThe University of North Carolina at Chapel Hill
InventorsRalph S. Baric, Boyd Yount
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Abstract
Full-length, functionally intact genomes or chromosomes are directionally assembled with partial cDNA or DNA subclones of a genome. This approach facilitates the reconstruction of genomes and chromosomes in vitro for reintroduction into a living host, and allows the selected mutagenesis and genetic manipulation of sequences in vitro prior to reassembly into a full length genome molecule for reintroduction into the same or different host. This approach also provides an alternative to recombination-mediated techniques to manipulate the genomes of higher plants and animals as well as bacteria and viruses.
Claims25
A method of directionally assembling a recombinant viral genome, comprising:
- obtaining a set of subclones of the viral genome, wherein each termini of each subclone is a restriction enzyme recognition site;
- and then
- ligating the subclones to assemble a recombinant viral genome.
A recombinant viral genome comprising a set of subclones, wherein each subclone has a 3′ terminus and 5′ terminus comprising a restriction enzyme recognition site, and wherein each terminus is ligated to the terminus of the adjacent subclone to form a junction, and wherein each junction has a unique nucleic acid sequence.
A method of infecting a host cell with a recombinant viral genome, comprising contacting the host cell with a viral genome that has been assembled from a set of subclones and wherein the terminus of each subclone is a restriction enzyme recognition site, and wherein each subclone is ligated to the adjacent subclone such that the viral genome is directionally assembled and infective.
Dependencies are inferred from the claim text.