Method for the complete chemical synthesis and assembly of genes and genomes
AssigneeEgea Biosciences, Inc.
InventorGlen A. Evans
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Abstract
The present invention relates generally to the fields of oligonucleotide synthesis. More particularly, it concerns the assembly of genes and genomes of completely synthetic artificial organisms. Thus, the present invention outlines a novel approach to utilizing the results of genomic sequence information by computer directed gene synthesis based on computing on the human genome database. Specifically, the present invention contemplates and describes the chemical synthesis and resynthesis of genes defined by the genome sequence in a host vector and transfer and expression of these sequences into suitable hosts.
Claims41
A method for the synthesis of a replication-competent, double-stranded polynucleotide, wherein said polynucleotide comprises an origin of replication, a first coding region and a first regulatory element directing the expression of said first coding region, comprising the steps of:
- (a) generating a first set of oligonucleotides corresponding to the entire plus strand of said double-stranded polynucleotide;
- (b) generating a second set of oligonucleotides corresponding to the entire minus strand of said double-stranded polynucleotide;
- and
- (c) annealing said first and said second set of oligonucleotides;
- wherein each of said oligonucleotides of said second set of oligonucleotides overlaps with and hybridizes to two complementary oligonucleotides of said first set of oligonucleotides, except that two oligonucleotides at a 5′ or 3′ end of said double-stranded polynucleotide will hybridize with only one complementary oligonucleotide.
The method of claim 13, wherein said pathway involves lipoic acid.
A method of producing a viral particle comprising the steps of:
- (a) providing a host cell;
- (b) transforming said host cell with an artificial viral genome prepared by:
- (i) generating a first set of oligonucleotides corresponding to the entire plus strand of said viral genome;
- (ii) generating a second set of oligonucleotides corresponding to the entire minus strand of said viral genome;
- and
- (iii) annealing said first and said second set of oligonucleotides;
- wherein each of said oligonucleotides of said second set of oligonucleotides overlaps with and hybridizes to two complementary oligonucleotides of said first set of oligonucleotides, except that two oligonucleotides at a 5′ or 3′ end of said viral genome will hybridize with only one complementary oligonucleotide;
- and
- (c) culturing said transformed host cell under conditions such that said viral particle is expressed.
A method of producing an artificial genome, wherein said chromosome comprises all coding regions and regulatory elements found in a corresponding natural chromosome, comprising the steps of:
- (a) generating a first set of oligonucleotides corresponding to the entire plus strand of said chromosome;
- (b) generating a second set of oligonucleotides corresponding to the entire minus strand of said chromosome;
- and
- (c) annealing said first and said second set of oligonucleotides;
- wherein each of said oligonucleotides of said second set of oligonucleotides overlaps with and hybridizes to two complementary oligonucleotides of said first set of oligonucleotides, except that two oligonucleotides at a 5′ or 3′ end of said chromosome will hybridize with only one complementary oligonucleotide.
A method of producing an artificial genetic system, wherein said system comprises all coding regions and regulatory elements found in a corresponding natural biochemical pathway, comprising the steps of:
- (a) generating a first set of oligonucleotides corresponding to the entire plus strand of said chromosome;
- (b) generating a second set of oligonucleotides corresponding to the entire minus strand of said chromosome;
- and
- (c) annealing said first and said second set of oligonucleotides;
- wherein each of said oligonucleotides of said second set of oligonucleotides overlaps with and hybridizes to two complementary oligonucleotides of said first set of oligonucleotides, except that two oligonucleotides at a 5′ or 3′ end of said chromosome will hybridize with only one complementary oligonucleotide
- wherein expression of said biochemical pathway coding regions results in the expression of a group of enzymes that serially metabolize a compound.
Dependencies are inferred from the claim text.