Reagents and methods for diversification of DNA
AssigneeFlinders Technologies Pty Ltd of Flinders University
InventorDavid E. Catcheside
Life of this patent
Estimating the term…
Abstract
The present invention provides methods and compositions for diversification of heterologous DNA sequences in vivo. The present invention employs a recombination hotspot functionally coupled to the heterologous DNA. The process of recombination generates new versions of the foreign sequences by recombining their differences in new combinations. Errors in recombination generate additional sequence diversity.
Claims127
A haploid fungal cell comprising a recombinant genome, the recombinant genome comprising a heterologous DNA functionally coupled to a recombination hotspot;
- the haploid fungal cell being capable of being converted to a diploid fungal cell;
- the heterologous DNA being adapted and configured within the recombinant genome for recombination in the diploid fungal cell.
A diploid fungal cell comprising a recombinant genome, the recombinant genome comprising a first heterologous DNA functionally coupled to a first recombination hotspot and a second heterologous DNA functionally coupled to a second recombination hotspot;
- the first heterologous DNA and second heterologous DNA being adapted and configured within the recombinant genome for recombination.
A plasmid comprising a tuncated Neurospora crassa his-3 gene and a Neurospora crassa recombination hot spot functionally coupled to a heterologous DNA, a multiple cloning site 3′ to the his-3 gene, and a marker gene;
- the plasmid being adapted and configured for transfection of a Neurospora crassa cell.
A method of preparing diversified DNA comprising the steps of:
- constructing a plurality of fertile strains of a fungus, each strain comprising a distinct heterologous DNA to be diversified, each heterologous DNA being functionally coupled to a recombination hot spot;
- mating a pair of the strains to form a dikaryon;
- establishing a diploid cell from the dikaryon;
- and
- inducing the diploid cell to undergo meiosis to produce a haploid spore, the meiosis comprising gene conversion, crossing over, errors in recombination, or a combination thereof;
- whereby the heterologous DNA is diversified.
A diversified DNA molecule made by a method comprising the steps of:
- constructing a plurality of fertile strains of a fungus, each strain comprising a distinct heterologous DNA to be diversified, each heterologous DNA being functionally coupled to a recombination hot spot;
- mating a pair of the strains to form a dikaryon;
- establishing a diploid cell from the dikaryon;
- and
- inducing the diploid cell to undergo meiosis to produce a haploid spore, the meiosis comprising gene conversion, crossing over, errors in recombination, or a combination thereof;
- whereby the heterologous DNA is diversified.
An isolated DNA molecule comprising a sequence of a recombination hotspot cog of Neurospora crassa.
A kit for preparing diversified DNA comprising:
- an fertile strain of fungus adapted and configured for housing a recombinant genome;
- a plasmid for preparing the recombinant genome comprising a recombination hotspot from the fungus strain, the plasmid being adapted and configured to receive heterologous DNA in a position functionally coupled to the recombination hot spot.
A method of preparing a strain of a fungus comprising the steps of:
- transfecting a first fungal cell having a first allele of a gene with a first vector including a first heterologous DNA and a second allele of the gene and establishing a first heterokaryon, the first and second allele each encode a defective version of the gene and are complementary alleles;
- growing the first heterokaryon to establish a first homokaryon containing the second allele of the gene and the first heterologous DNA;
- transfecting a second fungal cell having a third allele of the gene with a second vector including a second heterologous DNA and a fourth allele of the gene and establishing a second heterokaryon;
- the third and fourth alleles each encode a defective version of the gene and are complementary alleles;
- growing the second heterokaryon to establish a second homokaryon containing the fourth allele of a gene and the second heterologous DNA;
- and
- crossing the first and second homokaryons to establish the strain of the fungus.
Dependencies are inferred from the claim text.