Patent 04652639 - Manufacture and expression of structural genes > Claims
1. A method for synthesis of linear, double stranded DNA sequences from nucleotide bases comprising the steps of: (1) synthesizing from nucleotide bases two or more different, linear, duplex DNA strands, each duplex strand comprising a double stranded region of 12 or more selected complementary base pairs and further comprising at least one single stranded terminal sequence of from 3 to 7 selected bases at one end of the strand, each single stranded terminal sequence of each duplex DNA strand comprising the entire base complement of at most one single stranded terminal sequence of any duplex DNA strand prepared; and (2) annealing and ligating each duplex DNA strand prepared in step (1) to one or two different duplex strands prepared in step (1) having a complementary single stranded terminal sequence, thereby to form a single continuous double stranded DNA sequence which has a duplex region of at least 27 selected base pairs comprising at least 3 base pairs formed by complementary association of single stranded terminal sequences of duplex DNA strands prepared in step (1) and which has from 0 to 2 single stranded terminal regions of from 3 to 7 bases. 2. A method according to claim 1 wherein at least three different duplex DNA strands are prepared in step (1) and all strands so prepared are annealed and ligated concurrently in a single annealing and ligating reaction mixture to form a single continuous double stranded DNA sequence which has a duplex region of at least 42 selected base pairs comprising at least two non-adjacent sets of 3 or more base pairs formed by complementary association of single stranded terminal sequences of duplex strands prepared in step (1). 3. A method according to claim 1 wherein the duplex DNA strand preparation step (1) comprises the steps of: (a) constructing first and second linear oligodeoxynucleotide segments having 15 or more bases in a selected linear sequence, the linear sequence of bases of said second segment comprising the total complement of the sequence of bases of said first segment except that at least one end of said second segment shall either comprise an additional linear sequence of from 3 to 7 selected bases beyond those fully complementing said first segment, or shall lack a linear sequence of from 3 to 7 bases complementary to a terminal sequence of said first segment, provided, however, that said second segment shall not have an additional sequence of bases or be lacking a sequence of bases at both of its ends; and, (b) combining said first and second segments under conditions conducive to complementary association between segments to form a linear, duplex DNA strand. 4. A method according to claim 1 wherein the continuous double stranded DNA sequence formed in step (2) comprises an entire structural gene expressable in a selected host microorganism as a composition comprising a biologically functional protein. 5. A method according to claim 4 wherein, in the double-stranded DNA sequence formed, the sequence of bases comprises one or more codons selected, from among alternative codons specifying the same amino acid, on the basis of preferential expression characteristics of the codon in a projected host microorganism.
6. A method according to claim 1 further comprising the step of labelling the double-stranded DNA sequence formed in step (2) with a detectable signal for use as a reagent material. 7. The method according to claim 6 wherein said signal is .sup.125 I.