UNIT I

Recombinant DNA
Technology

Chapter 1: An Overview of Recombinant DNA Technology

Chapter 2: Host–Vector System

Chapter 3: Gene Cloning

Chapter 4: Application of Recombinant DNA Technology

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 Herbert Boyer

Herbert Wayne ‘Herb’ Boyer (born July 10, 1936) was a researcher
and entrepreneur in Biotechnology. Herb Boyer hails from
Derry, Pennsylvania. Boyer went on to graduate at the University
of Pittsburgh, where he specialised in microbial genetics. After
preliminary experiments in 1973, the Cohen-Boyer team was able
to cut open a plasmid loop from one species of bacteria, insert
a gene from different bacterial species and close the plasmid.
This created a recombinant—plasmid containing recombined
DNA from two different sources. The team had created the first
genetically modified organisms. He is the recipient of the 1990
National Medal of Science, co-recipient of the 1996 Lemelson–
MIT Prize, and a co-founder of Genentech. He was a professor
at the University of California, San Francisco (UCSF) and later
served as the Vice President of Genentech from 1976 until his
retirement in 1991.

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 Chapter 1 An Overview of Recombinant
DNA Technology

This chapter gives an overview of recombinant DNA (rDNA) 1.1 An Overview of

technology as to how the application of basic concepts of Recombinant
molecular biology, microbiology, genetics, biochemistry, DNA Technology
etc., led to initial development of rDNA technology.
Potential application of rDNA technology in the use of
medicine and agriculture is also discussed in conceptual
manner along with some noticeable examples of products
developed through rDNA technology.

1.1 AN OVERVIEW OF RECOMBINANT DNA

TECHNOLOGY
In the last century when scientists discovered that
nucleic acid (DNA) is the principal molecule responsible
for the expression of characters, attempts were made to
alter the genetic makeup of an organism by manipulating
nucleic acid directly. Various methods used for directly
manipulating nucleic acid/genome (DNA) of an organism
are collectively referred to as recombinant DNA (rDNA)
technology or genetic engineering.
rDNA technology has been possible due to rapid
progress in various fields of biology which spans from

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 biochemistry, genetics, cytology, microbiology, molecular
biology and others. Isolation and purification of nucleic
acids followed by the understanding of their structures,
properties, functions and finally their sequencing in the
last century are the most important contributions which
laid the foundation of development of rDNA technology.
The first breakthrough in this journey was to establish the
fact that DNA of an organism not only carries its genetic
information but also propagates it from one generation
to another. The next hallmark was the determination of
chemical and physical structure of DNA molecule and
double helical structure of DNA. Further, replication,
transcription and translation of DNA was understood in
detail by scientists. Scientists were also able to develop
various methods and techniques to isolate and purify
DNA from various organisms. Several enzymes were
simultaneously discovered using which one can precisely
manipulate a DNA molecule. Thus, new enzymes, such
as restriction enzymes (which act as scissors to cut the
molecules of DNA) by Werner Arbor, Hamilton Smith
and Daniel Nathan (during late 1960s and early 1970s)
and ligase (which joins two DNA fragments) by Gellert,
Lehman, Richardson and Hurwitz in the year 1967 were
discovered. During this period, scientists also noticed
that foreign DNA fragments can be taken by bacteria from
its surrounding environment where it can be integrated
into its genome. With all this knowledge, scientists asked
a question that is it possible to transfer the gene of
interest from one organism to another organism to get its
product? Stanley Cohen had the expertise in introducing
plasmid DNA into Escherichia coli (E. coli) and subsequent
propagation and cloning of plasmids in the bacteria. On
the other hand, Boyer had the expertise to cleave the
double stranded DNA to produce single stranded ends
with identical termini using restriction enzymes. Both
visualised the potential of combining the two discoveries
to what would later become rDNA technology or genetic
engineering.

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 Apart from its unquestionable contribution to basic
scientific endeavour, rDNA technology has played a
great role in shaping our life standards by its immense
contribution in diagnosis and treatment of various
diseases including genetic disorders and to improve and
develop disease free high yielding crops. The contribution
of rDNA technology in shaping our life can be judged
from the given examples. Earlier several tons of animal
pancreatic glands were needed to get a few milligrams of
insulin to treat diabetes, or thousands of animal pituitary
glands were required to isolate growth hormone to treat
dwarfism. Therefore, these products were available in
limited quantity as well as at a high cost. Nevertheless,
such purified therapeutic proteins from animal source
exhibited immunogenic reactions in humans. Needless
to say, scientists circumvent the above obstacles by
producing human insulin and growth hormone in bacterial
system using rDNA technology. Production of interferon
to treat cancer, plasminogen activator and urokinase to
dissolve blood clots are a few examples of the contribution
of rDNA technology to human society. In the last few
decades, by employing rDNA technology, scientists have
been able to introduce specific targeted modifications in
plant genome to get genetically modified crops. Thus, in
this way, crops have been developed which offer resistance
to diseases, thereby helping farmers to be free from worry
about damage of their crops. Similarly, drought resistant
or salinity tolerant crops were also developed so that
farmers can grow them in adverse environment. Such
modifications in genetic system of plants or crops by rDNA
technology not only improve the quality of production but
also enhance the value of products.
Days are not far, when a variety of important therapeutic
proteins, peptides and hormones will be produced from
plants employing rDNA technology. Such products will
have many advantages over animal-based products in
terms of costs and contamination. In general, animal-
based products are costlier and require extra care to be
free of virus and other animal protein contaminants.
Landmark discoveries which led to the development
of modern biotechnology (based on rDNA technology is
given in Box 1).

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 Box 1

Selected development in the history of biotechnology

1917 Karl Ereky coined the term ‘Biotechnology’

1944 Avery, MacLeod and McCarty demonstrated that ‘DNA is the genetic material’

1952 Joshua Lederberg Discovered ‘Plasmids’

1953 Watson and Crick proposed ‘Double Helical structure of DNA’

1960s Werner Arber, Matthew Meselson discovered ‘ Type I restriction enzymes’

1967 Gellert, Lehman, Richardson and Hurwitz discovered ‘ligases enzymes’

1970 Hamilton O. Smith and Thomas J. Kelly discovered ‘ Type II restriction enzymes’

Paul Berg assembled the first ‘Recombinant DNA’ from a bacterium into the
1972
virus

Stanley N. Cohen and Herbert Boyer developed ‘DNA cloning and rDNA
1973
technology’

Georges J.F. Köhler and César Milstein described the ‘Hybridoma Technique’
1975
for production of monoclonal antibodies

FDA approved world’s first recombinant DNA Therapeutic Product ‘Humulin’

1982
developed by Eli Lilly and Genentech

1983 Kary Mullis developed ‘Polymerase Chain Reaction’

1984 Sir Alec Jeffreys invented ‘DNA Fingerprinting’

The first recombinant vaccine ‘Recombivax HB’ for Hepatitis B was approved
1986
for human use

‘Human Genome Project’ officially initiated which was coordinated by the U.S.
1990
Department of Energy (DOE) and the National Institute of Health (NIH)

The first genetically engineered crop ‘Flavr Savr’ tomato was introduced which
1994
was produced by Calgene in 1992

Keith Campbell and Ian Wilmut cloned the first mammal from adult somatic
1996
cell using nuclear transfer ‘Dolly’ (Sheep)

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 Researchers at Monsanto developed ‘Bt cotton’ and first commercially released
1996 it in China and the United States in 1996, followed by its introduction in India
in 2003

2000 Ingo Potrykus and Peter Beyer developed ‘Golden Rice’

2003 The Human Genome Project (HGP) was completed

‘Avastin’, an anti-VEGF monoclonal antibody for cancer treatment was

2004
discovered

A recombinant vaccine ‘Gardasil’ against human papillomavirus (HPV) receives

2006
FDA approval

2006 Discovery of RNA interference ‘Gene Silencing’ by double stranded RNA

Robert Edwards awarded Nobel Prize for the development of human ‘in vitro
2010
fertilization’ (IVF) therapy

Shinya Yamanaka and John B. Gurdon discovered that mature differentiated

2012
cells can be reprogrammed into ‘Induced Pluripotent Stem Cells’

2019 Recent advances in the ‘CRISPR-Cas9’ genome editing tool

2020 Recombinant vaccines against COVID-19 was developed.

In the next chapters of the present Unit, the various

components and applications of rDNA technology are
discussed in detail.

SUMMARY
• The methods used for manipulating nucleic acid/
genome (DNA) of an organism are collectively referred
to as recombinant DNA (rDNA) technology or genetic
engineering.
• The fundamental theme of rDNA technology is the isolation
and propagation of a desired DNA molecule (gene) from a
source with an aim to have its product in ample quantity.
This technique is called gene cloning.

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 EXERCISES
1. Discuss in brief how recombinant DNA technology was
initially developed?
2. Briefly discuss the application of rDNA technology in
crop improvement and therapeutic.
3. Who discovered the Plasmid?
(a) Paul Berg
(b) Sir Alec Jeffreys
(c) Joshua Lederberg
(d) Kary Mullis
4. Plasminogen activator and Urokinase are used as:
(a) Antiviral agent
(b) Blood clot dissolving drug
(c) Sugar lowering agent
(d) Cholesterol lowering agent
5. Assertion: Restriction endonuclease cuts DNA and
isolated mostly from bacteria.
Reason: Restriction endonuclease is a type of nuclease.
(a) Both assertion and reason are true and the reason
is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason
is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
6. Assertion: E.coli divides in 20 minutes while
replicates its DNA in about 60 minutes.
Reason: E.coli follows multifork replication mecha-
nism.
(a) Both assertion and reason are true and the reason
is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason
is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.

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