Recombinant DNA

Definition

Recombinant DNA is a form of DNA constructed in the laboratory. It is generated by transferring selected pieces of DNA from one organism to another.

The vial shown in the photograph contains human insulin, one of the first therapeutic proteins that was genetically cloned. The drug is used to treat diabetes. Credit: Wellcome Library, London.

Importance

Genetic engineering is used for many different purposes in research, medicine, agriculture and industry. The technology is important because it enables the creation of multiple copies of genes and the insertion of foreign genes into other organisms to give them new traits, such as antibiotic resistance or a new colour. One of the first ways in which the technology was deployed was to re-engineer microbial cells to produce foreign proteins. This facilitated the manufacture of human proteins on an unprecedented scale at minimum cost, thereby opening the way to study the function of proteins in greater detail and to their therapeutic use. By 2001 over 80 recombinant DNA based products had been approved for treating disease and for vaccination and a further 350 recombinant DNA-based drugs were being tested for safety and efficacy. The technology is also an important tool in agriculture, being used to improve plants' resistance to pests and increase crop yields.

Discovery

While the structure of DNA was first determined in 1953, it was to take another two decades before scientists had the means to generate recombinant DNA. This was aided by firstly the realisation in the 1950s that plasmids, small mobile pieces of DNA, could replicate in huge quantities independently of chromosomal bacteria DNA and that they could transfer genetic information. It was this process that gave host bacteria the capacity to inherit new genes and therefore new functions such as resistance to antibiotics. Another important tool for creating recombinant DNA was the discovery in the 1960s by the Swiss microbiologist Werner Arber and American biochemist Stuart Linn that bacteria could protect themselves from attack by viruses the production of endonucleases, known as restriction enzymes, which could seek out a single DNA sequence in a virus and cut it precisely in one place. This process prevented the replication of viruses and hence the death of virally infected bacteria. The first restriction enzyme, Escscherichia coli K, was isolated and purified in 1968 by Matthew Meselson and Robert Yuan at Harvard University. Two years later Hamilton O Smith, Thomas Kelly and Kent Welcox at Johns Hopkins University isolated and characterised the first site-specific restriction enzyme, later named HindII. This was demonstrated by Daniel Nathans to be a useful tool for cutting and pasting specific DNA segments. The first protocol for creating recombinant DNA was put forward in the early 1970s by Peter Lobban and Armin Dale Kaiser at Stanford University Medical School. In 1971 Paul Berg, attached to Stanford University, demonstrated the feasibility of splicing and recombining genes for the first time. Two years later, Stanley Cohen and Herbert Boyer, based respectively at Stanford University and University of California at San Francisco, successfully inserted recombinant DNA into bacteria for replication.

Application

Gene cloning has a diverse range of applications. Where it has proven particularly useful has been in mapping out the human genome, the creation of transgenic animals, and the development of insect-resistant crops. It is also pivotal to genetic tests carried out in forensic science and archaeology as well as in tests for determining hereditary disease and paternity. The technology also forms the backbone of hepatitis and human immunodeficiency virus (HIV) diagnostic tests. Recombinant DNA technology has also proven important to the production of vaccines and protein therapies such as human insulin, interferon and human growth hormone. It is also used to produce clotting factors for treating haemophilia and in the development of gene therapy.

Recombinant DNA: timeline of key events

Speigelman developed the nucleic acid hybridization technique that enables specific DNA and RNA strands to be removed from cells and is the foundation of present day recombinant DNA technology. 1914-12-14T00:00:00+0000Berg helped pioneer recombinant DNA and set up the Asimolar Conference which established guidelines for experiments using the technology.1926-06-30T00:00:00+0000Werner Arber shared the 1978 Nobel Prize in Medicine for the discovery of 'restriction enzymes and their application to problems in molecular genetics.'1929-06-03T00:00:00+0000Michael Smith shared the 1993 Nobel Prize for Chemistry for a technique that enables researchers to introduce specific mutations into genes and, thus, to the proteins that they encode.1932-04-26T00:00:00+0000Cohen helped pioneer recombinant DNA.1935-06-30T00:00:00+0000Term first used by A. Jost, a Danish microbiologist, in lecture on sexual reproduction in yeast presented to the Technical Institute in Lwow, Poland 1941-01-01T00:00:00+0000Noted by Salvador Luria and his graduate student Mary Human while conducting experiments into the break-up of DNA in phage-infected bateria.1952-01-01T00:00:00+0000The enzyme was discovered in Escherichia Coli. Its isolation paved the way to understanding how DNA is replicated, repaired and transcribed and the development of recombinant DNA. A collective group of scientists made the discovery: Arthur Kornberg, Maurice Bessman, Ernie Simms, I R Lehman.1955-12-01T00:00:00+0000Achieved by Arthur Kornberg, the experiment was published in the Journal of Biological Chemsitry in May 1958.1957-10-01T00:00:00+0000Werner Arber, Swiss microbiologist and geneticist, and his doctoral student Daisy Dussoix propose bacteria produce restriction and modification enzymes to counter invading viruses. 1962-01-01T00:00:00+0000W. Arber, 'Host-controlled modification of bacteriophage', Annual Review Microbiology 19 (1965), 365-78.1965-01-01T00:00:00+00001968-01-01T00:00:00+0000This was developed by Peter Lobhan, a graduate student of Dale Kaiser at Stanford University.1969-01-01T00:00:00+0000Achived by Har Gobind Khorana at the University of Wisconsin-Madison1970-01-01T00:00:00+0000Hamilton O Smith, Kent W Wilcox, Journal of Molecular Biology 51/2 (1970), 379-91. 1970-07-01T00:00:00+0000This was done in Dale Kaiser's laboratory by Douglas Berg together with Janet Mertz and David Jackson1971-01-01T00:00:00+0000Robert Pollack contacted Paul Berg to raise concerns about the potential biohazards of experiments his doctoral research plans to do involving the introduction of genes from the oncovirus SV40 in the human gut bacteria, E-Coli. Following this Berg self-imposed a moratorium on experiments in his laboratory involving the cloning of SV40 in E-Coli. 1971-06-01T00:00:00+0000Kathleen Danna and Daniel Nathans, PNAS, 68/12 (1971), 2913-17.1971-12-01T00:00:00+0000This took place during an unscheduled extra session held at a three-day EMBO workshop on DNA restriction and modification. The session was chaired by Norton Zinder.1972-09-26T00:00:00+0000D A Jackson, R H Symons, P Berg, 'Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli', PNAS USA, 69/10 (1972), 2904-09.1972-10-01T00:00:00+0000Janet Mertz and Ronald Davis publish an easy-to-use technique for constructing recombinant DNA. 1972-11-01T00:00:00+0000This was prompted by the publication of the recombinant DNA experpiments published by Berg, Jackson and Symons1972-11-01T00:00:00+0000Stanley Cohen and Herbert Boyer publish genetic engineering techniques to cut and paste DNA (using restriction enzymes and ligases) and reproduce the new DNA in bacteria1973-01-01T00:00:00+0000The National Institutes of Health forms a Recombinant DNA Advisory Committee to oversee recombinant genetic research.1974-01-01T00:00:00+0000Berg, P, Baltimore, D, Boyer, J W, Cohen, S N, et al, 'Biohazards of Recombinant DNA,' Science, 185 (1974): 3034.1974-07-05T00:00:00+0000Asilomar Conference in California declares moratorium on genetic engineering research in order to have time to estimate the biohazard risks of recombinant DNA research and develop guidelines.1975-01-01T00:00:00+0000Robert Swanson, venture capitalist and Herbert Boyer, American biochemist, establish Genentech in San Francisco. It is the first biotechnology company established specifically dedicated to commercialising recombinant DNA. Its founding marks the start of what is to become a burgeoning biotechnology industry. 1976-04-01T00:00:00+0000Genetically engineered bacteria are used to synthesize human growth protein.1977-01-01T00:00:00+0000Genentech scientists succeed in genetically engineering human insulin in E-Coli.1978-01-01T00:00:00+0000Awarded to Werner Arber, Daniel Nathans and Hamilton O Smith.1978-10-01T00:00:00+0000US Supreme Court, in the landmark case Diamond v. Chakrabarty, approves the principle of patenting genetically engineered life forms1980-01-01T00:00:00+0000The American scientists Stanley Cohen and Herbet Boyer are awarded the first US patent for gene cloning.1980-01-01T00:00:00+0000Milstein suggests at a Wellcome Foundation lecture that by using genetic engineering scientists might be able to design tailor-made monoclonal antibodies that mimic antibodies made by the human body. This would free them up from a dependence on rodents for producing monoclonal antibodies. He publishes the idea in C. Milstein, 'Monoclonal antibodies from hybrid myelomas: Wellcome Foundation Lecture 1980', Proceedings Royal Society of London, 211 (1981), 393-412.1980-01-01T00:00:00+0000Gordon, J W, Scangos, G A, Plotkin, D J, Barbosa, J A, Ruddle, F H, 'Genetic transformation of mouse embryos by microinjection of purified DNA', PNAS USA 77 (1980), 7380–4.1980-09-01T00:00:00+0000First genetically-engineered plant is reported1981-01-01T00:00:00+0000First mice genetically cloned1981-01-01T00:00:00+0000The first drug (human insulin), based on recombinant DNA, is marketed. 1982-10-01T00:00:00+0000Speigelman developed the nucleic acid hybridization technique that enables specific DNA and RNA strands to be removed from cells and is the foundation of present day recombinant DNA technology. 1983-01-20T00:00:00+0000Two teams of scientists publish methods for the generation of chimeric monoclonal antibodies, that is antibodies possessing genes that are half-human and half mouse. Each team had developed their techniques separate from each other. The first team was lead by Michael Neuberger together with Terence Rabbitts and other colleagues at the Laboratory of Molecular Biology, Cambridge. The second team consisted of Sherie Morrison and colleagues at Stanford University together with Gabrielle Boulianne and others at the University of Toronto. 1984-12-01T00:00:00+0000Greg Winter together with other colleagues from the Laboratory Molecular Biology demonstrate the feasibility of building a new more human-like monoclonal antibody by grafting on to the humab antibody portions of a variable region from a mouse antibody. This reduced the mouse component of the monoclonal antibody to just 5%, making the monoclonal antibody safer and more effective for use in humans. The technique was published in PT Jones, PH Dear, J Foote, MS Neuberger, G Winter, 'Replacing the complementarity-determining regions in a mouse antibody with those from a mouse', Nature, 321 (29 May 1986), 522-5.1986-05-01T00:00:00+0000Hoffmann-LaRoche and Schering-Plough gain FDA permission to market genetically engineered alpha interferon for use as treatment hairy cell leukaemia. The development of interferon rested on the application of both genetic cloning and monoclonal antibodies. 1986-06-04T00:00:00+0000FDA grants a BLA to Merck & Co. for Recombivax HB, the first recombinant vaccine for hepatitis B. It is seen as a breakthrough because it carries no risk of infecting healthy people,1986-07-23T00:00:00+0000Campath-1G is humanised, resulting in Campath-1H. It is accomplished with technology developed by Greg Winter.1988-01-01T00:00:00+0000The drug, a genetically engineered enzyme, was developed by the Genentech researcher Steven Shak. It was the first new treatment for cystic fibrosis in 30 years. The enzyme was engineered to dissolve mucus plugs in the lungs of cystic fibrosis patients.1993-12-30T00:00:00+0000Abciximab (ReoPro) approved by the FDA and European regulatory authorities to prevent blot clots during coronary artery procedures like angioplasty. The monoclonal antibody was originally developed by Barry Coller at State University of New York and commercially developed by Centocor. The drug showed for the first time that monoclonal antibodies could be used for the treatment of acute disease conditions. 1994-12-01T00:00:00+0000Daclizumab was approved by the FDA for the preventition of acute rejection of kidney transplants. The monoclonal antibody was developed by Protein Design Labs using a humanising method devised by Cary Queen and marketed together with F. Hoffmann-La Roche. 1997-12-01T00:00:00+0000Nathans was an American molecular biologist who co-discovered restriction enzymes and their applications to break DNA molecules. 1999-11-16T00:00:00+0000A Canadian biochemist, Michael Smith helped develop site-direct mutagenesis, a technique that allows for a mutation to be created at a specific defined site in a DNA molecule. This technique is pivotal to genetic and protein research and engineering. 2000-10-04T00:00:00+0000Twelve patients with HIV treated between 2009 and 2014 report benefits from genetically engineered virus with a rare mutatiuon known to protect against HIV (CCR5 deficiency).2014-03-01T00:00:00+0000
Date Event People Places
14 Dec 1914Solomon Spiegelman was born in Brooklyn, NY, USASpiegelmanUniversity of Minnesota
30 Jun 1926Paul Berg was born in New York NY, USABergStanford University
3 Jun 1929Werner Arber was born in Granichen, SwitzerlandArberUniversity of Geneva
26 Apr 1932Michael Smith was born in Blackpool, United KingdomSmithUniversity of British Columbia
30 Jun 1935Stanley Norman Cohen was born in Perth Amboy, NJ, USACohenStanford University
1941Term 'genetic engineering' first coinedJost 
1952First observation of the modification of viruses by bacteriaLuria, HumanUniversity of Illinois
December 1955First discovery of the enzyme DNA polymeraseKornberg, Bessman, Simms, LehmanWashington University in St. Louis
October 1957First synthesis of DNA in a test tubeKornbergWashington University in St. Louis
1962Concept of restriction and modification enzymes bornArber, DussoixUniversity of Geneva
1965Werner Arber predicts restriction enzymes could be used as a labortory tool to cleave DNAArberUniversity of Geneva
1968Paul Berg started experiments to generate recombinant DNA moleculesBergStanford University
1969New idea for generating recombinant DNA conceivedLobhanStanford University
1970First complete gene synthesised KhoranaUniversity of Wisconsin
July 1970First restriction enzyme isolated and characterisedSmith, WilcoxJohns Hopkins University
1971First plasmid bacterial cloning vector constructedBerg, Mertz, JacksonStanford University
June 1971First time potential biohazards of recombinant DNA raisedMertz, Berg, PollackStanford University
December 1971First experiments published demonstrating the use of restriction enzymes to cut DNADanna, NathansJohns Hopkins University
September 1972 - Sep 1972First time possible biohazards of recombinant DNA technology publicly discussedZinder 
October 1972First paper published on generating recombinant DNABerg, Jackson, SymonsStanford University
November 1972First easy-to-use technique published for constucting recombinant DNA. J. Mertz, R. Davis, Proceedings of the National Academy of Science, USA 69/11, pp. 2270-74.Berg, MertzStanford University Medical School
1 Nov 1972Nature editorial voiced concern about generating recombinant DNABerg, Jackson, SymonsStanford University
1973Recombinant DNA produced in bacteriaCohen, BoyerStanford University Medical School, University of California San Francisco
1974Regulation begins for recombinant genetic research 
July 1974First concerns about potential biohazards of recombinant DNA publishedBerg, Baltimore, Boyer, Cohen 
1975Temporary moratorium on genetic engineeringBerg 
April 1976Genentech foundedSwanson, BoyerGenentech Inc
1977Human growth hormone genetically engineered 
1978Human insulin produced in E-coliGenentech
October 1978Nobel Prize for discovery and understanding of restriction enzymesArber, Nathans, SmithJohns Hopkins University, University of Geneva
1980Genetic engineering recognised for patenting 
1980First patent awarded for gene cloningCohen, BoyerStanford University Medical School
1980Cesar Milstein proposed the use of recombinant DNA to improve monoclonal antibodiesMilsteinLaboratory of Molecular Biology
September 1980First transgenic mice made with recombinant DNA announced Barbosa, Gordon, Plotkin, Ruddle, ScangosYale University
1981First genetically-engineered plant reported 
1981First genetically cloned mice 
October 1982First recombinant DNA based drug approvedGenentech Inc
20 Jan 1983Solomon Spiegelman diedSpiegelmanUniversity of Minnesota
1984First chimeric monoclonal antibodies developed which lays foundation for safer and more effective monoclonal antibody therapeuticsNeuberger, Rabbitts, Morrison, Oi, Herzenberg, Boulianne, Schulman, HozumiLaboratory of Molecular Biology, Stanford Univerity Medical School
May 1986First humanised monoclonal antibody createdDear, Foote, Jones, Neuberger, WinterLaboratory of Molecular Biology
June 1986Interferon approved for treating hairy cell leukaemia 
23 Jul 1986First recombinant vaccine licensedMerck & Co
1988Campath-1H is created - the first clinically useful humanised monoclonal antibody.Winter, Waldmann, Reichmann, ClarkCambridge University, Laboratory of Molecular Biology
December 1993Recombinant human deoxyribonuclease approved by FDA for cystic fibrosisGenentech
1994First chimeric monoclonal antibody therapeutic approved for marketCollerCentocor, State University of New York
December 1997First humanised monoclonal antibody approved for marketQueenProtein Design Labs, Roche
16 Nov 1999Daniel Nathans diedNathans Johns Hopkins University
4 Oct 2000Michael Smith diedUniversity of British Columbia
March 2014Promising results announced from trial conducted with HIV patients 

14 Dec 1914

Solomon Spiegelman was born in Brooklyn, NY, USA

30 Jun 1926

Paul Berg was born in New York NY, USA

3 Jun 1929

Werner Arber was born in Granichen, Switzerland

26 Apr 1932

Michael Smith was born in Blackpool, United Kingdom

30 Jun 1935

Stanley Norman Cohen was born in Perth Amboy, NJ, USA

1941

Term 'genetic engineering' first coined

1952

First observation of the modification of viruses by bacteria

Dec 1955

First discovery of the enzyme DNA polymerase

Oct 1957

First synthesis of DNA in a test tube

1962

Concept of restriction and modification enzymes born

1965

Werner Arber predicts restriction enzymes could be used as a labortory tool to cleave DNA

1968

Paul Berg started experiments to generate recombinant DNA molecules

1969

New idea for generating recombinant DNA conceived

1970

First complete gene synthesised

Jul 1970

First restriction enzyme isolated and characterised

1971

First plasmid bacterial cloning vector constructed

Jun 1971

First time potential biohazards of recombinant DNA raised

Dec 1971

First experiments published demonstrating the use of restriction enzymes to cut DNA

Sep 1972 - Sep 1972

First time possible biohazards of recombinant DNA technology publicly discussed

Oct 1972

First paper published on generating recombinant DNA

Nov 1972

First easy-to-use technique published for constucting recombinant DNA. J. Mertz, R. Davis, Proceedings of the National Academy of Science, USA 69/11, pp. 2270-74.

1 Nov 1972

Nature editorial voiced concern about generating recombinant DNA

1973

Recombinant DNA produced in bacteria

1974

Regulation begins for recombinant genetic research

Jul 1974

First concerns about potential biohazards of recombinant DNA published

1975

Temporary moratorium on genetic engineering

Apr 1976

Genentech founded

1977

Human growth hormone genetically engineered

1978

Human insulin produced in E-coli

Oct 1978

Nobel Prize for discovery and understanding of restriction enzymes

1980

Genetic engineering recognised for patenting

1980

First patent awarded for gene cloning

1980

Cesar Milstein proposed the use of recombinant DNA to improve monoclonal antibodies

Sep 1980

First transgenic mice made with recombinant DNA announced

1981

First genetically-engineered plant reported

1981

First genetically cloned mice

Oct 1982

First recombinant DNA based drug approved

20 Jan 1983

Solomon Spiegelman died

1984

First chimeric monoclonal antibodies developed which lays foundation for safer and more effective monoclonal antibody therapeutics

May 1986

First humanised monoclonal antibody created

Jun 1986

Interferon approved for treating hairy cell leukaemia

Jun 1986

First recombinant vaccine licensed

1988

Campath-1H is created - the first clinically useful humanised monoclonal antibody.

Dec 1993

Recombinant human deoxyribonuclease approved by FDA for cystic fibrosis

1994

First chimeric monoclonal antibody therapeutic approved for market

Dec 1997

First humanised monoclonal antibody approved for market

16 Nov 1999

Daniel Nathans died

4 Oct 2000

Michael Smith died

Mar 2014

Promising results announced from trial conducted with HIV patients