Methods for preparing synthetic repetitive DNA

Claim 1

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (4) excising said monomer from said cloning vector by restriction enzyme digestion; and (5) oligomerizing said monomer to provide a multimer comprising at least two monomers.

Claim 2

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (4) excising said monomer from said cloning vector by restriction enzyme digestion; and (5) oligomerizing said monomer to provide a multimer comprising at least two monomers.

Claim 10

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least three different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments each having from 21 to 90 bases to provide at least three segments, each segment having a different nucleic acid sequence and having complementary ends to contiguous segments; (3) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (4) excising said monomer from said cloning vector by restriction enzyme digestion; and (5) oligomerizing said monomer to provide a multimer comprising at least two monomers.

Claim 11

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least three different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments each having from 21 to 90 bases to provide at least three segments, each segment having a different nucleic acid sequence and having complementary ends to contiguous segments; (3) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (4) excising said monomer from said cloning vector by restriction enzyme digestion; and (5) oligomerizing said monomer to provide a multimer comprising at least two monomers.

Claim 14

A method of preparing a DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least three different pairs of single stranded oligomers of from about 30 to 100 bases, wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing said pairs of oligomers to provide segments; (3) inserting a first segment into a linearized cloning vector; (4) sequencing said first segment to ensure the fidelity of said sequence; (5) sequentially linearizing said vector with a restriction enzyme cleaving proximal to a terminus of said segment, and adding additional segments at a terminus of the prior segment, by digesting said cloning vector with a restriction enzyme cutting at a site proximal to a terminus of the preceding segment, inserting each successive segment in reading frame with the prior segment, and cloning the cloning vector comprising said next segment, to provide a monomer; (6) excising said monomer from said cloning vector; (7) oligomerizing said monomer to provide at least one multimer comprising at least two monomers; wherein the sequences of said segments and vector are selected to permit insertion of said segments and excision of said monomer by restriction enzyme digestion with restriction enzymes which cleave at asymmetric consensus sequences or distal from the consensus sequence.

Claim 15

A method of preparing a DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least three different pairs of single stranded oligomers of from about 30 to 100 bases, wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing said pairs of oligomers to provide segments; (3) inserting a first segment into a linearized cloning vector; (4) sequencing said first segment to ensure the fidelity of said sequence; (5) sequentially linearizing said vector with a restriction enzyme cleaving proximal to a terminus of said segment, and adding additional segments at a terminus of the prior segment, by digesting said cloning vector with a restriction enzyme cutting at a site proximal to a terminus of the preceding segment, inserting each successive segment in reading frame with the prior segment, and cloning the cloning vector comprising said next segment, to provide a monomer; (6) excising said monomer from said cloning vector; (7) oligomerizing said monomer to provide at least one multimer comprising at least two monomers; wherein the sequences of said segments and vector are selected to permit insertion of said segments and excision of said monomer by restriction enzyme digestion with restriction enzymes which cleave at asymmetric consensus sequences or distal from the consensus sequence.

Claim 18

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) cloning a first segment in a cloning vector and analyzing the cloned first segment to determine the fidelity of the sequence, discarding any segment having an erroneous sequence; (4) either: (a) adding each successive segment in reading frame to prior segments to provide a monomer and determining the fidelity of the sequence of each successive segment; or (b) cloning each successive segment in a cloning vector and analyzing each successive segment to determine the fidelity of the sequence and combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (5) excising said monomer from said cloning vector by restriction enzyme digestion; and (6) oligomerizing said monomer to provide a multimer comprising at least two monomers.

Claim 19

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) cloning a first segment in a cloning vector and analyzing the cloned first segment to determine the fidelity of the sequence, discarding any segment having an erroneous sequence; (4) either: (a) adding each successive segment in reading frame to prior segments to provide a monomer and determining the fidelity of the sequence of each successive segment; or (b) cloning each successive segment in a cloning vector and analyzing each successive segment to determine the fidelity of the sequence and combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (5) excising said monomer from said cloning vector by restriction enzyme digestion; and (6) oligomerizing said monomer to provide a multimer comprising at least two monomers.

Claim 22

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) isolating at least one double stranded segment from a previously synthesized monomer; (4) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (5) excising said monomer from said cloning vector by restriction enzyme digestion; and (6) oligomerizing said monomer to provide a multimer comprising at least two monomers; wherein synthesized segments are sequenced to ensure the fidelity of replication.

Claim 23

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) isolating at least one double stranded segment from a previously synthesized monomer; (4) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (5) excising said monomer from said cloning vector by restriction enzyme digestion; and (6) oligomerizing said monomer to provide a multimer comprising at least two monomers; wherein synthesized segments are sequenced to ensure the fidelity of replication.

Claim 26

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing a single strand encoding a monomer comprising from about 100 to 300 bases comprising repeating units of from about 3 to 30 codons, wherein said single strand comprises 5' and 3' flanking primer binding site sequences; (2) preparing a complementary strand and amplifying the resultant dsDNA monomer by polymerase chain reaction using primers of from 1 5 to 50 nt having a sequence comprising a sequence which hybridizes with the respective flanking primer binding site sequences, wherein said primers are different for each end of the dsDNA; (3) cloning said dsDNA monomer and selecting for a monomer having the correct sequence; and (4) oligomerizing said monomer to provide at least one multimer comprising at least two monomers.

Claim 27

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing a single strand encoding a monomer comprising from about 100 to 300 bases comprising repeating units of from about 3 to 30 codons, wherein said single strand comprises 5' and 3' flanking primer binding site sequences; (2) preparing a complementary strand and amplifying the resultant dsDNA monomer by polymerase chain reaction using primers of from 1 5 to 50 nt having a sequence comprising a sequence which hybridizes with the respective flanking primer binding site sequences, wherein said primers are different for each end of the dsDNA; (3) cloning said dsDNA monomer and selecting for a monomer having the correct sequence; and (4) oligomerizing said monomer to provide at least one multimer comprising at least two monomers.

Claim 33

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers of wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (4) excising said monomer from said cloning vector by restriction enzyme digestion; (5) oligomerizing said monomer to provide a multimer comprising at least two monomers; wherein the sequences of said segments and vector are selected to permit insertion of said segments and excision of said monomer by restriction enzyme digestion; (6) inserting said multimer in an expression vector functional for expression in an expression host; (7) introducing said expression vector into said expression host; and growing said expression host, whereby said protein polymer is expressed.

Claim 34

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing at least two different pairs of single stranded oligomers of wherein each of the oligomers of a pair overlap except as to any protruding ends; (2) hybridizing each pair of single stranded oligomers to provide double stranded segments; (3) combining said segments or amplified copies thereof in a cloning vector to form a monomer, where the combined segments are in reading frame; (4) excising said monomer from said cloning vector by restriction enzyme digestion; (5) oligomerizing said monomer to provide a multimer comprising at least two monomers; wherein the sequences of said segments and vector are selected to permit insertion of said segments and excision of said monomer by restriction enzyme digestion; (6) inserting said multimer in an expression vector functional for expression in an expression host; (7) introducing said expression vector into said expression host; and growing said expression host, whereby said protein polymer is expressed.

Claim 38

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing a single strand encoding a monomer comprising from about 100 to 300 bases comprising repeating units of from about 3 to 30 codons, wherein said single strand comprises 5' and 3' flanking primer binding site sequences; (2) preparing a complementary strand and amplifying the resultant dsDNA monomer by polymerase chain reaction using primers of from 20 to 45 nt having a sequence which hybridizes with the respective flanking primer binding site sequences, wherein said primers are different for each end of the dsDNA; (3) cloning said dsDNA monomer and selecting for a monomer having the correct sequence; (4) oligomerizing said monomer to provide at least one multimer comprising at least two monomers; (5) inserting said multimer in an expression vector functional for expression in an expression host; (6) introducing said expression vector into said expression host; and growing said expression host, whereby said protein polymer is expressed.

Claim 39

A method of preparing a synthetic DNA sequence having repeating units of from about 3 to 30 codons and encoding a protein of at least about 30 kDal, said method comprising: (1) synthesizing a single strand encoding a monomer comprising from about 100 to 300 bases comprising repeating units of from about 3 to 30 codons, wherein said single strand comprises 5' and 3' flanking primer binding site sequences; (2) preparing a complementary strand and amplifying the resultant dsDNA monomer by polymerase chain reaction using primers of from 20 to 45 nt having a sequence which hybridizes with the respective flanking primer binding site sequences, wherein said primers are different for each end of the dsDNA; (3) cloning said dsDNA monomer and selecting for a monomer having the correct sequence; (4) oligomerizing said monomer to provide at least one multimer comprising at least two monomers; (5) inserting said multimer in an expression vector functional for expression in an expression host; (6) introducing said expression vector into said expression host; and growing said expression host, whereby said protein polymer is expressed.

Claim 41

A method according to 31, wherein at least one repeating unit encodes an amino acid sequence selected from the group consisting of: Gαβ, GAGAGS (SEQ ID NO:6), GVGVP (SEQ ID NO:4), VPGVG (SEQ ID NO:3), SGAGAG (SEQ ID NO:1), and AGAGSG (SEQ ID NO:20), wherein α and β are any amino acid, α and β being selected so that from about 10 to 45 number % of the total number of amino acid residues of the protein are proline.

Claim 42

A method according to 31, wherein at least one repeating unit encodes an amino acid sequence selected from the group consisting of: Gαβ, GAGAGS (SEQ ID NO:6), GVGVP (SEQ ID NO:4), VPGVG (SEQ ID NO:3), SGAGAG (SEQ ID NO:1), and AGAGSG (SEQ ID NO:20), wherein α and β are any amino acid, α and β being selected so that from about 10 to 45 number % of the total number of amino acid residues of the protein are proline.