Method of producing a gene cassette coding for polypeptides with repeating amino acid sequences

Claim 1

A method of producing a gene cassette coding for polypeptides comprised of one or more repeating amino acid sequences, said method comprising the steps of (a) annealing at least one mixture of complementary but circularly permuted oligodeoxynucleotides which have phosphorylated 5' ends and which code for one or more repeating amino acid sequences, to form DNA 1 fragments, each having (i) an internal double stranded sequence of paired bases and (ii) a single-stranded sequence of unpaired bases of both ends of the double stranded internal sequence, said mixture being annealed by heating said mixture and thereafter cooling said heated mixture to allow formation of said DNA 1 fragments; (b) annealing a mixture of complementary but circularly permuted oligodeoxynucleotides which have phosphorylated 5' ends to form DNA 2 linker fragments, each having at least one restriction enzyme recognition site which does not occur within said DNA 1 fragments but occurs within the DNA of a plasmid vector, said DNA 2 linker fragments having (i) a single-stranded sequence of unpaired bases at both ends thereof which is complementary to a single-stranded sequence of a corresponding DNA 1 fragment and (ii) an internal double stranded sequence of paired bases, said linker DNA 2 fragments adapted to maintain the reading frame and to maintain the repeating amino acid sequence of the internal sequence of the DNA 1 fragments when said DNA 2 linker fragments and said DNA 1 fragments are attached enzymatically in tandem in said plasmid vector, said mixture of oligodeoxynucleotides being annealed by heating said mixture and thereafter cooling said heated mixture to allow formation of said DNA 2 fragments; (c) combining all or a portion of said mixture of DNA 1 fragments and all or a portion of said mixture of DNA 2 linker fragments to form a combined mixture and further cooling said combined mixture to allow oligomerization, by annealing of complementary single-stranded sequences, of said DNA 2 linker fragments and of said DNA 1 fragments with themselves and with each other to form nicked DNA 3 fragments consisting of DNA 1 fragments interspersed DNA 2 linker fragments; (d) treating said cooled combined mixture with an enzyme to covalently join adjacent oligodeoxynuxleotides in said nicked DNA 3 fragments to form DNA 4 fragments; (e) treating said DNA 4 fragments with a restriction endonuclease to cleave said fragments at said restriction site to form gene cassettes which code for polypeptides having one or more repeating amino acid sequences.

Claim 2

A method of producing a gene cassette coding for polypeptides comprised of one or more repeating amino acid sequences, said method comprising the steps of (a) annealing at least one mixture of complementary but circularly permuted oligodeoxynucleotides which have phosphorylated 5' ends and which code for one or more repeating amino acid sequences, to form DNA 1 fragments, each having (i) an internal double stranded sequence of paired bases and (ii) a single-stranded sequence of unpaired bases of both ends of the double stranded internal sequence, said mixture being annealed by heating said mixture and thereafter cooling said heated mixture to allow formation of said DNA 1 fragments; (b) annealing a mixture of complementary but circularly permuted oligodeoxynucleotides which have phosphorylated 5' ends to form DNA 2 linker fragments, each having at least one restriction enzyme recognition site which does not occur within said DNA 1 fragments but occurs within the DNA of a plasmid vector, said DNA 2 linker fragments having (i) a single-stranded sequence of unpaired bases at both ends thereof which is complementary to a single-stranded sequence of a corresponding DNA 1 fragment and (ii) an internal double stranded sequence of paired bases, said linker DNA 2 fragments adapted to maintain the reading frame and to maintain the repeating amino acid sequence of the internal sequence of the DNA 1 fragments when said DNA 2 linker fragments and said DNA 1 fragments are attached enzymatically in tandem in said plasmid vector, said mixture of oligodeoxynucleotides being annealed by heating said mixture and thereafter cooling said heated mixture to allow formation of said DNA 2 fragments; (c) combining all or a portion of said mixture of DNA 1 fragments and all or a portion of said mixture of DNA 2 linker fragments to form a combined mixture and further cooling said combined mixture to allow oligomerization, by annealing of complementary single-stranded sequences, of said DNA 2 linker fragments and of said DNA 1 fragments with themselves and with each other to form nicked DNA 3 fragments consisting of DNA 1 fragments interspersed DNA 2 linker fragments; (d) treating said cooled combined mixture with an enzyme to covalently join adjacent oligodeoxynuxleotides in said nicked DNA 3 fragments to form DNA 4 fragments; (e) treating said DNA 4 fragments with a restriction endonuclease to cleave said fragments at said restriction site to form gene cassettes which code for polypeptides having one or more repeating amino acid sequences.