C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
195/1.14, 195/1.
C12N 15/00 (2006.01) C12P 19/34 (2006.01) C12R 1/19 (1980.01)
Patent
CA 1156166
ABSTRACT OF THE DISCLOSURE The disclosure sets forth a process for isolating a specific nucleotide sequence containing genetic information, synthesis of DNA having the specific nucleotide sequence and transfer of the DNA to a host microorganism The process involves isolation of a selected cell population, extraction of intact mRNA from the cells, purification of intact messenger RNA from the extract and subjection of the purified extract to the action of reverse transcriptase in the presence of the four deoxynucleoside triphosphates needed to synthesize a complementary (cDNA) strand. The ribonucleotide sequence is selectively removed and the remaining deoxynucleotide sequence is incubated with reverse transcriptase or DNA polymerase in the presence of the four deoxynucleoside triphosphates to form a duplex cDNA structure having its comple menary strands joined together at one end by a single stranded loop. This product then is treated with single strand specific nuclease to cleave the single stranded loop, the resulting double stranded cDNA is extended in length by the addition at both ends of a specific DNA containing a restriction enzyme recognition site sequence, and the extended cDNA is treated with a restriction endonuclease to produce self-complementary single stranded ends at the five-prime termini of each strand in the duplex. A plasmid DNA having a recognition site for the same endonuclease is treated with the enzyme to cleave the poly- nucleotide strand, the 5' terminal phosphate groups from the resulting self-complementary single strand nucleotide sequences at the five-prime termini are removed and the prepared cDNA and plasmid DNA are incubated in the presence of DNA ligase, so that a viable closed ring of plasmid DNA can only occur if a segment of cDNA is included the cDNA-containing plasmid then being introduced to an appropriate host cell. Pure bacterial strains containing the recombinant plasmid are grown up and isolated. By this procedure, large amounts of recombi- nant plasmid DNA may be prepared and the specific cDNA sequence reisolated therefrom by endonucleolytic cleavage.
303974
Chirgwin John
Goodman Howard M.
Pictet Raymond
Rutter William J.
Shine John
Sim & Mcburney
The Regents Of The University Of California
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