Methods and materials for obtaining microbial expression of polypeptides including bovine prolactin
AssigneeAmgen Inc.
InventorLawrence M. Souza
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Abstract
Disclosed are methods for selective modification of double-stranded DNA sequences facilitating their storage and incorporation into expression vectors. Manufactured partially double-stranded DNA primers are hybridized to complementary regions of single-stranded forms of DNA sequences to be altered. Desired, selectively modified double-stranded DNA sequences are then formed by DNA polymerization and specific nuclease digestion of undesired double- and single-stranded regions. Illustratively provided are DNA sequences useful in the microbial expression of bovine prolactin and other valuable polypeptides such as avian growth hormone.
Claims12
A method for selective modification of a double-stranded DNA sequence, comprising the steps:
- (1) forming a first selected single-stranded DNA copy of said double-stranded DNA sequence;
- (2) hybridizing said first selected single-stranded copy to a manufactured DNA sequence which comprises
- (a) a single-stranded portion complementary to a selected portion of said first selected single-stranded copy, and
- (b) a double-stranded portion having a free end and an end wherein one strand is joined to the single-stranded portion, which double-stranded portion includes base pairs forming a recognition site for cleavage by a restriction endonuclease enzyme or includes at its free end base pairs forming half of a recognition site for cleavage by a blunt-end-forming restriction endonuclease;
- (3) incubating the hybridized product of step (2) with nucleotide triphosphates and DNA polymerase under conditions facilitative of 3' extension of the single-stranded portion of said manufactured sequence with bases complementary to said first selected single-stranded DNA copy, so that said double-stranded DNA sequence is reconstructed except for that portion of its sequence 3' to the site of hybridization of the selected single-stranded copy to the 5' end of the single-stranded portion of said manufactured sequence;
- (4) treating the product of step (3) to delete single-stranded sequences 3' to the site of hybridization of the selected single-stranded copy to the 5' end of the single-stranded portion of said manufactured sequence;
- and
- (5) recovering the modified double-stranded DNA sequence.
Dependencies are inferred from the claim text.