Recipes for Recombining DNA. A History of ‘Molecular Cloning: A Laboratory Manual’

Since Warren Weaver coined the term “molecular biology” in the late 1930s, technological innovation has driven the life sciences, from the analytical ultracentrifuge to high-throughput DNA sequencing. Within this long history, the invention of recombinant DNA techniques in the early 1970s proved to be especially pivotal. The ability to manipulate DNA consolidated the high-profile focus on molecular genetics, a trend underway since Watson and Crick’s double-helical model in 1953. But the ramifications of this technology extended far beyond investigating heredity itself. Biologists doing research on a wide variety of molecules, including enzymes, hormones, muscle proteins, RNAs, as well as chromosomal DNA, could harness genetic engineering to copy the gene that encoded their molecule of interest, from whatever organism they worked on, and put that copy in a bacterial cell, from which it might be expressed, purified, and characterized. Many life scientists who wanted to use recombinant DNA techniques were not trained in molecular biology. They sought technical know-how on their own in order to bring their labs into the vanguard of gene cloners. Manuals became a key part of this dissemination of expertise.

What did it mean to clone a gene? Simply put, cloning is copying, and a gene is usually copied onto a vector that can replicate in a cell, so that the copied gene can be propagated and studied. In seeking to make copies of genes and move them around from organism to organism, biologists were inspired by bacteria, whose ability to exchange genetic material had been recognized in 1946 by Joshua Lederberg and Edward Tatum. It turned out that there were numerous genetic units that enabled gene exchange in bacteria, including lysogenic viruses and fertility factors. In 1952 Lederberg christened the entities “plasmids.”

By the 1960s, researchers were using these naturally-occurring gene shuttles in microbes to identify, map, and characterize bacterial genes.[1] Unsurprisingly, many biologists were more interested in tracking genes found in humans and other “higher organisms” (eukaryotes—plants, animals, and fungi—as opposed to the one-celled prokaryotes, mostly bacteria). The discovery of bacterial restriction enzymes, which sever DNA strands at specific base-pair combinations, inspired molecular biologists to attempt to use these as microscopic scissors. In principle, if a researcher could identify and locate a particular eukaryotic gene, she could use a restriction enzyme to “cut” it out of chromosomal DNA and insert it into a circular bacterial plasmid (Figure 1). Cloning eukaryotic genes was an immensely difficult task, and several early attempts faltered. Other efforts did not go forward due to the potential public health hazards of placing genes from widely-studied tumor viruses into E. coli, a bacterium that usually inhabits the gut of humans. No one knew whether exposure to bacteria toting these tumor-associated genes could give people cancer.

In 1973, a group of scientists at UCSF and Stanford, led by Herbert Boyer and Stanley Cohen, succeeded in placing a copy of a frog gene (one that encoded ribosomal RNA) into a bacterial plasmid. Not only was the inserted gene on its plasmid vector taken up and replicated by E. coli, but also the foreign DNA was expressed into the corresponding product RNA. Their 1974 publication became the much-cited proof that genes from a higher organism could be cloned and expressed in a bacterium.

Few scientists, however, had the specialized materials with which to achieve such a feat. Richard Roberts at Cold Spring Harbor discovered and purified many of the restriction enzymes essential for this work. He recalls that “Summer visitors would stop by with a tube of their favorite DNA in their pocket, just to see if we had an enzyme that would convert it into some useful fragments.” Unable to persuade his own institution to start manufacturing and selling restriction enzymes, Roberts helped the newly-founded New England Biolabs corner this market. The first company catalog was issued in 1975; their enzymes became indispensable to the early gene cloners. Biologists who worked on bacteria were able to rapidly exploit these newly commercialized enzymes and customized plasmids, so that the cloning of genes from microbes took off.

However, cloning of genes from higher organisms remained in the hands of the experts who could make the difficult techniques work. In 1977, Shirley Tilghman and other members of Philip Leder’s group cloned the first mammalian genes from mice.[2] In addition to academic researchers, biotech entrepreneurs were keenly interested in cloning eukaryotic genes. Simply obtaining genetic material from higher organisms in a form that could be searched for a specific gene was a formidable challenge. Tom Maniatis, part of the group that cloned the first human gene, created a human genomic “library” and shared it with other biologists.[3] But researchers also needed protocols and know-how. Courses (for practitioners, not only university students) became a popular way to meet this demand.

Cold Spring Harbor Laboratory had been offering summer courses on new laboratory techniques since the 1940s. One popular course, “Advanced Bacterial Genetics,” already offered researchers a chance to learn how to identify, map, and copy genes from prokaryotes. In 1980, Cold Spring Harbor Laboratory (CSHL) began offering a postgraduate summer course called “Molecular Cloning of Eukaryotic Genes.” James Watson, director of CSHL, asked Maniatis to teach this course, and others joined the effort. Nancy Hopkins, who had taught a tumor virology course that had just ended, stayed on for the cloning course. Ed Fritsch, a postdoc in Maniatis’s lab, put together the laboratory materials, and Helen Donis-Keller and Catherine O’Connell served as course assistants.[4]

The coursebook was made up of “consensus protocols” defining the field at the time (many of which were already circulating informally).[5] Upon advertising the postgraduate training course, “Molecular Cloning of Eukaryotic Genes,” more than 300 applied to take it. Only sixteen students could enroll. Watson immediately saw the opportunity to make cloning know-how available to a wider base of users through publication. Issuing an instructional guide from Cold Spring Harbor Laboratory would further consolidate the institution’s reputation for being at the vanguard of molecular biology—and there was already a tradition there of publishing course manuals as books.

Watson wanted Maniatis on the team of authors, as his reputation in cloning genes was already formidable. But he had recently moved to Caltech, where he was busy chairing an NIH study section and running his own lab. He only agreed to prepare a manual based on the course if he had significant help.[6] Watson persuaded Joe Sambrook, a long-time tumor virologist at the lab, to join the effort. Although Sambrook had not taught the summer course, he did have extensive relevant knowledge, and he would do a lion’s share of the manual-writing.[7] Fritsch, who was about to leave for a tenure-track faculty position at Michigan State University, remained involved with the project having helped teach the course twice.[8] In the end, the collaboration was productive, and the first edition was published in 1982 (Figure 2). Maniatis handed off teaching of the “Molecular Cloning of Eukaryotic Genes” summer course at CSHL to others the same year as the manual came out.

The three authors explain in the Preface that because “the manual was originally written to serve as a guide to those who had little experience in molecular cloning, it contains much basic material.”[9] Indeed, the book was full of both recipes and tips. That said, part of its success, according to one early user, was that it communicated enough about the science behind the recipes that users were able to trouble-shoot the problems they ran into.[10] And part of the utility of the book was that, by virtue of its plastic-ring binding, it could be laid flat on a laboratory bench (Figure 3).[11]

Just as Watson had suspected, Molecular Cloning met widespread demand. There were orders for more than 5,000 copies before the publication date. Consequently, the press sold 5,113 copies the first month of its appearance, in July 1982 (as compared with its original number for sales projected by the press: 210 copies). In August 988 copies were sold, in September 2,487, in October 1863, and in November 768. That fall, Molecular Cloning was outselling every other book in the press’s line-up.[12] As a reviewer for the British Society for Developmental Biology put it, “no laboratory with any serious interest in molecular biology of development and their cloning should be without it.”[13] By late June 1983, more than 18,000 copies had been sold.[14] Plans for a second edition, initially scheduled for 1984, were already underway.[15] The second edition, which actually appeared in 1989, was received just as enthusiastically as the first. As a reviewer in Nature put it,

Few molecular biologists welcome publication of any of the many protocol books that promise to be the single source for their laboratory methods. For the most part, such laboratory methods fall far short of this goal. So why the excitement surrounding the long-awaited second edition of the classic guide, Molecular Cloning, which first appeared in 1982? The original version immediately filled the need for an anthology of laboratory procedures pertinent to the emerging field of recombinant DNA. With the 545-page spiral-bound paperback in hand, virtually any experimentalist could make a stab at cloning and have a reasonable expectation of success.[16]

In short, the Cold Spring Harbor Laboratory publication became the canonical manual—or “Bible”—for gene cloners. Extending this common metaphor, one biochemist made reference to “those who daily workshop the Cold Spring Harbor idol.”[17] But the deity had rivals. Its strongest competitor was Current Protocols in Molecular Biology, introduced in 1987 by a group of researchers based at Massachusetts General Hospital.[18] Sarah Greene was the original publisher, but the series was soon bought by Wiley. Rather than being written by three authors, this manual was produced by an entire team of scientists, who contributed individual pieces on various techniques. In addition, Current Protocols had a very different way of dealing with the rapid growth (and obsolescence) of techniques—the book was designed to be expanded via subscription. Through a quarterly update service, subscribers received supplements to insert into the original loose-leaf binder, which was separated into sections by preprinted dividers (Figures 4 and 5). This meant that the Table of Contents also needed frequent updating. Five thick binders were published in the original series (Figure 6).

The loose-leaf format proved unwieldy, and in 1989 Wiley published Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. This single volume work was bound as a traditional text, with wide pages in a format that would prop open easily on the back of a lab bench. The challenge of updating was more easily accommodated by the growth of multimedia technologies in the 1990s. The 2001 edition came with a CD-ROM “Lab Book.” By the third edition (2001), Molecular Cloning: A Laboratory Manual also had an associated website for its publication. Moving manuals online put knowledge at one’s fingertips in a new way, yet the demand for guides that can be plopped open on a lab bench has meant that print versions retain value, as evidenced by the publication of a fourth edition of Molecular Cloning in 2012. Most fields of life science today, including bioinformatics, cell biology, immunology, neuroscience, stem cell science, and toxicology, have their go-to manuals and protocol books, in print and online.

These “cookbooks” occupy the shelves, benches, and hard-drives of most biology labs, important if unnoticed. Their ubiquity enriches our understanding of the scientific process. An obsession with innovation may blind us to the importance of procedure, repeatability, and tried-and-true methods. Manuals make discovery possible, by leading scientists through the routine steps of their experiments and (if the manual is good) helping them trouble-shoot when experiments fail. In a world of hyper-specialized research, guide books are bridges, carrying technical know-how between laboratories and enabling researchers to master the latest methods without going back to school.

This is the first of four pieces in our pre-conference blog series that we are posting in tandem with The Recipes Project: Food, Magic, Art, Science, and Medicine.

Angela N. H. Creager is the Thomas M. Siebel Professor in the History of Science at Princeton University.