PURIFICATION OF NUCLEOTIDE SEQUENCES SUITABLE FOR EXPRESSION IN BACTERIA

• Main Authors: HOWARD MICHAEL GOODMAN, JOHN SHINE, PETER HORST SEEBURG
• Format: Patent
• Language: English
• Published: 01.07.1978
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; DERIVATIVES THEREOF; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; HUMAN NECESSITIES; HYGIENE; MEDICAL OR VETERINARY SCIENCE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; NUCLEIC ACIDS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC CHEMISTRY; PEPTIDES; PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; SUGARS; VINEGAR; WINE

A method for obtaining a strain of microorganisms modified to contain a fragment of a specific deoxyribonucleotide sequence, which method comprises: (I) purifying a fragment of a deoxyribonucleotide sequence by the following steps: (a) providing a population of cdna transcripts of the polyribonucleotides. (b) submit the cdna transcripts to the action of a restriction endonuclease preparation capable of catalyzing the hydrolysis of the cdna transcripts at each of the restriction sites. (c) fractionating the fragments produced by the action of the restriction endonuclease according to its length. (d) pretreating the dna to remove any 5''-phosphate terminal group. (e) incubating the dna with a restriction endonucelase capable of acting on the desired specific nucleotide sequence to produce two linear sub-fragments thereof. (f) fractionating the sub-fragments according to their length. (g) isolate the two sub-fragments, (h) assemble the sub-fragments covalently, in a reaction catalyzed by dna ligase, (I) fractionate the assembled dna molecules according to their length, (II) recombine the resulting product of the above sequence with a dna transfer vector and transfer it to a microorganism. (Machine-translation by Google Translate, not legally binding)