DNA cloning: A personal view after 40 years

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.restriction enzyme | pSC101 | EcoRI | genetic engineering | gene cloning

The art of vector engineering: towards the construction of next‐generation genetic tools

“…Extrachromosomal genetic elements, now widely known as plasmids, were first recognized in bacteria over 60 years ago (Cohen, 2013;Kado, 2014). The development of recombinant DNA techniques in the 1970s (Lobban and Kaiser, 1973;Novick et al, 1976;Bolivar et al, 1977;Sinsheimer, 1977;Cohen et al, 1992;Cohen, 2013;Kado, 2014) led to a multitude of possibilities for manipulating those natural plasmids, turning them into vector systems which could be useful in several applications. Early vector systems were based on natural ColE1 derivatives and were primarily restricted to E. coli owing to their replication machinery (Hershfield et al, 1974;Bolivar et al, 1977).…”

Even therapeutic antimicrobial use in animal husbandry may generate environmental hazards to human health

“…The advent of DNA restriction enzymes and recombinant DNA methods permitted confirmation of plasmid modularity, construction of physical maps and further study of the functions of many of their genes in isolation (Timmis et al ., ; Cohen, ). Epidemiological information indicating that antimicrobial use in animal husbandry selected for antimicrobial resistance in the animal environment coupled to the genetic and molecular information regarding the DNA organization of plasmids explained their ready dissemination among bacteria of different species and genera (Datta, ; Anderson, ; Clowes, ; Smith, ; Timmis et al ., ; Cohen, ). It also underscored the potential of antimicrobial use in animals to harm human health through transfer of multiple antimicrobial‐resistant zoonotic bacteria and antimicrobial resistance genes to human pathogens (Datta, ; Anderson, ; Clowes, ; Smith, ; Marshall and Levy, ; Cohen, ).…”

“…Subsequent molecular work in multiple laboratories confirmed the DNA composition of antimicrobial resistance plasmids (R‐plasmids), their extrachromosomal nature and their modular architecture with genes devoted to replication, transfer and antimicrobial resistance (Falkow et al ., 1964; 1966; Womble and Rownd, ; Cohen, ). The advent of DNA restriction enzymes and recombinant DNA methods permitted confirmation of plasmid modularity, construction of physical maps and further study of the functions of many of their genes in isolation (Timmis et al ., ; Cohen, ).…”

“…Subsequent molecular work in multiple laboratories confirmed the DNA composition of antimicrobial resistance plasmids (R‐plasmids), their extrachromosomal nature and their modular architecture with genes devoted to replication, transfer and antimicrobial resistance (Falkow et al ., 1964; 1966; Womble and Rownd, ; Cohen, ). The advent of DNA restriction enzymes and recombinant DNA methods permitted confirmation of plasmid modularity, construction of physical maps and further study of the functions of many of their genes in isolation (Timmis et al ., ; Cohen, ). Epidemiological information indicating that antimicrobial use in animal husbandry selected for antimicrobial resistance in the animal environment coupled to the genetic and molecular information regarding the DNA organization of plasmids explained their ready dissemination among bacteria of different species and genera (Datta, ; Anderson, ; Clowes, ; Smith, ; Timmis et al ., ; Cohen, ).…”

Protein crystallography from the perspective of technology developments

“…In the early years of protein crystallography, proteins and nucleic acids studied were all purified from naturally abundant sources. Rare but important proteins could only be studied after the invention of recombinant DNA techniques by Stanley Cohen in 1970s [12, 13, 14] and the resulting progress in expression of exotic proteins in hosts such as E. coli . Other crucial techniques responsible for the exponential growth of PX included synchrotron radiation (SR), computer algorithms for obtaining crystallographic phase information, and structural genomics (SG) as demonstrated in Figure 1.…”