Manufacture and expression of structural genes
AssigneeAmgen Inc.
InventorYitzhak Stabinsky
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Abstract
Described are rapid and highly efficient procedures for the total synthesis of linear, double stranded DNA sequences of up to about 200 base pairs, which sequences may comprise entire structural genes. Illustratively disclosed is the preparation and expression of manufactured genes, including fusion genes, capable of directing synthesis of human .beta.-endorphin and of proteins which differ from human .beta.-endorphin in terms of the identity or relative position of one or more amino acids. Manufactured genes preferably include codons selected from among alternative codons specifying the same amino acid on the basis of preferential expression in a projected host microorganism (e.g., E. coli) to be transformed.
Claims7
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.
Dependencies are inferred from the claim text.