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, 110 (39), 15521-9

DNA Cloning: A Personal View After 40 Years

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DNA Cloning: A Personal View After 40 Years

Stanley N Cohen. Proc Natl Acad Sci U S A.

Abstract

In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer's laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning.

Keywords: EcoRI; gene cloning; genetic engineering; pSC101; restriction enzyme.

Conflict of interest statement

The author declares no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cartoon by D. Adair in the Honolulu Advertiser newspaper, September 26, 1988 accompanying an article reporting demolition of the Waikiki beach delicatessen where the initial DNA cloning experiments were planned. The persons depicted clockwise are presumed to be H. Boyer (12 o’clock), S. Cohen, G. Brinton, C. Brinton, and S. Falkow. Reprinted by permission.
Fig. 2.
Fig. 2.
Schematic diagram of the strategy used for construction of biologically functional plasmids (1). R6-5 plasmid DNA fragments generated by cleavage using the EcoRI endonuclease were allowed to associate randomly in vitro and were then covalently joined by DNA ligase. DNA in the resulting mixture was introduced into calcium chloride-treated E. coli, and bacterial colonies expressing individual antibiotic resistance phenotypes encoded by R6-5 were selected on media containing antibiotics. Plasmid constructs isolated from these E. coli clones contained DNA fragments carrying specific resistance genes.
Fig. 3.
Fig. 3.
DNA analysis in the initial DNA cloning experiments. (Top) Agarose-gel electrophoresis of (lane a) the pSC102 plasmid containing three of the multiple EcoRI-generated fragments of R6-5 DNA (lane b). Lane c shows that EcoRI cleavage of the pSC101 vector produces a single DNA fragment of the expected size. (Middle) Electron photomicrograph of pSC101, the first plasmid used successfully as a vector for DNA cloning. (Bottom) Agarose gel electrophoresis showing cloning of the kanamycin resistance gene of R6-5: (lane d) EcoRI-cleaved DNA of the pSC101 plasmid vector, (lane c) EcoRI-generated fragments of a novel plasmid (pSC102) that had been constructed from R6-5 (see Top) and that expresses the kanamycin resistance determinant of the parental R6-5 replicon, (lane b) mixture of the DNAs shown in lanes c and d, and (lane a) EcoRI-generated fragments of a novel plasmid (pSC105) expressing both the tetracycline resistance gene of the pSC101 vector and the kanamycin resistance gene, which had been cloned from pSC102 by attaching it to pSC101. Top and Bottom are from ref. .
Fig. 4.
Fig. 4.
Cloning of S. aureus plasmid DNA in E. coli. (Upper) Schematic diagram of strategy used for testing the viability of interspecies DNA hybrids (2). DNA of the pI258 plasmid, which carries a β lactamase gene encoding resistance to penicillins in S. aureus was cleaved by EcoRI endonuclease and mixed with similarly cleaved DNA of the pSC101 vector encoding tetracycline resistance. After ligation, the mixture was introduced into E. coli cells, and colonies that expressed both resistance phenotypes were identified. (Lower) Centrifugation analysis in isopycnic density gradient of plasmid DNA (pSC112) isolated from an E. coli clone expressing both resistances and showing DNA species that band at buoyant densities characteristic of E. coli (ρ = 1.710) and S. aureus (ρ = 1.68–1.69) DNAs and reflect the distinctly different A+T/G+C nucleotide ratios of these unrelated bacterial species. Lower is from ref. .
Fig. 5.
Fig. 5.
Electric photomicrograph of heteroduplex showing homology between DNA isolated from X. laevis oocytes and plasmid DNA isolated from bacteria and containing fragments of ribosomal RNA genes that had been cloned by attaching the eukaryotic cell DNA to the pSC101 vector. (A) Single strand of X. laevis rDNA. (B) Double-stranded regions of homology between X. laevis rDNA and plasmid DNA isolated from E. coli. (C), Single-strand DNA regions corresponding in length to the pSC101 vector, which shares no homology with X. laevis rDNA. (Scale bar: 1 μm.) Figure is from ref. .

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