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Nucleic Acids Research, 1978, Vol. 5, No. 3 905-924
© 1978

Articles

Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA

Peter Humphries, Robert Old, Lesley W. Coggins, Theresa McShane, Christine Watson and John Paul

Beatson Institute for Cancer Research, Wolfson Laboratory for Molecular Pathology Garscube Estate, Switchback Road, Glasgow G61, UK

Received January 12, 1978. Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment ‘A’ of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH terimini of the synthetic globin gene sequences were extended with short tracts of oligo dCMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E.coli 32 Plasmids carrying globin sequences were identified by hybridization of P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show tnat extensive DNA copies of Xenopus globiri mRNA have been integrated into recombinant plasmids.


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