Introduction and Historical

Background- Part-1 Lecture No.1

G. S. Randhawa
Department of
Biotechnology
1
Indian Institute
of
Technology

 Our planet Earth is the only planet known

to have life.
As per the Encyclopedia of Life, about 1.9
million living species are known to
science.
Each of these species has unique
characteristics because of the presence of
unique genes in its cells.

Alfred Hershey and Martha

Chase in 1952 showed that
genes are made up of DNA.
Soon after this discovery the
structure
of
DNA
was
elucidated by James Watson
and Francis Crick in 1953.

 Every
species
of
living
organisms
has
been
maintaining its identity over a
very
long
period
of
time
because it does not allow its
DNA to recombine with the DNA
of other species and transfers
its distinct DNA to its next
generation.
The exceptions in which a
foreign DNA molecule enters
a
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Moreover in most of these exceptions the recombinant DNA molecules

are formed in the somatic cells and hence are not passed to the next
generation.
Even scientists were not able to recombine DNA molecules from two
different organisms under in vitro conditions.
But the scenario changed dramatically in 1972 with the trend-setting
research of Paul Berg and his coworkers who, for the first time,
constructed a recombinant DNA molecule outside a living cell.
In the subsequent year, the research groups led by Stanley Cohen and
Herbert Boyer together developed a method of gene cloning (making
copies of a gene) by constructing recombinant DNA.
These two events marked the birth of a new technology called
recombinant DNA technology which has the potential to change the
5
genetic make-up of any living being on Earth.

Birth of Recombinant DNA Technology

In the year 1972, D. A. Jackson, R. H. Symons and Paul Berg
reported the construction of first recombinant DNA under in
vitro conditions in a test tube.
The constructed recombinant DNA molecule consisted of
SV40 DNA, bacteriophage lambda genes and the galactose
operon of E. coli. For this discovery Paul Berg was awarded
Nobel Prize in Chemistry in 1980. In 1973, S. N. Cohen, A. C.
Y. Chang, H. W. Boyer and R. B. Helling inserted kanamycin
resistance gene of E. coli in the plasmid pSC101 and
introduced the recombinant plasmid in the E. coli cells.
Hence they were able to obtain multiple copies of
kanamycin resistance gene. This was the first successful
gene cloning experiment. For this discovery Stanley Cohen
was awarded Nobel Prize in Physiology or Medicine in 1986.
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Steps Involved in DNA/Gene Cloning by Host Vector

Technique
If cloning of an isolated DNA fragment is desired, it can be
achieved by the host vector technique in which the isolated DNA
fragment is inserted in a vector DNA. The recombinant vector is
introduced into a host. The various steps involved are as follows:
(i) Cutting of the DNA fragment to be cloned with a specific
enzyme
(ii) Isolation and cutting of the vector DNA with a specific
enzyme
(iii) Joining of the DNA fragment with the vector DNA to form a
recombinant vector
(iv) Introduction of the recombinant vector into the appropriate
host cells
(v) Selection of the host cells containing recombinant vector
and the growth of the selected cells of the7 host

If the gene to be cloned has not been isolated

from the organism, first of all, total DNA is
isolated from this organism or cDNA (copy or
complementary DNA) is synthesized by reverse
transcription of the RNA isolated from this
organism.
Random fragments of the total DNA or fulllength cDNAs are introduced, with the help of a
vector, into a suitable host. The resulting host
cells are then screened to obtain the desired
gene.
8

2004
1973
1972
1953
1952
1944
1912
1928
1927
1909
1900

1868
1866

Factor (Gene)

Nuclein (DNA)
9

1944

Oswald Avery Colin MacLeod Maclyn McCarty

Oct 21, 1877-1955

(January 28, 1909- Feb, 11, 1972)

(Jun 9, 1911 Jan 2, 2005)

Showed that Griffiths transforming principle was

DNA
10

1952

lfred Hershey

showed that
the genetic
material of
bacteriophage
T2 is DNA
Martha Chase

ec 4, 1908 May 22, 1997)

(1927-2003)

ershey was awarded Nobel Prize in Physiology or Medicine

in 1969

11

1953

James Watson

Francis Crick

(April 6 1928-)

(Jun 8, 1916-Jul

28, 2004)

proposed
12
Double helical model for DNA
Nobel Prize In Physiology or Medicine in 1962

Watson and Crick with their model of

DNA
13

1958

discovered
DNA polymerase

Arthur Kornberg

March 3, 1918 October 26, 2007

Nobel Prize in Physiology or Medicine 1959
14

1966

Vin Thorne
introduced agar gels
to analyze DNA
separated superhelical , nicked and linear forms of
radiolabelled polyomavius DNA

15

1967

Martin Gellert
and three other groups
independently isolated
DNA ligase enzyme

Martin Gellert
16

1968

showed
in vitro restriction
of
fd phage DNA
by E. coli extract
Werner Arber
(Jun 3, 1929 - )
Nobel Prize in Physiology or Medicine 1978

17

1970

Isolated and characterized

the first restriction enzyme
Endonuclease R
( later renamed HindII )
Hamilton O.
Smith

(Aug
1931-) or Medicine 1978
obel Prize
in 23,
Physiology

18

1970

David Baltimore
(Mar 7, 1938 - )

Howard
Temin

(Dec 10, 1934 Feb 9,

discovered
1994)
reverse transcriptase

Nobel Prize in Physiology or Medicine 1975

19

1970

M. Mandel and A. Higa

developed
a transfection method for E. coli

20

1971

Daniel Nathans
(Oct 30, 1928 Nov 16, 1999)

Kathleen Janet Danna

Pioneered the application of

restriction enzymes :
Specific cleavage of SV 40 DNA

obel Prize in Physiology or Medicine 1978

21

1972

Cohen and coworkers

achieved
genetic transformation of
E. coli

by R-factor DNA (purified plasmid DNA

Stanley Cohen
(Nov 17, 1922 - )

22

1972
constructed first
recombinant DNA
molecule in vitro

Paul Berg
(Jun 30, 1926 - )

Nobel Prize in Chemistry 1980

23

1973

Stanley Cohen

Herbert Boyer

(Nov 17, 1922 - )

(1936 - )

In the first gene cloning experiment, Cohen, Chang, Boyer

& Helling inserted a bacterial kanamycinR gene into a
plasmid vector
First use of plasmid cloning vector
Boyer and Cohen were awarded Nobel Prize in Physiology or Medicine 1986
24

1974

Development of phage (viral) cloning vector

by three research groups

25

1975
developed the Southern
blotting technique

Edward Mellor Southern

(1938 - )

26

1975

Jeff Schell
(Jul 20, 1935-Apr 17, 2003)

Mark van Montagu

(Nov 10, 1933 - )

Identified
tumour inducing (Ti) plasmid in
27
Agrobacterium tumefaciens

1975

M. Grunstein and D.S. Hogness

developed
colony hybridization method

28

1977

F. Bolivar and co workers constructed the

plasmid vector pBR322

29

1977

Joachin Messing and co workers

reported
the construction of
bacteriophage M13 cloning vector (M13 mp1)

30

1977

Walter Gilbert
(Mar 21, 1932 - )

Frederick Sanger
(Aug 13, 1918 Nov 19, 2013 )

devised methods for DNA sequencing

Nobel Prize in Chemistry 1980

31

1977

Obtained the
complete nucleotide
sequence of a virus,
bacteriophage X174

Frederick Sanger
(Aug 13, 1918 Nov 19, 2013 )
32

1977

Paul Berg and

coworkers introduced
nick translation technique
for radiolabelling of DNA

33

1977

Herbert BoyerArthur D. Riggs

(1936 - )

Somatostatin
becomes the
first human
hormone
produced by
using
Recombinant
DNA technology
34

University of Basel
Switzerland

1978

Construction of
Cosmid cloning
vectors

Barbara Lenore Hohn

35

1978

Transformation of Yeast

Gerald R. Fink
36

1980

Discovery of Fluorescent In Situ Hybridization (FISH

(Direct method)
direct labeling of nucleic acid with fluorophores

37

1980
D. Botstein, R. L. White, M. Skolnick and
R. W. Davis
gave
Restriction Fragment Length Polymorphism
(RFLP)
Concept

38

1981

Garry Ruvkun

Frederick M Ausube

developed a general method for

39
site- directed mutagenesis
in

1982

Human insulin produced by recombinant

DNA methods goes on the market under
the trade name Humulin

40

198
2

Allen C. Spradling
Genetic transformation of Drosophila
41

198
2

Obtained the
complete nucleotide
sequence of
bacteriophage
lambda

Frederick Sanger
(Aug 13, 1918 - )
42

1982

discovered self-splicing of an intron RNA

Thomas Cech
(Dec 8, 1947 - )

Nobel Prize in Chemistry 1989 43

1983

Showed enzymatic cleavage

of RNA by RNA
Discovery of ribozymes
Sidney Altman
(May 7, 1939 - )
Nobel Prize in Chemistry 1989 44

1983

Jeff Schell
(Jul 20, 1935-Apr 17, 2003)

Mark van Montagu

(Nov 10, 1933 - )

Expression of chimaeric genes

transferred to the plant cells
using
45
a Ti plasmid derived vector

1983

Construction of suicide plasmid

vectors
for
transposon mutagenesis
in Gram-negative bacteria

Alfred Phler
46

1984

D.C. Schwartz & C.R. Cantor

Introduced
Pulse Field Gradient Gel Electrophoresis

47