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Recipes for recombining DNA: A history of Molecular Cloning: A Laboratory Manual

Abstract

Laboratory instructions and recipes are sometimes edited into books with a wide circulation. Even in the late twentieth century, publications of this nature remained influential. For example, protocols from a 1980 summer course on gene cloning at Cold Spring Harbor Laboratory provided the basis for a bestselling laboratory manual by Tom Maniatis, Ed Fritsch and Joe Sambrook. Not only did the Molecular Cloning: A Laboratory Manual become a standard reference for molecular biologists (commonly called the 'bible'), but also its recipes and clear instructions made gene cloning and recombinant DNA technologies accessible to non-specialists. Consequently, this laboratory manual contributed to the rapid spread of genetic-engineering techniques throughout the life sciences, as well as in industry. As is often the case with how-to books, however, finding a way to update methods in this rapidly changing field posed a challenge, and various molecular-biology reference books had different ways of dealing with knowledge obsolescence. This paper explores the origins of this manual, its publication history, its reception and its rivals – as well as the more recent migration of such laboratory manuals to the Internet.

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RESEARCH ARTICLE

Recipes for recombining DNA: A history of Molecular

Cloning: A Laboratory Manual

Angela N.H. Creager*

Department of History, Princeton University, 129 Dickinson Hall, Princeton, NJ 08544-1174, USA.

*E-mail: creager@princeton.edu

Abstract

Laboratory instructions and recipes are sometimes edited into books with a wide circulation. Even in

the late twentieth century, publications of this nature remained influential. For example, protocols

from a 1980 summer course on gene cloning at Cold Spring Harbor Laboratory provided the basis for

a bestselling laboratory manual by Tom Maniatis, Ed Fritsch and Joe Sambrook. Not only did the

Molecular Cloning: A Laboratory Manual become a standard reference for molecular biologists (com-

monly called the ' bible' ), but also its recipes and clear instructions made gene cloning and recom-

binant DNA technologies accessible to non-specialists. Consequently, this laboratory manual

contributed to the rapid spread of genetic-engineering techniques throughout the life sciences,

as well as in industry. As is often the case with how-to books, however, finding a way to update

methods in this rapidly changing field posed a challenge, and various molecular-biology reference

books had different ways of dealing with knowledge obsolescence. This paper explores the origins of

this manual, its publication history, its reception and its rivals – as well as the more recent migra-

tion of such laboratory manuals to the Internet.

The invention of recombinant DNA techniques in the early 1970s consolidated the high-

profile focus on molecular genetics, a trend under way since Watson and Crick' s double-

helical DNA model in 1953. With the tools of genetic engineering, biologists doing

research on a wide variety of molecules (including enzymes, hormones, muscle proteins

and RNAs, as well as chromosomal DNA), and any organism, could identify and copy the

gene containing its 'code ' and place that copy in a bacterial cell. At this point, the copied

gene could be amplified, sequenced or analysed, or its product (usually a protein)

expressed, purified and characterized. Initially, only a few molecular biologists and

biochemists had the materials and know-how to do this. Many other life scientists sought

this practical knowledge, 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. A gene would be

isolated from all of the other genes in a cell, then inserted into a DNA ' cloning vector'

that could replicate in a bacterial cell, so that the copied gene could be propagated indef-

initely in a culture of the host cell, usually E. coli . In seeking to make copies of genes and

move them around from organism to organism, biologists were inspired by bacteria,

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whose ability to exchange genetic material had been recognized in 1946.

It turned out

that there were numerous genetic units (dubbed plasmids) that enabled gene exchange

among bacteria in the real world.

By the 1960s, researchers were using these naturally

occurring gene shuttles in microbes to identify, map and characterize bacterial genes.

Unsurprisingly, many biologists were more interested in studying 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 cleave DNA strands at specific base-pair combinations, inspired molecular

biologists to attempt to use these as submicroscopic 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, insert it into a circular bacterial plasmid, then intro-

duce the ' recombinant' plasmid into bacteria (see Figure 1).

That this could be done with

genetic material from an animal was demonstrated first in 1973 by a collaborating group

of scientists from the laboratories of Herbert Boyer (University of California,

San Francisco – UCSF) and Stanley Cohen (Stanford). They inserted a frog ribosomal

RNA gene into a customized bacterial plasmid. Not only was the inserted gene on its plas-

mid vector taken up and replicated by E. coli , but also the foreign DNA was transcribed into

the corresponding rRNA product.

However, cloning genes from higher organisms remained immensely challenging, for

both intrinsic and extrinsic reasons. Intrinsically, it was technically difficult to locate spe-

cific genes in higher organisms. Both the human and mouse genomes, for instance, are

650 times larger than that of E. coli , so a given mammalian gene might comprise a ten-

millionth of that organism' s DNA.

The best way to identify the gene of interest was

with a matching piece of nucleic acid, obtained by isolating the messenger RNA

(mRNA) and using it to generate a DNA copy (cDNA). This was hard enough, but if a

nucleic acid probe could not be produced, gene screening was even more arduous:

every candidate clone had to be put into a protein expression vector, to search with anti-

bodies or enzyme assay for the identifiable product.

Extrinsically, concerns about

public-health hazards from genetically engineered pathogens led the National Institutes

1 Joshua Lederberg and Edward L. Tatum, ' Novel genotypes in mixed cultures of biochemical mutants of bac-

teria' , Cold Spring Harbor Symposia on Quantitative Biology (1946) 11, pp. 113 –14.

2 Joshua Lederberg, ' Cell genetics and hereditary symbiosis' , Physiological Reviews (1952) 32, pp. 403–30.

3 E.g. E.L. Wollman, F. Jacob and W. Hayes, ' Conjugation and genetic recombination in Escherichia coli K-12' , Cold

Spring Harbor Symposium on Quantitative Biology (1956) 21, pp. 141–62.

4 Richard J. Roberts, ' How restriction enzymes became the workhouses of molecular biology' , Proceedings of the

National Academy of Sciences of the United States of America (2005) 102, pp. 5905–8.

5 These plasmids were often modified from naturally occurring extrachromosomal hereditary units in bac-

teria, often adding antibiotic resistance genes that could be used to screen for copies with the desired DNA.

6 John F. Morrow, Stanley N. Cohen, Annie C.Y. Chang, Herbert W. Boyer, Howard M. Goodman and Robert

B. Helling, ' Replication and transcription of eukaryotic DNA in Escherichia coli' , Proceedings of the National

Academy of Sciences of the United States of America (1974) 71, pp. 1743– 7. Boyer and Cohen filed patents as well

as publishing results: Rajendra K. Bera, ' The story of the Boyer– Cohen patents' , Current Science (2009) 96,

pp. 760– 3; Doogab Yi, ' Who owns what? Private ownership and the public interest in recombinant DNA technol-

ogy in the 1970s' , Isis (2011) 102, pp. 446– 74. For background to Boyer and Cohen' s work, Harrison Echols,

Operators and Promoters: The Story of Molecular Biology and Its Creators (ed. Carol A. Gross), Berkeley: University

of California Press, 2001, pp. 333– 40; Doogab Yi, The Recombinant University: Genetic Engineering and the

Emergence of Stanford Biotechnology, Chicago: The University of Chicago Press, 2015.

7 S.M. Tilghman, D.C. Tiemeier, F. Polsky, M.H. Edgell, J.G. Seidman, A. Leder, L.W. Enquist, B. Norman and

P. Leder, ' Cloning specific segments of the mammalian genome: bacteriophage λ containing mouse globin and

surrounding gene sequences' , Proceedings of the National Academy of Sciences of the United States of America

(1977) 74, pp. 4406– 10, 4406.

8 Stephanie Broome and Walter Gilbert, ' Immunological screening method to detect specific translation pro-

ducts' , Proceedings of the National Academy of Sciences of the United States of America (1978) 75, pp. 2746 –9.

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of Health (NIH) to require that recombinant work on higher organisms be done in biocon-

tainment facilities with special ventilation systems and protective clothing, masks and

gloves. Few such facilities existed in universities in the 1970s, as compared with military

laboratories such as Fort Dietrich, set up for working with biological-warfare agents.

The

impact on the field was dramatic, because the NIH funded nearly all of the leading aca-

demic molecular-biology laboratories in the US.

The first complete mammalian gene, that for mouse globin, was cloned in 1977 by

scientists in Philip Leder' s laboratory at NIH.

Shirley Tilghman, then a postdoc in the

lab, recalls going with colleague David Tiemeier into a biocontainment facility with

their cumbersome protective gear, and picking out tens of thousands of candidate clones,

Figure 1. A typical recombinant DNA experiment depicting the cloning of eukaryotic genomic DNA fragments

into a plasmid that is transformed into E. coli . Drawing by Georgia Creager.

9 Complying with the new NIH guidelines brought a wide range of complications, including at the community

level. In Cambridge, MA, the city council did not approve Harvard' s proposal in 1976 to build a biocontainment

facility for its molecular biologists, instead enacting a three-month moratorium on recombinant DNA experi-

ments. See James D. Watson and John Tooze, The DNA Story: A Documentary History of Gene Cloning,

San Francisco: W.H. Freeman, 1981, pp. 91– 135; Sheldon Krimsky, Genetic Alchemy: The Social History of the

Recombinant DNA Controversy, Cambridge, MA: MIT Press, 1982, pp. 294–311.

10 Susan Wright, Molecular Politics: Developing British and American Regulatory Policy for Genetic Engineering,

Chicago: The University of Chicago Press, 1994, pp. 160– 218; J. Benjamin Hurlbut, ' Remembering the future: sci-

ence, law, and the legacy of Asilomar' , in Sheila Jasanoff and Sang-Hyun Kim (eds.), Dreamscapes of Modernity:

Sociotechnical Imaginaries and the Fabrication of Power, Chicago: The University of Chicago Press, 2015, pp. 126–

51; Melanie Armstrong, Germ Wars: The Politics of Microbes and America' s Landscape of Fear, Oakland, CA:

University of California Press, 2017.

11 Tilghman et al., op. cit. (7); D.C. Tiemeier, S.M. Tilghman and P. Leder, ' Purification and cloning of a mouse

ribosomal gene fragment in coliphage lambda' , Gene (1977) 2, pp. 173– 91. The group first cloned part of a ribo-

somal RNA gene, to test their vector, before cloning the mouse globin gene. The ribosomal RNA gene had the

advantage of existing in more than one copy per genome.

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which appeared on Petri dishes of E. coli as plaques. Each plaque (a specially constructed

lambda vector containing a random bit of mouse DNA) had to be transferred onto a nitro-

cellulose filter which would then be hybridized to a radioactively labelled globin cDNA

probe.

They successfully identified the complete beta globin gene on a

seven-thousand-base pair fragment of mouse DNA, but Tilghman remarked of the gruel-

ling labour required, ' it was ugly'.

Conditions for would-be genetic engineers improved in 1978, when NIH began relaxing

the safety guidelines for recombinant DNA work on higher organisms. This meant that

most work with recombinant DNA from higher organisms could be conducted in ordinary

labs. Many more biologists wanted to learn these techniques, not only in academia but

also in the burgeoning biotechnology industry.

One book became a canonical guide to

this field, Molecular Cloning: A Laboratory Manual by Tom Maniatis, Ed Fritsch and Joe

Sambrook, first published in 1982 (Figure 2 ). Including subsequent editions, this manual

sold over 200,000 copies, making it a bestseller in the cottage industry of methods pub-

lications.

Drawing on both documentary sources and oral histories, this paper examines

how protocols, recipes and pragmatic tips for gene cloning were shared, highlighting the

role of published manuals as sources of practical knowledge.

Learning how

By the late 1970s, scientists and journalists alike spoke of the ' recombinant revolution'.

Most of the excitement revolved around the ability to clone and characterize individual

genes from animals and plants, not least humans. The company Genentech, founded in

1976, led the race to clone genes and produce therapeutic proteins such as human insu-

lin.

But the bounty was not restricted to the biotech industry. The techniques of genetic

engineering had the potential to transform nearly every area of biomedical research. As

one neuroscientist expressed it,

Imagine setting out to purify and characterize all of the proteins in a rat brain: each

protein would require a specifically designed purification scheme, perhaps kilogram

12 Lynn Enquist, also working at NIH in Robert Weisberg' s laboratory, constructed the lambda vector

(λgtWES - λ B) used in this cloning experiment. D. Tiemeier, L.W. Enquist and P. Leder, ' An improved derivative

of a bacteriophage λ EK-2 vector useful in the cloning of DNA molecules' , Nature (1976) 263, pp. 526– 7; Lynn

Enquist, interview, 25 June 2018.

13 Shirley M. Tilghman, interview, 25 May 2018. Their group was using two methods after digesting the mouse

genome into fragments to enrich the genetic material of interest about two-thousand-fold, making their brute-

force screening of candidates more feasible. Otherwise they could have had to screen more than a million pos-

sible clones.

14 Nicholas Wade, ' Cloning gold rush turns basic biology into big business' , Science (1980) 208, pp. 688–92.

15 There is some literal truth to this expression: the same year Molecular Cloning appeared, Cold Spring Harbor

Laboratory Press moved to Urey Cottage at Cold Spring Harbor Laboratory. Nancy Ford, 'Publications ' , in Cold

Spring Harbor Laboratory, Annual Report 1983, Cold Spring Harbor Laboratory Archives, p. 12. On the number

of copies sold see Jan A. Witkowski, The Road to Discovery: A Short History of Cold Spring Harbor Laboratory , Cold

Spring Harbor: Cold Spring Harbor Laboratory Press, 2016, p. 267.

16 Oral histories were conducted with two authors of Molecular Cloning : A Laboratory Manual , participants in the

Cold Spring Harbor Laboratory course from which the manual emerged, and other molecular biologists who used

this manual in the 1980s. When possible, factual information was checked against documentary sources.

Unpublished interview quotes are included with permission.

17 Susan Wright, ' Recombinant DNA technology and its social transformation, 1972–1982 ' , Osiris (1986) 2,

pp. 302–60.

18 Stephen S. Hall, Invisible Frontiers: The Race to Synthesize a Human Gene , New York: Atlantic Monthly Press,

1987; Nicolas Rasmussen, Gene Jockeys: Life Science and the Rise of Biotech Enterprise , Baltimore: Johns Hopkins

University Press, 2014.

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quantities of starting material, and the determination of each primary amino-acid

sequence would be complex and time-consuming. To do this for each of the many

thousands of proteins in a mammalian brain would be a daunting task indeed! Yet

a few micrograms of rat brain mRNA contain molecules coding for every protein

in the brain: by means of recombinant DNA techniques we can use the information

encoded in each mRNA to investigate not only the structure of the corresponding

protein but also its genetic regulation … This simple cloning procedure is the essence

of recombinant DNA technology.

For biologists new to genetic engineering, assembling the needed materials required

effort and expense. An array of specialized reagents, enzymes and plasmids began to be

sold by companies such as New England Biolabs, which issued their first catalog in

1975. As mentioned earlier, finding a specific gene in chromosomal DNA was a major hur-

dle. The method used by Tilghman and colleagues to clone the first mouse gene effect-

ively used a purification approach, fractionating the chromosomal DNA and then

searching for the desired gene. Another approach was to construct a stable collection

of DNA fragments from a particular organism, which could be screened for any individual

gene. Such a collection was dubbed a genomic ' library' . Tom Maniatis at Caltech, in col-

laboration with Arg Efstratiadis and others at Harvard, pioneered the methods for

Figure 2. Photograph of Tom Maniatis, Ed

Fritsch and Joe Sambrook, Molecular

Cloning: A Laboratory Manual, Cold Spring

Harbor: Cold Spring Harbor Laboratory

Press, 1982.

19 Robert J. Milner, ' Recombinant DNA strategies and techniques' , Trends in Neurosciences (1982) 5, pp. 297–300,

297.

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creating such a library and cloning genes from it.

He made his organism-specific librar-

ies available to other researchers, even though each library was a biologically limited

resource.

When Ed Fritsch, at that time a postdoc in Maniatis' s lab, succeeded in devel-

oping a library of human DNA, it was reported in the Boston Globe , and researchers from

many institutions began requesting it.

Even if one had all the materials at hand, however, reliable protocols and bench know-

how could be just as challenging to procure. Even the cognoscenti struggled to keep up

with new knowledge. As one of the cloners of the first mouse gene put it, ' There was a

lot of sharing "tricks " back then – it was pre-email of course. We all had folders or file

boxes or notes stuck in notebooks of the various techniques that came as letters,

phone messages, or notes from meeting talks.'

One key resource became available in

1979: volume 68 of the serial Methods in Enzymology , edited by Raymond Wu, was on

Recombinant DNA.

Since 1955, Methods in Enzymology had provided standard protocols

for biomedical researchers. This volume brought together the innovators of many of

the key techniques for cloning genes. It provided a definitive set of methods, from

their originators, for trained biochemists.

For DNA novices, courses were developed to teach recombinant techniques. In the

autumn of 1979, Raymond L. Rodriguez, Robert C. Tait and other colleagues at the

Department of Genetics at University of California, Davis offered a ten-week course

entitled Advanced Molecular Genetics Laboratory. Rodriguez had been a pioneer in the

field, having worked in Herbert Boyer' s UCSF group that designed the most widely

used plasmid cloning vector of that era.

His Davis course enrolled both undergraduate

and graduate students, but there was, in addition, ' a heavy demand for copies of labora-

tory handouts and protocols' . This inspired the publication of those in a course manual,

Recombinant DNA Techniques: An Introduction, in 1983.

As it turned out, while the Davis

course may have been the first to offer training in recombinant DNA techniques, its

book was not the first entry onto the market.

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 microbes.

These courses resulted in several important manuals for bacterial genetics, making the

20 Tom Maniatis, Ross C. Hardison, Elizabeth Lacy, Joyce Lauer, Catherine O' Connell, Diana Quon, Gek Kee Sim

and Argiris Efstratiadis, ' The isolation of structural genes from libraries of eucaryotic DNA' , Cell (1978) 15,

pp. 687– 701. There were two kinds of library that simplified gene cloning: (1) a ' cDNA library 'of cloned copies

of every mRNA in a cell, and (2) a genomic DNA library, created from chromosomal DNA, not mRNA. Maniatis was

involved in developing both.

21 Ron Davis at Stanford also made libraries and shared them. Echols, op. cit. (6), p. 346, lauds the non-

proprietary shipping of these ' libraries … in a test tube' to labs worldwide.

22 Robert Cooke, ' Biologists start full "library " of human genes ',Boston Globe, 22 December 1978, p. 7; Richard

M. Lawn, Edward F. Fritsch, Richard C. Parker, Geoffrey Blake and Tom Maniatis, ' The isolation and characteriza-

tion of linked δ - and β -globin genes from a cloned library of human DNA' , Cell (1978) 15, pp. 1157– 74. On the

sharing of the library, Edward Fritsch, interview, 6 September 2018.

23 Lynn Enquist, personal communication, 23 December 2019.

24 Raymond Wu (ed.), Recombinant DNA , Methods in Enzymology (1979) 68.

25 The plasmid was called pBR322 after Bolivar and Rodriguez. Francisco Bolivar, Raymond L. Rodriguez,

Patricia J. Greene, Mary C. Betlach, Herbert L. Heyneker, Herbert W. Boyer, Jorge H. Crosa and Stanley Falkow,

'Construction and characterization of new cloning vehicle. II. A multipurpose cloning system' , Gene (1977) 2,

pp. 95–113.

26 Raymond L. Rodriguez and Robert C. Tait, Recombinant DNA Techniques: An Introduction , Reading:

Addison-Wesley Publishing Company, 1983, p. xvii.

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instruction available to many more scientists than could come to Long Island.

Since

1969, molecular genetic techniques for higher organisms were taught in an Animal

(Tumor) Virus Course.

Nancy Hopkins co-taught the version of this course in 1979,

but the enrolment was relatively low, in part because tumour virus know-how was avail-

able in ' most academic centers'.

She argued that what was most needed at CSHL was a

course on molecular cloning of eukaryotic genes, and that it had to involve Maniatis,

whose lab was leading the development of recombinant DNA methods.

Maniatis, agree-

ing to teach it, recruited his postdoc Ed Fritsch to join as an instructor; Hopkins remained

on board as the third member of the team that first year. Fritsch would put together all of

the reagents and supplies for the course and adapted protocols from the Maniatis lab and

the literature – no small task.

Helen Donis-Keller and Catherine O' Connell served as

course assistants.

When CSHL advertised the postgraduate training course, Molecular Cloning of

Eukaryotic Genes, 172 people applied for the sixteen spots. Thus even before it began,

it immediately became the most popular course ever offered at CSHL.

Among the sixteen

students selected for 1980, Robert Waterston would go on to a renowned career mapping

the genome of a model worm (C. elegans ) and, ultimately, playing a leadership role in the

Human Genome Project.

Happily for the historian, he also kept his immaculately orga-

nized course notebook, as did Steve Goodbourn, now a renowned British virologist who

took the course the second year.

Like other CSHL summer courses, the three-week

schedule consisted of laboratory sessions and lectures by leading scientists in the

field – in this case, twenty-one lectures (about half at 9 a.m., the others at 8 p.m.).

Days were reserved for benchwork. Situated on a beautiful stretch on the coast of Long

Island, Cold Spring Harbor Laboratory had a summer camp feel.

Both Hopkins and Donis-Keller recall that the night before the first class, just after they

had finished setting up the teaching laboratory, there was a fire that filled the entire room

27 J.H. Miller, Experiments in Molecular Genetics , Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1972;

Ronald W. Davis, David Botstein and John R. Roth, Advanced Bacterial Genetics: A Manual for Genetic Engineering , Cold

Spring Harbor: Cold Spring Harbor Laboratory Press, 1980; Thomas J. Silhavy, Michael L. Berman and Lynn

W. Enquist, Experiments with Gene Fusions , Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1984.

28 From 1969 to 1976, CSHL had two consecutive postgraduate courses, one on Animal Viruses and the other

on Tumor Viruses (or, in later years, Immunogenetics and Tumor Immunology). In 1977 Animal Viruses was

replaced by Oncogenic Viruses; in 1979 this became RNA Tumor Viruses. Cold Spring Harbor Laboratory,

Annual Report 1968– Annual Report 1979, Cold Spring Harbor Laboratory Archives.

29 James D. Watson, 'Director ' s report' , in Cold Spring Harbor Laboratory, Annual Report 1980 , Cold Spring

Harbor Laboratory Archives, pp. 6– 18, 13.

30 Nancy Hopkins, interview, 7 September 2018. As Hopkins remembers it, she made this case about a cloning

course to Joe Sambrook, who had her approach Maniatis about teaching it, which he agreed to do. Maniatis recalls

being asked by Watson. Tom Maniatis, ' Tom Maniatis on writing and science: molecular cloning' , Oral History

Collection, Cold Spring Harbor Digital Archives, 22 March 2003, http://library.cshl.edu/oralhistory/interview/

james-d-watson/writer/writing-and-science-molecular-cloning, accessed 2 January 2020.

31 Fritsch interview, op. cit. (22).

32 Tom Maniatis, interview, 25 October 2016.

33 Watson, op. cit. (29), p. 13.

34 Kathryn Maxson Jones, Rachel A. Ankeny and Robert Cook-Deegan, ' The Bermuda triangle: the pragmatics,

policies, and principles for data sharing in the history of the Human Genome Project' , Journal of the History of

Biology (2018) 51, pp. 693–805.

35 Bob Waterston and Steve Goodbourn kindly loaned me their notebooks; both are now deposited in the Cold

Spring Harbor Laboratory Archives. Whereas Waterston' s notebook consists mainly of his notes and handouts

from the lectures, Goodbourn' s includes photocopied protocols and some experimental notes and results. For

using lab notebooks as historical sources, Frederic L. Holmes, Jürgen Renn and Hans-Jörg Rheinberger (eds.),

Reworking the Bench: Research Notebooks in the History of Science, Dordrecht: Kluwer Academic Publishers, 2003.

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with smoke, ignited by frayed wires in a piece of equipment.

They stayed late into the

night cleaning up the mess. As Donis-Keller recalls of the rest of the course, while the

lectures were great, it was a lot of trouble to get the experiments working, even with

the expertise of the instructors.

The students were supposed to learn how to clone

like the pros, construct a library in lambda phage, screen plaques with radioactively

labelled nucleic acid probes and make a cDNA clone from messenger RNA, among other

techniques. Likely reflecting the precedent of the tumour virology course, there were a

number of lectures on that field' s exemplars, such as SV40, adenovirus, RNA retroviruses

and oncogenes. In contrast to these lectures on animal viruses, the laboratory exercises

focused on cloning and manipulating eukaryotic DNA.

A student in the 1981 summer course (who had reapplied after not being selected in

1980), Gert-Jan van Ommen, remarked that socializing was a key part of what made the

course so successful – amidst the long hours in the labs were breaks to swim in the

Banbury pool, play volleyball games and attend barbeques (see Figure 3).

But it was

the information conveyed in the course, going far beyond what was available in the pub-

lished literature, that made the experience so valuable. Van Ommen recalls that ' we were

taught lots of tricks like cDNA synthesis, lambda cloning, making packaging mix, and cos-

mid cloning'.

Upon returning to the Netherlands, Van Ommen wrote up an extensive

report ' on all the techniques and tricks I learned there' , and sent it around the molecular-

biology community in the Netherlands. ' People were really over the moon with the

report.'

From course to book

Watson saw the opportunity to make this cloning know-how available to a wider base of

users through publication.

Issuing an instructional guide on gene cloning 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 from

the Advanced Bacterial Genetics summer school of publishing course manuals as

books.

Watson wanted Maniatis anchoring the team of authors, not only as an instructor

but also on account of his renown in the world of cloning. But Maniatis had recently

moved to Caltech, where he was busy with chairing an NIH study section, teaching and

running his own lab. He only agreed to prepare a manual based on the course if he

had significant help.

Watson persuaded Joe Sambrook, a talented and combative

British tumour virologist at the lab, to join the effort. Although Sambrook never served

as a formal instructor for the summer course, he was a leading research scientist at

36 Hopkins interview, op. cit. (30); Helen Donis-Keller, interview, 8 September 2018.

37 Donis-Keller interview, op. cit. (36).

38 Robert Waterston, CSH Molecular Cloning notebook, 1980; Steve Goodbourn, CSH course notes, 1981.

39 Gert-Jan van Ommen, interview, 12 July 2018.

40 Van Ommen interview, op. cit. (39).

41 Van Ommen interview, op. cit. (39). Van Ommen was more advanced than many students taking the course;

he even brought along to CSHL the DNA, from a goat with an inborn genetic thyroid disease, from which he

hoped to clone the thyroglobulin gene – an effort at which he succeeded. Goodbourn was also more advanced,

and the two of them were labmates at the course.

42 ' Tom Maniatis: gene expression, cloning and beyond' , 16 November 2004, www.mcb.harvard.edu/archive/

tom-maniatis-gene-expression-cloning-and-beyond, accessed 2 January 2020.

43 The course-based generation of manuals at CSHL has continued, notably with the ' Mouse Book' in 1986.

Brigid Hogan, Franklin Costantini and Elizabeth Lacy, Manipulating the Mouse Embryo: A Laboratory Manual, Cold

Spring Harbor: Cold Spring Harbor Laboratory Press, 1986; Dmitriy Myelnikov, ' Transforming mice: technique

and communication in the making of transgenic animals' , PhD dissertation, University of Cambridge, 2015, p. 176.

44 Maniatis interview, op. cit. (32); Maniatis, op. cit. (30).

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CSHL and a superb writer.

Fritsch, who was beginning a tenure-track faculty position at

Michigan State University, remained centrally involved in the project.

The writing was

already under way before Maniatis and Fritsch taught the course a second time with Doug

Engels, in the summer of 1981.

None of the authors imagined the impact the ' Maniatis manual' , as it came to be

known, would have when it came out in 1982. For the second edition, Sambrook took a

lead role and became first author, but the book' s nickname stuck, irritating him.

Nancy Hopkins feels she should have been invited to participate as an author, having

taught the 1980 course, though Fritsch and Maniatis are puzzled as to why she did not

raise this at the time.

As the Preface makes clear, there were in fact a number of con-

tributors who were not listed as authors. Maniatis, Fritsch and Sambrook thanked

Figure 3. Instructors and students working in the lab during the Molecular Cloning of Eukaryotic Genes course at

Cold Spring Harbor Laboratory, summer 1981. The lack of lab coats reflects the casual atmosphere. There were

more men than women students in the 1981 course, but almost equal numbers in 1980. Seated is Tom Maniatis;

the man in the striped red shirt is Ed Fritsch. Photo courtesy of Gert-Jan van Ommen.

45 James D. Watson, ' James D. Watson on Joe M. Sambrook' , Oral History Collection, Cold Spring Harbor Digital

Archives, http://library.cshl.edu/oralhistory/interview/scientific-experience/molecular-biologists/j-sambrook,

accessed 2 January 2020. Maniatis met Sambrook when doing his cloning work at CSHL in the 1970s. On

Sambrook' s research see Gregory J. Morgan, Cancer Virus Hunters: How Tumor Virology Influenced a Century of

Biology and Medicine, under review, Johns Hopkins University Press.

46 Maniatis interview, op. cit. (32). After teaching twice with Maniatis, Fritsch taught the course for one more

year, in 1982.

47 T. Maniatis, E. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor: Cold

Spring Harbor Laboratory Press, 1982, p. iii.

48 Sambrook never expressed this frustration directly to Maniatis and Fritsch. Personal communication, 13

January 2020.

49 Nancy Hopkins, personal communication, 1 July 2018; Hopkins interview, op. cit. (30). As Hopkins observes,

she might not have agreed to become an author even if asked – writing the manual was an enormous task, and

cloning technology was not her area of expertise.

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individual scientists (e.g. Brian Seed, Doug Melton) for providing particular protocols or for

supplying the anthology of methods for a chapter (Nina Irwin).

In addition, Maniatis,

Fritsch and Sambrook provided full references for methods adapted from the published lit-

erature.

Attribution was harder to ascertain for other protocols. As they stated,

We have tried to give credit at appropriate places in the text to the people who ori-

ginally developed the procedures presented here, but in many cases tracing a par-

ticular method to its undisputed roots has proved to be impossible. We therefore

wish to apologize –and to express gratitude – to those we have been unable to

acknowledge for an idea, procedure, or recipe. Our major function has been to com-

pile, to verify, and, we hope to clarify; less frequently we have introduced modifica-

tions, and only in rare instances have we devised new protocols.

In short, manuals raise the same problems of credit as other compilations, such as atlases

and databases.

Though the authors (especially Maniatis and Fritsch) had personally

developed key methods in the book, their role as authors of the manual made them

stand-ins for many other scientists who had pioneered techniques. Not surprisingly,

researchers began citing the manual rather than the original literature.

Sometimes,

methods acquire an eponymous name, such as Maxam– Gilbert sequencing or Southern

blotting. But for most methods, the successful circulation (and adaptation) of a lab recipe

or method subverted conventional notions of authorship and credit.

That said, Molecular Cloning achieved a distinctive authorial voice, one aimed at the nov-

ice, but not the amateur. The three authors explain in the Preface that because ' the man-

ual was originally written to serve as a guide to those who had little experience in

molecular cloning, it contains much basic material'.

Through chapter introductions

that functioned in some respects like a textbook, the manual communicated enough

about the science behind the recipes that users could troubleshoot the problems they

encountered.

For instance, the first chapter on plasmids provided general information

about these cloning vectors and genetic maps of the most commonly used ones, and out-

lined the three most popular methods of inserting a gene. As the authors note, ' In prin-

ciple, cloning in plasmid vectors is very straightforward'.

They then go on to enumerate

the usual complications, and include a special section covering ' Problems in cloning large

DNA fragments in plasmids'.

This leads to sections on vectors used to handle larger

50 Maniatis, Fritsch and Sambrook, op. cit. (47), p. iv.

51 Of course, neither the scientists thanked in the Preface nor those who had published the original methods

received royalties from the manual, even when attribution was maintained.

52 Maniatis, Fritsch and Sambrook, op. cit. (47), p. iii.

53 Bruno J. Strasser, ' Collecting, comparing, and computing sequences: the making of Margaret O. Dayhoff's

Atlas of Protein Sequence and Structure, 1954–65 ' , Journal of the History of Biology (2010) 43, pp. 623– 60, 644; Bruno

J. Strasser, ' The experimenter' s museum: GenBank, natural history, and the moral economies of biomedicine,'

Isis (2011) 102, pp. 60–96.

54 For one early example: Lance S. Davidow, Diane Apostolakos, Michele M. O' Donnell, Alan R. Proctor, David

M. Ogrydziak, Rod A. Wing, Irene Stasko and John R. DeZeeuw, ' Integrative transformation of the yeast Yarrowia

lipolytica', Current Genetics (1985) 10, pp. 39 –48.

55 For more on protocols as a genre of collective or quasi-anonymous scientific text see Hans-Jörg

Rheinberger, '" Discourses of circumstance" : a note on the author in science' , in Mario Biagioli and Peter

Galison (eds.), Scientific Authorship: Credit and Intellectual Property in Science , New York: Routledge, 2003,

pp. 309– 23, 318.

56 Maniatis, Fritsch and Sambrook, op. cit. (47), p. iii.

57 Michael S. Levine, interview, 13 September 2016; Jan Witkowski, personal communication, 31 March 2018.

58 Maniatis, Fritsch and Sambrook, op. cit. (47), p. 11.

59 Maniatis, Fritsch and Sambrook, op. cit. (47), p. 14.

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pieces of DNA. A table lays out which of the four vectors was best for various experimen-

tal goals, such as cloning large DNA fragments, sequencing DNA or expressing foreign

genes in E. coli.

This chapter repeats, and expands upon, material presented in the

1980 CSHL summer course.

Each of the other eleven chapters similarly instructs the

reader on the relevant biology as well as giving practical advice in selecting methods.

The text is also clear on omissions, such as ' enzymes that find occasional use in molecular

cloning but that are not necessary to carry out any of the procedures described in this

manual'.

By contrast, chapters in the Methods in Enzymology volume 68 on Recombinant

DNA, which also provided protocols, tended to be more like review articles in their com-

prehensive coverage. Readers might need to consult chapters by several different authors

to understand the alternative strategies available for reaching their experimental goal.

The chapters of Molecular Cloning are organized around specific materials or procedures

(e.g. Chapter 4, ' Enzymes used in molecular cloning' ; Chapter 7, ' Synthesis and cloning of

cDNA' ). The overall sequence of information reflects the key choices involved in identify-

ing and manipulating DNA, in the general order of steps required for cloning a gene. The

second chapter, ' Propagation and maintenance of bacterial strains and viruses' , provides

basic instruction on isolating single colonies of bacteria and verifying strains through gen-

etic markers, as might be taught in a microbiology course, as well as techniques more spe-

cific to molecular cloning, such as large-scale preparation of the vector bacteriophage

lambda. This combination of instruction on basic lab methods with more specialized tech-

niques distinguished Molecular Cloning from both textbooks and methods papers in the sci-

entific literature.

The book includes numerous drawings showing how to perform various procedures,

such as recovering purified bacteriophage from a caesium chloride gradient, or how to

pour an electrophoresis gel, in both cases showing where and how to position the

hands (see Figure 4).

Integrated into the text are hundreds of tables and diagrams,

including detailed genetic maps, as well as sample photographs of results. A well-

furnished molecular biology lab of the time relied on a local machine shop to fabricate

gel electrophoresis tanks; the manual included all the needed information in engineering

drafting, with complete measurements.

Not all materials had to be custom-made, of

course, and the manual mentioned many lab supplies by brand, such as Kodak X-Omat

AR film, Whatman-52 filter paper, Sigma Type-III sodium salt DNA, and Dowex XG-8

mixed-bed resin. In addition, the authors included specific tips on how to keep costs

down on restriction enzymes, the most expensive commercial reagents for gene cloning.

As a physical object, the 545-page manual brought together text, photographs, tables

and diagrams, as well as countless technical symbols and some equations. The editing and

layout of such a complicated publication must have been laborious, but the final product is

clear and easy to read. Notably, the design is much more sophisticated and pleasing than

that of the Advanced Bacterial Genetics manual that Cold Spring Harbor brought out just

two years prior, which looked more like a print-out than a book, lacking any integration of

images into the text.

Molecular Cloning did borrow one feature of that earlier manual,

60 Maniatis, Fritsch and Sambrook, op. cit. (47), p. 54.

61 Waterston, op. cit. (38), notes on ' Overview of plasmids,' 30 June 1980.

62 Maniatis, Fritsch and Sambrook, op. cit. (47), p. 107.

63 J.C. Wotton, ' Review of Methods in Enzymology , vol. 65, Nucleic Acids and Molecular Biology , and vol. 68,

Recombinant DNA', Heredity (1981) 46, pp. 142–4.

64 Maniatis, Fritsch and Sambrook, op. cit. (47), pp. 105, 154.

65 Maniatis, Fritsch and Sambrook, op. cit. (47), pp. 154–5.

66 Maniatis, Fritsch and Sambrook, op. cit. (47), p. 108.

67 Davis, Botstein and Roth, op. cit. (27). This is in contrast to the CSHL' s Advanced Bacterial Genetics manual

by Miller, op. cit. (27), which is professionally produced, including colour images.

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though: a plastic ring-comb binding (see Figures 2 and 4).

As David Crotty observed,

some users complained that this binding served to ' deliberately get the book to fall

apart forcing you to buy a new copy'.

The advantage, however, was that the manual

could be laid flat on a laboratory bench, a feature that the majority of users praised.

The much-expanded second edition of Molecular Cloning appeared as three thick volumes

in 1989; the plastic ring-comb binding remained.

Sales and rivals

Just as Watson expected, Molecular Cloning met a strong demand. There were orders for

more than five thousand copies before the publication date.

Consequently, the press

Figure 4. Instructions and diagram on how to pour an agarose gel for electrophoresis. In this set-up apparatus,

agarose is used to separate a mixed population of nucleic acid fragments by length (in base pairs). Ethidium bromide

is added to the gel to make the DNA visible under ultraviolet light. These descriptions include tips on the ' combs'

that create the wells for DNA samples. Molecular Cloning , op. cit., pp. 158–9.

68 Stephanie Radner, Yong Li, Mary Manglapus and William J. Brunken, ' Joy of cloning: updated recipes',

Trends in Neuroscience (2002) 25, pp. 594–5.

69 David Crotty, ' Molecular Cloning (AKA Maniatis, AKA The Bible) at 25' , 22 October 2007, Bench Marks , https://

cshbenchmarks.wordpress.com/2007/10/22/molecular-cloning-aka-maniatis-aka-the-bible-at-25, accessed 2

January 2019.

70 J. Sambrook, E.F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual , 2nd edn, Cold Spring

Harbor: Cold Spring Harbor Laboratory Press, 1989. As Karen-Beth Scholthof has noted, you could also easily

lay the manual flat on a photocopy machine.

71 James D. Watson, ' Report' , in Cold Spring Harbor Laboratory, Annual Report 1982 , Cold Spring Harbor

Laboratory Archives, pp. 5– 16, 12.

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sold 5,113 copies the first month of its appearance, July 1982.

Sales remained robust that

autumn, and in 1983 Cold Spring Harbor Laboratory Press sold more copies of Molecular

Cloning than all of its other titles together (37,337 versus 24,234). The book went through

four printings during its first six months, and three more were issued the following year.

In explaining the surprisingly high sales number, the Annual Report 1982 points to the man-

ual' s' rapid adoption by many, many university courses '.

To be clear, students were not the only ones driving sales; working biologists were also

buying the book in droves.

As a reviewer for the British Society for Developmental

Biology put it, ' no laboratory with any serious interest in molecular biology of develop-

ment and their [sic ] cloning should be without it'.

The testimony of historian of science

Nick Hopwood confirms this view:

When I worked in developmental biology between 1986 and 1991 only two books had

permanent places on my bench. The first, ' Maniatis' , was a manual of the molecular

cloning methods that our Cambridge laboratory was using to identify genes that spe-

cify muscle development in the South African clawed frog … I know the ring-bound

recipes of Tom Maniatis et al. inside out.

The reliability of the protocols was a major reason for the book' s popularity. As one

admirer put it, ' Just like the cookbooks of Betty Crocker and Fannie Farmer, the molecular

cloning manual was chock full of recipes that worked'.

Another reviewer proclaimed, 'In

our laboratory, mirabile dictu, the procedures in this manual nearly always work'.

The trustworthiness of Molecular Cloning , however, did not solve the problem of keeping

abreast of new and improved methods. Some researchers complained that the manual was

out of date as soon as it was published.

Plans for a second edition, scheduled for 1984,

were under way, but it was deferred for five more years as the first edition went through

sixteen printings.

The rapid pace of change in molecular biology was blamed for the dif-

ficulty in updating the book. To take the most prominent example, the development of

polymerase-chain reaction (PCR) by Kary Mullis and others at Cetus Corporation in

1985 made it possible to amplify a piece of DNA in a test tube, transforming cloning meth-

ods.

Thereafter, Crotty contended, a new Molecular Cloning manual without it would be

'pointless'.

72 Memorandum from Susan Gensel to Jim Watson, 10 December 1982, re: sales at the American Society for

Cell Biology meeting, James D. Watson Collection, Cold Spring Harbor Laboratory Archives, Record Group III,

Series 4 (hereafter Watson Papers).

73 Watson, op. cit. (71), p. 12.

74 One can see this from sales at professional meetings: memorandum from Gensel to Watson, op. cit. (72). At

that meeting Molecular Cloning sold eighty-three copies, and all the other CSHL Press sales together, twenty-two

titles in all, made up 102 copies.

75 British Society for Developmental Biology Newsletter VII, October 1982, review of Molecular Cloning: A

Laboratory Manual, copy in Watson Papers.

76 Nick Hopwood, ' Visual standards and disciplinary change: normal plates, tables and stages in embryology',

History of Science (2005) 43, pp. 239– 303, 239.

77 Steven L. McKnight, ' Pure genes, pure genius' , Cell (2012) 150, pp. 1100– 2, 1102. For more on Molecular

Cloning and ' cookbook technologies' see Joan Fujimura, Crafting Science: A Sociohistory of the Quest for the Genetics

of Cancer, Cambridge, MA: Harvard University Press, 1996, pp. 84–6.

78 George McCorkle, ' Review of Molecular Cloning: A Laboratory Manual' , American Scientist (1983) 71, p. 418; also

quoted in Crotty, op. cit. (69).

79 Crotty, op. cit. (69).

80 Mala Mazzullo, Cold Spring Harbor Laboratory Press, personal communication, 3 April 2019.

81 Paul Rabinow, Making PCR: A Story of Biotechnology , Chicago: The University of Chicago Press, 1996.

82 Crotty, op. cit. (69).

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When the second edition of Molecular Cloning appeared in 1989, with the authors now

listed as Sambrook, Fritsch and Maniatis, it 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.

Both the first and second editions were reviewed in other languages, and translations

appeared. A Russian translation of the first edition appeared in 1984; Chinese translations

of the first edition came out in 1987 and of the second in 1992. Maniatis was told by trav-

elling scientists in the early 1990s that ' every lab they go to in China has the cloning man-

ual on their desk'.

Reflecting its canonical status, Molecular Cloning was generally referred to by its users as

the 'bible ' .

Extending this common metaphor, one biochemist made reference to 'those

who daily worship the Cold Spring Harbor idol'.

But the deity had rivals. Its main com-

petitor in the first decade was Current Protocols in Molecular Biology , introduced in 1987 by a

group of researchers based at Massachusetts General Hospital.

Sarah Greene was the ori-

ginal 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 (see Figure 5 ). This meant that the table of contents

also needed frequent updating. Five thick binders were published in the original series.

The first volume covered most of the topics and methods in Molecular Cloning.

Maintaining the three-ring binders of Current Protocols as supplements arrived was

inconvenient for users. Karen-Beth Scholthof, who was a postdoc in plant virology in

Berkeley at the time, was responsible for updating the Current Protocols binders in the

lab she worked in:

Your writing about Current Protocols reminded me of what a terrible system it was. I

was assigned to add the quarterly updates (and remove the ' old' pages). This was

such a hassle – having to take the entire binder apart to insert the new pages.

83 Sambrook, Fritsch and Maniatis, op. cit. (70). This edition was in three volumes.

84 Stuart Orkin, ' By the book' , Nature (1990) 343, pp. 604– 5, 604.

85 Mike Fortun interview with Tom Maniatis, February 1992, as quoted in Fujimura, Crafting Science , p. 84.

86 David Crotty found the first such reference in a New York Newsday piece from 1984, which I have not been

able to recover. Crotty, op. cit. (69). One prominent reviewer commented, ' This book is omnipresent in molecular

biology laboratories and is utilized to the point where it is frequently referred to as " The Bible" .' Kevin Struhl,

'Cloning cookbook for the laboratory',Nature (1985) 316, p. 222.

87 S.J.W. Busby, ' Comprehensive cloning' , Trends in Genetics (1988) 4, p. 352.

88 The Harvard-affiliated editors were Frederick Ausubel, Robert Kingston, Jonathan Seidman and Kevin

Struhl.

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The spiral-bound Maniatis was so much more user-friendly and the group annota-

tions made it a kind of living document for the lab.

Given the unwieldiness of the Current Protocols loose-leaf format, 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. That same

year the second edition of Molecular Cloning appeared, and more than seven thousand sci-

entific articles cited the title that year alone. From 1991 to 2000, editions of Molecular

Cloning were being cited over ten thousand times per year; by comparison, Current

Protocols in Molecular Biology peaked at 2,520 citations in 1998.

There were many other manuals besides the rivals Molecular Cloning and Current

Protocols – some geared towards students, with discrete lab exercises, and some to more

advanced practitioners aiming to incorporate new techniques (e.g. PCR Cloning

Protocols).

Others sought a place on the bench as essential reference tools. Bernard

Perbal' s A Practical Guide to Molecular Cloning combined up-to-date protocols with useful

information on commercially available enzymes and cloning vectors.

In effect, this

Figure 5. Volume 1 of Current Protocols in

Molecular Biology opened to reveal how the

protocols are organized by dividers in each

three-ring binder. The topics in this volume

are similar to those covered in Molecular

Cloning. Frederick M. Ausubel, Roger

Brent, Robert E. Kingston, David

D. Moore, J.G. Seidman, John A. Smith and

Kevin Struhl (eds.), Current Protocols in

Molecular Biology , 5 vols., New York: John

Wiley & Sons, 1987.

89 Karen-Beth G. Scholthof, personal communication, 5 May 2018.

90 Citation analysis done using the Cited Reference search (with the appropriate abbreviations) on Web of

Science Core Collection.

91 Bruce A. White (ed.), PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering , Totowa: Humana,

1997. This was published as vol. 67 of Methods in Molecular Biology.

92 Bernard V. Perbal, A Practical Guide to Molecular Cloning , New York: Wiley, 1984 (2nd edn 1988).

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guide compiled what was available, but scattered, in free handbooks from companies such

as Amersham, Boehringer and Pharmacia, as well as listing equipment needed for molecu-

lar biology and safety guidelines. Along similar lines, Terence A. Brown at the Department

of Biomolecular Sciences at Manchester issued a book called Molecular Biology Labfax ,a

'compendium of essential information –on genotypes, reagents, enzymes, reaction condi-

tions, cloning vectors, and suchlike – that is needed to plan and carry out molecular biol-

ogy research' . As the author explained,

Some of this information is already available in cloning manuals, catalogues and pos-

sibly on pieces of paper kept somewhere safe, but tracking down exactly what you

need to know takes time and can be a frustrating experience. With molecular biology

becoming an interesting sophisticated science, an acute need has arisen for a data-

book to complement the traditional cloning manuals.

The notion of a ' databook' stands in an odd relationship to database, and the updating of

such hard-copy publications attests to how slowly search engines came to replace other

information-hunting methods.

The true test of any such reference work, however useful, was to become indispensable.

As one reviewer remarked of Labfax,

To use books such as these to the best advantage, you need to spend a long time

becoming familiar with them and, frankly, life is too short to master every book. I

suspect that most mortals learn to feel their way around just one or two of these

massive works and then use them over and over again: my favourites are Molecular

Cloning: A Laboratory Manual and the 1989 Pharmacia Molecular Biology Catalogue

(which, incidentally, is much better organized than the current catalogue!). Of

course, Labfax contains a lot more facts than either of my two favourite works,

but are they facts that I am ever going to want to know?

This comment crystallized the task facing publishers of compendia on cloning: how to

determine exactly the current information most needed by biologists, in a format they

would be happy to pay for, more than once as editions became obsolete?

The challenge of updating was more easily accommodated by the growth of multimedia

technologies in the 1990s. By its third edition in 2001, Molecular Cloning: A Laboratory

Manual came with a CD-ROM ' Lab Book' . It also had an associated website,

MolecularCloning.com, with the book' s protocols and ' discussion space for those asking

questions or making alterations to techniques'.

However, the site did not have a 'mech-

anism for continuously adding updated new material' , which was considered a major

drawback given the technical feasibility of doing this online.

But who was to be the

maintainer of up-to-date knowledge? The authors? The readers? In reality, there was

not much community participation in the manual' s online forum. Efforts by other pub-

lishers to create ' yet another " myspace for biologists"' also met with disappointment.

93 T.A. Brown (ed.), Molecular Biology Labfax , Oxford: BIOS Scientific Publishers, 1991, p. v, emphasis added.

94 Steve Busby, ' Drowning by numbers: review of Molecular Biology Labfax , edited by T.A. Brown' , Trends in

Genetics (1992) 8, p. 77.

95 Crotty, op. cit. (69).

96 Crotty, op. cit. (69).

97 Crotty, op. cit. (69). In this respect, Molecular Cloning seems different from feminist health manuals for

which readers volunteered their own perspectives and knowledge. See Wendy Kline, '" Please include this in

your book" : readers respond to Our Bodies, Ourselves' , Bulletin of the History of Medicine (2005) 79, pp. 81–110.

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By contrast, ongoing maintenance of methods, recipes and protocols was vibrant at the

level of individual laboratories. In fact, the more manuals and handbooks expanded to

include more information, the greater was the need for abstracting the most essential

instructions. In this sense authors of manuals face the same dilemma as those of hand-

books, which tend to grow fatter with each edition. In response, CSHL Press issued a single

volume called The Condensed Protocols from Molecular Cloning: A Laboratory Manual.

Some

laboratories created their own solutions. As Karen-Beth Scholthof recounts about

Molecular Cloning, 'After it went to three volumes (Sambrook, although we continued to

refer to it as Maniatis), we started formally writing and sharing lab recipes for common

protocols. I also had an index card/recipe box with my favorite concoctions and cheat

sheets for various calculations.'

At Washington University, Helen Donis-Keller developed

a customized laboratory manual, specifically modelled on Molecular Cloning , with the

methods and protocols her group relied upon.

100

As she commented,

We found that even though protocols were published by other labs or in the litera-

ture, we needed to test them ourselves and prepare updated standardized methods

for use by everyone in the lab. The amounts of the various reagents we typically

used might be different from the published protocols as well or we might have

found that a different reagent than that specified might be more appropriate for

our purposes. I really wanted everyone to work from the same set of protocols to

maintain consistency and in order to make troubleshooting easier when things

went wrong.

101

Her in-house manual, while ensuring uniformity in methods in a large (forty-person) lab,

also made sure that know-how was not lost when people left. Each protocol was formatted

in the same way, with the name of the person who wrote and tested it, the date, instruc-

tions, time needed, any special reagents or equipment required, and, at the end, the

sources (which included, for example, Molecular Cloning).

102

As the annotations of ' NOT

DONE' in the table of contents suggest, a customized manual of this sort was usable as

a work-in-progress, updated and reimagined as needed.

The original challenge that had prompted Molecular Cloning , the need for instructions

on how to copy genes from unsequenced chromosomal DNA, was simplified by the

Human Genome Project, which was funded by the US Congress in 1988.

103

By the early

2000s, the full genomic sequences of several organisms were online and publicly available

to researchers; biologists could retrieve their genes of interest more easily, and the com-

parisons available through sequence data inspired a new generation of in silico rather than

in vitro experiments.

104

To be sure, Molecular Cloning expanded its reach for the age of

genomics, and remains a relevant resource. But the massive shift of reference works to

the Internet – including serials like Methods in Molecular Biology , started in 1983 –made

98 Joseph Sambrook and David Russell, The Condensed Protocols from Molecular Cloning: A Laboratory Manual , Cold

Spring Harbor: Cold Spring Harbor Laboratory Press, 2006.

99 Scholthof, op. cit. (89).

100 Donis-Keller interview, op. cit. (36); and Helen Donis-Keller, personal communication, 28 September 2018.

101 Helen Donis-Keller, personal communication, 28 December 2018.

102 Helen Donis-Keller, personal communication, 28 September 2018.

103 For a recent overview of the large literature on the Human Genome Project see Jones, Ankeny and

Cook-Deegan, op. cit. (34).

104 Hallam Stevens, Life out of Sequence: A Data-Driven History of Bioinformatics, Chicago: The University of

Chicago Press, 2013.

BJHS Themes 241

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the publication of manuals, and the release of new editions, less eventful.

105

In addition,

the publication of protocols has also migrated back to journal publication, now also

online. As Crotty notes, ' The lessons learned from MolecularCloning.com led to CSH

Protocols. We realized that if we were going to continuously add new material, we had

to think of the project as a journal, and give it a full editorial staff'.

106

This new journal

(and many other similar titles, such as Nature Methods ) effectively moved the development

and dissemination of molecular biology protocols back to the research literature, a full

circle after the gene cloning manual' s success.

Conclusions

Even among scientific methods, the history of gene cloning is unusual. This set of tech-

niques went in the short space of ten years from being considered dangerous and in

need of biocontainment to becoming adopted in laboratories throughout the world.

There are many reasons for this radical and rapid shift, not least the politics of deregu-

lation and the massive infusion of venture capital into biotechnology. But changing moti-

vations for the spread of knowledge cannot explain how the practices spread so quickly,

through a generation of technical workers, most of whom had completed their formal

education. Manuals and other sources of how-to knowledge provided guidance to count-

less life scientists and technicians who mastered these cutting-edge techniques.

Among the manuals available, Molecular Cloning succeeded remarkably in becoming an

indispensable element of the biology lab, often figured as the genetic ' kitchen' . As one

enthusiastic reviewer of the third edition, which was published in 2000, put it,

In every kitchen there is at least one indispensable cookbook. Sambrook and Russell's

Molecular Cloning: A Laboratory Manual fills the same niche in the laboratory. Like its

kitchen counterparts (e.g. Rombeck' s Joy of Cooking ) Sambrook' s Molecular Cloning has

information to help both the inexperienced and advanced user.

107

These comparisons are even more apt when one considers some of the supplies in

molecular biology labs. Since the 1950s, bacterial geneticists had been using blenders

to disrupt bacterial infection or mating. Methods developed in the 1970s for identifying

genes using radioactively labelled nucleic acid probes employed ' Seal-a-Meal' bags for

the hybridization step.

108

Carnation non-fat dry milk became the blocking agent of choice

for Western blots.

109

There are countless other such mundane connections, though of

course the traffic between lab chemistry and cooking is hardly new.

110

Yet seeing the manual as one' s standby cookbook for gene cloning obscures a key dif-

ference, namely the rapid pace of change in DNA methods that molecular biologists faced

in the 1980s. Of course, researchers had and have clever strategies for exploiting trust-

worthy protocols while also trying to master the latest methods. But there remains a

105 Although the first volume of Methods in Molecular Biology appeared in 1983, citations of its many volumes

(now over two thousand) took off in the 2000s, as assessed by searches on Web of Science Core Collection.

106 Crotty, op. cit. (69).

107 Radner et al., op. cit. (68).

108 Maniatis, Fristch and Sambrook, op. cit. (47), p. 587.

109 Karen-Beth G. Scholthof, personal communication, 21 May 2019.

110 Ho Ping-Yü and Joseph Needham, ' The laboratory equipment of early medieval Chinese alchemists' , Ambix

(1959) 7, 58– 112; Elizabeth Spiller, ' Recipes for knowledge: maker' s knowledge traditions, Paracelsian recipes, and

the invention of the cookbook' , in Joan Fitzpatrick (ed.), Renaissance Food from Rabelais to Shakespeare , London:

Ashgate, 2009, pp. 55– 72; Elaine Leong, Recipes and Everyday Knowledge: Medicine, Science, and the Household in

Early Modern England, Chicago: The University of Chicago Press, 2018.

242 Angela N. H. Creager

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central trade-off in laboratory how-to literature between reliability and state-of-the-art

technique. During the height of the recombinant revolution, Molecular Cloning seemed

able to provide both. But in biology, even the bible has to contend with obsolescence.

Acknowledgements. I thank Tom Maniatis, Ed Fritsch, Nancy Hopkins, Helen Donis-Keller, Gert-Jan van

Ommen, Bob Waterston, Steve Goodbourn, Shirley Tilghman, Mike Levine, Karen Scholthof, Lynn Enquist and

Arnold Levine for their generosity with time, information, materials and corrections; Kat Maxson Jones and

Gina Surita for assistance with research and editing; and Jan Witkowski, Alex Gann, Marta Hanson, Mathias

Grote and two referees for helpful suggestions. Gert-Jan von Ommen and Nancy Hopkins have allowed copies

of their wonderful personal photographs from the 1980 and 1981 summer courses to be deposited in Cold

Spring Harbor Laboratory Archives, along with the notebooks of Bob Waterston and Steve Goodbourn.

Cite this article: Creager ANH (2020). Recipes for recombining DNA: A history of Molecular Cloning: A Laboratory

Manual. BJHS Themes 5, 225 –243. https://doi.org/10.1017/bjt.2020.5

BJHS Themes 243

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... To determine the optimal amino acid dosage, 400 μL of 20 mg/mL geniposide solution, 50 μL of recombinant protein solution, and a gradient volume of 140 mg/mL glycine solution (1,3,5,7,9,10,11,12,13,15, or 17 μL) were added to a 1-mL reaction tube. ...

... In this study, bglA was codonoptimized for efficient expression in E. coli [11]. However, there remains a need to improve the expression of the thermophilic enzyme in the heterologous host by introducing homologous transcription factors to improve enzyme yield [12]. ...

Wenxi Li
Jielin Li
Ying Xu
Weizhao Chen

Gardenia blue is a natural blue pigment that is environmentally friendly, non-toxic, and stable. The hydrolysis of geniposide, catalyzed by β-glucosidase, is a critical step in the production process of gardenia blue. However, β-glucosidase is not resistant to high temperatures, limiting the production of gardenia blue. In this study, we investigated the effectiveness of a heat-resistant glucosidase obtained from Thermotoga maritima in the production of gardenia blue. The enzyme exhibited a maximum activity of 10.60 U/mL at 90 °C. Single-factor and orthogonal analyses showed that exogenously expressed heat-resistant glucosidase reacted with 470.3 μg/mL geniposide and 13.5 μg/mL glycine at 94.2 °C, producing a maximum yield of 26.2857 μg/mL of gardenia blue after 156.6 min. When applied to the dyeing of denim, gardenia blue produced by this method yielded excellent results; the best color-fastness was achieved when an iron ion mordant was used. This study revealed the feasibility and application potential of microbial production of gardenia blue.

Hend A Abdelfatah

This book addresses the basics for Medical Biotechnology. It is suitable for 12 -18-year-old readers in Middle and High school. This curriculum, approach, and way of presenting information have been chosen after reading many books in the field of Medical Biotechnology and discerned the pros and cons of them from the point of view of two students in High School. This book is instructed as follows: it introduces the definition of Medical Biotechnology, gives some ethical questions that Medical Biotechnology raises and highlights the importance of the field that continues to grow, people from all types of industries are going to be required to make decisions to help regulate this field.

Kathryn Maxson Jones
Rachel A. Ankeny
Robert Cook-Deegan

The Bermuda Principles for DNA sequence data sharing are an enduring legacy of the Human Genome Project (HGP). They were adopted by the HGP at a strategy meeting in Bermuda in February of 1996 and implemented in formal policies by early 1998, mandating daily release of HGP-funded DNA sequences into the public domain. The idea of daily sharing, we argue, emanated directly from strategies for large, goal-directed molecular biology projects first tested within the "community" of C. elegans researchers, and were introduced and defended for the HGP by the nematode biologists John Sulston and Robert Waterston. In the C. elegans community, and subsequently in the HGP, daily sharing served the pragmatic goals of quality control and project coordination. Yet in the HGP human genome, we also argue, the Bermuda Principles addressed concerns about gene patents impeding scientific advancement, and were aspirational and flexible in implementation and justification. They endured as an archetype for how rapid data sharing could be realized and rationalized, and permitted adaptation to the needs of various scientific communities. Yet in addition to the support of Sulston and Waterston, their adoption also depended on the clout of administrators at the US National Institutes of Health (NIH) and the UK nonprofit charity the Wellcome Trust, which together funded 90% of the HGP human sequencing effort. The other nations wishing to remain in the HGP consortium had to accommodate to the Bermuda Principles, requiring exceptions from incompatible existing or pending data access policies for publicly funded research in Germany, Japan, and France. We begin this story in 1963, with the biologist Sydney Brenner's proposal for a nematode research program at the Laboratory of Molecular Biology (LMB) at the University of Cambridge. We continue through 2003, with the completion of the HGP human reference genome, and conclude with observations about policy and the historiography of molecular biology.

Melanie Armstrong

The United States government has spent billions of dollars to prepare the nation for bioterrorism despite the extremely rare occurrence of biological attacks in modern American history. Germ Wars argues that bioterrorism has emerged as a prominent fear in the modern age, arising with the production of new forms of microbial nature and the changing practices of warfare. In the last century, revolutions in biological science have made visible a vast microscopic world, and in this same era we have watched the rise of a global war on terror. Germ Wars demonstrates that these movements did not occur separately but are instead deeply entwined-new scientific knowledge of microbes makes possible new mechanisms of war. Whether to eliminate disease or create weapons, the work to harness and control germs and the history of these endeavors provide an important opportunity for investigating how biological natures shape modern life. Germ Wars aims to convince students and scholars as well as policymakers and activists that the ways in which bioterrorism has been produced have consequences for how people live in this world of unspecifiable risks.

Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace "α" with "alpha"). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.

Robert J. Milner

Imagine setting out to purify and characterize all of the proteins in a rat brain: each protein would require a specifically designed purification scheme, perhaps kilogram quantities of starting material, and the determination of each primary amino-acid sequence would be complex and time consuming. To do this for each of the many thousands of proteins in a mammalian brain would be a daunting task indeed! Yet a few micrograms of rat brain mRNA contain molecules coding for every protein in the brain: by means of recombinant DNA techniques we can use the information encoded in each mRNA to investigate not only the structure of the corresponding protein but also its genetic regulation. The mRNA molecules are first converted into a form which can be inserted into a bacterial plasmid. The population of plasmids, each containing a different piece of inserted material, is then used to infect bacteria, in such a way that each bacterium receives a different plasmid. The complex mixture of plasmids, and hence the original mRNA population, can now be very easily separated into its different components by growing up each bacterium which received a plasmid as an individual colony or clone. This simple cloning procedure is the essence of recombinant DNA technology.

Source: https://www.researchgate.net/publication/347545743_Recipes_for_recombining_DNA_A_history_of_Molecular_Cloning_A_Laboratory_Manual

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