Directional assembly of large viral genomes and chromosomes
AssigneeThe University of North Carolina at Chapel Hill
InventorsRalph S. Baric, Boyd Yount
Life of this patent
Estimating the term…
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.
Claims53
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.
A method of directionally assembling a recombinant viral genome, comprising:
- obtaining a set of at least three subclones representing the viral genome, wherein each termini of each subclone is a single-stranded sticky end that is only complementary within a recursive assembly pathway to a sticky end of a subclone representing an adjacent portion of the genome;
- and then
- ligating the subclones to directionally assemble a recombinant viral genome.
A method of directionally assembling a recombinant coronavirus genome, comprising:
- obtaining a set of at least three subclones representing the genome, wherein each termini of each subclone is a single stranded sticky end that only complementary within a recursive assembly pathway to a sticky end of a subclone representing an adjacent portion of the genome;
- and then
- ligating the subclones to directionally assemble a recombinant genome.
A method of directionally assembling a recombinant transmissible gastroenteritis virus genome, comprising:
- obtaining a set of at least three subclones representing the genome, wherein each termini of each subclone is a single-stranded sticky end that is only complementary within a recursive assembly pathway to a sticky end of a subclone representing an adjacent portion of the genome;
- and then
- ligating the subclones to directionally assemble a recombinant genome.
Dependencies are inferred from the claim text.