Genentech, Inc.

City of Hope Research Institute

Systec Corporation

Keiichi Itakura

Arthur D Riggs

John J Rossi

Carl W Sims

Bruce E Kaplan

Richard J Zelinka

A first polynucleotide is formed with a first particular sequence of nucleotides such as by providing a solid-phase support and adding nucleotides to the support. A second polynucleotide is formed in a similar manner with a second particular sequence of nucleotides which are complementary to the nucleotides in the first polynucleotide at first particular ends of the polynucleotides. The first particular ends may be the 3'-ends of the polynucleotides. The polynucleotides are hybridized or annealed at their 3'-ends to form a base-pair complementary stretch between the first and second polynucleotides. A repair synthesis is then provided in the first and second polynucleotides at second ends (such as the 5'-ends) opposite the first particular ends to provide a double-stranded polynucleotide. The repair synthesis may be monitored by labelling the second end (or 5'-end) of one or both of the nucleotides with radioactivity and then subjecting the nucleotides to an enzymatic reaction.

DNA polymerase mediated synthesis of double stranded nucleic acids

Method and means for microbial polypeptide expression

Recombinant cloning vehicle microbial polypeptide expression

A microprocessor controlled, general purpose gene synthesizer for programmably synthesizing selected nucleotide sequences. Depending upon a programmed chemistry, metered volumes of selected bases and reagent/solvents are sequentially metered into the bottom of a reaction cell containing solid-supported nucleotides, where desired linkages are achieved in an agitated suspension. Self-refilling syringe metering and electrically initiated, pneumatically operated valving insures economical, non-contaminated synthesis. Preprogrammed, selectable chemistries and operator programmed sequences are operatively coupled under microprocessor control to ensure a general purpose synthesizer.

General purpose gene synthesizer

Mammalian gene for microbial expression

A resin support formed from a suitable resin such as polystyrene or a silica gel may be provided by forming an amino resin and by reacting the amino resin with an activated ester of a nucleoside to obtain the dimethoxytrityl resin. To add each successive nucleotide to the resin support, the nucleoside is phosphorylated to provide a monotriazolide. The phosphorylation may be provided by the reaction of a phosphoditriazolide with a 3'-hydroxyl neucleoside. The dimethoxytrityl resin is then hydroxylated to substitute the hydroxyl group for the dimethoxytrityl group in the resin support. The monotriazolide is then reacted with the hydroxylated resin support to provide the dimethoxytrityl resin. This reaction may occur in the presence of a nucleophilic catalyst, such as a catalyst selected from a group consisting of dimethylaminopyridine, N'-methylimidazole and tetrazole. A hydroxyl group may be then substituted again for the dimethoxytrityl group in the dimethoxytrityl resin to provide for the addition of other nucleotides to the resin support. The procedure may be repeated to provide polynucleotides with extended chains of nucleotides.

Solid-phase synthesis of polynucleotides

A commercially available resin such as polystyrene is converted into a phthalimidomethyl-resin by treatment with potassium phthalimide. The phthalimidomethyl-resin is converted into an amino resin with hydrazine in ethanol. The amino resin is then combined with an activated ester of a nucleoside to obtain an amide-bonded dimethoxytrityl resin. The activated ester of the nucleoside is obtained by reacting a nucleoside with succinic anhydride in the presence of 4-(dimethylamino) pyridine in pyridine to provide a monosuccinate derivative, which is in turn treated with pentachlorophenol and dicyclohexylcarbodimide in dimethylformamide. Any unreacted amino groups in the amide-bonded dimethoxytrityl resin may be masked and the dimethoxytrityl group may be removed by treatment of the amide-bonded resin with a solution of benzenesulfonic acid to obtain a hydroxylated resin support. Additional mononucleotides, dinucleotides and trinucleotides may be added to the resin support in the presence of a coupling reagent such as 2,4,6-triisopropylbenzenesulfonyl tetrazolide. Any unreacted 5'-hydroxyl group may be masked with acetic anhydride. The steps described in the previous two sentences may be repeated to form polynucleotides of any desired sequence. The resin may also be a polyacrylmorpholide. This resin may be converted into an amino resin with ethylene diamine in ethylene glycol. The resin may also be a silica gel. The silica gel may be treated by any of the above methods, to provide a solid-phase resin support for the nucleotides.

The Specification discloses: 1. Recombinant microbial cloning vehicles comprising heterologous DNA coding for the expression of mammalian hormone (e.g., somatostatin) and other polypeptides, including plasmids suited for the transformation of bacterial hosts. The latter incorporate a regulon homologous to the host in its untransformed state, in reading phase with the structural gene for the heterologous DNA; 2. Cloning vehicles coding for the microbial expression of a protein variously comprising (a) a polypeptide hapten and additional protein sufficient in size to confer immunogenicity on the product of expression, which may find use in raising antibodies to the hapten for assay use or in the manufacture of vaccines; and (b) a desired polypeptide product and additional protein from which the desired product may be cleaved; and 3. Methods of preparing synthetic structural genes coding for the expression of mammalian polypeptides in microbial cloning systems.

Growing community of inventors

Arcadia, CA, United States of America

Keiichi Itakura