A DNA transfer vector for human pre-growth hormone, a microorganism transformed thereby, and a method of cloning therefor

Claim 1

A DNA transfer vector comprising a deoxynucleotide sequence coding for human pre-growth hormone.

Claim 18

A method for cloning a deoxynucleotide sequence coding for human pre-growth hormone from pituitary gland tissue of an individual human, comprising the steps of a) extracting mRNA coding for human pre-growth hormone from an individual human pituitary gland, b) separating the mRNA substantially free of protein, DNA and other RNA, c) synthesizing a single-stranded cDNA having a nucleotide sequence complementary to that of the mRNA, in a series of procedures including incubation with an enzyme, extraction to remove protein from the reaction mixture, alcohol precipitation, and alkaline hydrolysis, all of said procedures being carried out in the same reaction tube, in the absence of added carrier DNA, d) purifying the single-stranded cDNA by chromatography wherein those fractions containing the single-stranded DNA are located by a radioassay which does not consume the reaction product, e) synthesizing double-stranded cDNA having one strand with a nucleotide sequence corresponding to that of the mRNA, in a series of procedures including incubation with an enzyme, and extraction to remove protein, said procedures being carried out in a single reaction tube in the absence of added carrier DNA, f) purifying the double-stranded cDNA by chromatography as in step d), g) further treating the double-stranded cDNA with enzyme, essentially as described in steps e) and f) to provide end groups suitable for inserting the cDNA into a DNA transfer vector, h) inserting the double-stranded cDNA into a DNA transfer vector, thereby forming a recombinant transfer vector, i) transforming a microorganism with the recombinant transfer vector, and j) selecting a microorgamism strain derived from a single transformed cell transformed by a recombinant transfer vector containing cDNA coding for human pre-growth hormone, thereby cloning a deoxynucleotide sequence coding for human pre-growth hormone.

Claim 19

A method for cloning a deoxynucleotide sequence coding for human pre-growth hormone from pituitary gland tissue of an individual human, comprising the steps of a) extracting mRNA coding for human pre-growth hormone from an individual human pituitary gland, b) separating the mRNA substantially free of protein, DNA and other RNA, c) synthesizing a single-stranded cDNA having a nucleotide sequence complementary to that of the mRNA, in a series of procedures including incubation with an enzyme, extraction to remove protein from the reaction mixture, alcohol precipitation, and alkaline hydrolysis, all of said procedures being carried out in the same reaction tube, in the absence of added carrier DNA, d) purifying the single-stranded cDNA by chromatography wherein those fractions containing the single-stranded DNA are located by a radioassay which does not consume the reaction product, e) synthesizing double-stranded cDNA having one strand with a nucleotide sequence corresponding to that of the mRNA, in a series of procedures including incubation with an enzyme, and extraction to remove protein, said procedures being carried out in a single reaction tube in the absence of added carrier DNA, f) purifying the double-stranded cDNA by chromatography as in step d), g) further treating the double-stranded cDNA with enzyme, essentially as described in steps e) and f) to provide end groups suitable for inserting the cDNA into a DNA transfer vector, h) inserting the double-stranded cDNA into a DNA transfer vector, thereby forming a recombinant transfer vector, i) transforming a microorganism with the recombinant transfer vector, and j) selecting a microorgamism strain derived from a single transformed cell transformed by a recombinant transfer vector containing cDNA coding for human pre-growth hormone, thereby cloning a deoxynucleotide sequence coding for human pre-growth hormone.