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How did Stanley N. Cohen and Herbert W. Boyer insert new genes into E. coli bacteria?

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A step-by-step diagram shows donor DNA and vector DNA cut with restriction enzymes to create overhangs, mixed and sealed with ligase to form recombinant DNA, which is introduced into bacterial cells. The recombinant DNA replicates as the cells divide, producing clones. — biotechnology Steps involved in the engineering of a recombinant DNA molecule.

Stanley N. Cohen and Herbert W. Boyer pioneered the insertion of new genes into E. coli by developing methods to create and utilize recombinant plasmids.1

Their process relied on the use of restriction enzymes, which act as "molecular scissors" to cleave DNA at specific target sequences. By using the same restriction enzyme to cut both the donor DNA and the plasmid vector, they created complementary, single-stranded "overhangs" that allowed the donor fragment and the vector to splice together. The enzyme DNA ligase was then applied to seal the sugar-phosphate backbone, resulting in a stable, circular recombinant DNA molecule.123

Finally, this recombinant DNA was introduced into E. coli cells through a process known as transformation, for which the bacteria were specially treated to increase their permeability. Once inside, the recombinant plasmid replicated autonomously as the bacteria divided, effectively cloning the inserted gene.23