Method for the complete chemical synthesis and assembly of genes and genomes

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.

1 . 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.    
     
     
         2 . The method of  claim 1 , further comprising the step of treating said annealed oligonucleotides with a ligating enzyme to generate continuous strands of said double-stranded polynucleotide.  
     
     
         3 . The method of  claim 1 , further comprising the step of amplifying said double-stranded polynucleotide.  
     
     
         4 . The method of  claim 1 , wherein said double-stranded polynucleotide comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10×10 3 , 20×10 3 , 30×10 3 , 40×10 3 , 50×10 3 , 60×10 3 , 70×10 3 , 80×10 3 , 90×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9  or 1×10 10  base pairs in length.  
     
     
         5 . The method of  claim 1 , wherein said first regulatory element is a promoter.  
     
     
         6 . The method of  claim 5 , wherein said double-stranded polynucleotide comprises a second regulatory element, said second regulatory element being a polyadenylation signal.  
     
     
         7 . The method of  claim 1 , wherein said double-stranded polynucleotide comprises a plurality of coding regions and a plurality of regulatory elements.  
     
     
         8 . The method of  claim 7 , wherein said coding regions encode products that comprise a biochemical pathway.  
     
     
         9 . The method of  claim 8 , wherein said biochemical pathway is glycolysis.  
     
     
         10 . The method of  claim 9 , wherein said coding regions encode enzymes selected from the group consisting of hexokinase, phosphohexose isomerase, phosphofructokinase-1, aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase.  
     
     
         11 . The method of  claim 8 , wherein said biochemical pathway is lipid synthesis.  
     
     
         12 . The method  claim 7 , wherein said biochemical pathway is cofactor synthesis.  
     
     
         13 . The method of  claim 13 , wherein said pathway involves lipoic acid.  
     
     
         14 . The method of  claim 13 , wherein said biochemical pathway is riboflavin synthesis.  
     
     
         15 . The method of  claim 7 , wherein said biochemical pathway is nucleotide synthesis.  
     
     
         16 . The method of  claim 15 , wherein said nucleotide is a purine.  
     
     
         17 . The method of  claim 15 , wherein said nucleotide is a pyrimidine.  
     
     
         18 . The method of  claim 7 , wherein said coding regions encode enzymes involved in a cellular process selected from the group consisting of cell division, chaperone, detoxification, peptide secretion, energy metabolism, regulatory function, DNA replication, transcription, RNA processing and tRNA modification.  
     
     
         19 . The method of  claim 18 , wherein said energy metabolism is oxidative phosphorylation.  
     
     
         20 . The method of  claim 1 , wherein said double-stranded polynucleotide is a DNA.  
     
     
         21 . The method of  claim 1 , wherein said double-stranded polynucleotide is an RNA.  
     
     
         22 . The method of  claim 1 , wherein said double-stranded polynucleotide is an expression construct.  
     
     
         23 . The method of  claim 22 , wherein said expression construct is a bacterial expression construct.  
     
     
         24 . The method of  claim 22 , wherein said expression construct is a mammalian expression construct.  
     
     
         25 . The method of  claim 17 , wherein said expression construct is a viral expression construct.  
     
     
         26 . The method of  claim 1 , wherein said double-stranded polynucleotide comprises a genome selected from the group consisting of bacterial genome, yeast genome, viral genome, mammalian genome, amphibian genome and avian genome.  
     
     
         27 . The method of  claim 1 , wherein said overlap between the oligonucleotides of said first and said second set of oligonucleotides is between about 5 base pairs and about 75 base pairs.  
     
     
         28 . The method of  claim 1 , wherein said overlap is about 10 base pairs, about 15 base pairs, about 20 base pairs, about 25 base pairs, about 30 base pairs, about 35 base pairs, about 40 base pairs, about 45 base pairs, about 50 base pairs, about 55 base pairs, about 60 base pairs, about 65 base pairs, or about 70 base pairs.  
     
     
         29 . The method of  claim 5 , wherein said promoter is selected from the group consisting of CMV IE, SV40 IE, RSV, β-actin, tetracycline regulatable and ecdysone regulatable.  
     
     
         30 . The method of  claim 26 , wherein said genome is a viral genome.  
     
     
         31 . The method of  claim 30 , wherein said viral genome is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.  
     
     
         32 . The method of  claim 1 , wherein said double-stranded polynucleotide is a chromosome.  
     
     
         33 . 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.    
     
     
         34 . The method of  claim 33 , wherein said viral genome is selected from the group consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-associated virus.  
     
     
         35 . 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.    
     
     
         36 . The method of  claim 35 , wherein said corresponding natural chromosome is a human mitochondrial genome.  
     
     
         37 . The method of  claim 35 , wherein said corresponding natural chromosome is a chloroplast genome.  
     
     
         38 . 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.    
     
     
         39 . The method of  claim 38 , wherein said biochemical pathway comprises the activities required for glycolysis.  
     
     
         40 . The method of  claim 38 , wherein said biochemical pathway comprises the enzymes required for electron transport.  
     
     
         41 . The method of  claim 38 , wherein said biochemical pathway comprises the enzyme activities required for photosynthesis.