Biotechnology Engineering

1. Introduction and Historical
Background- Part-1 Lecture No.1
1

2. • 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.
2

3. 3
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.

4. 4
•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 living cell of a
species are very rare.

5. 5
•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
genetic make-up of any living being on Earth.

6. 6
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.

7. 7
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 the host

8. 8
•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 full-
length 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.

9. Factor (Gene)
1866 Nuclein (DNA)
1868
1944
1952
1953
1972
1973
1900
1928
1927
1909
1912
2004
9

10. Oswald Avery Colin MacLeod Maclyn McCarty
Oct 21, 1877-1955 (January 28, 1909- Feb, 11, 1972) (Jun 9, 1911 – Jan 2, 2005)
Showed that Griffith’s transforming principle was
DNA
1944
10

11. 1952
Alfred Hershey Martha Chase
(Dec 4, 1908 – May 22, 1997) (1927-2003)
Hershey was awarded Nobel Prize in Physiology or Medicine
in 1969
showed that
the genetic
material of
bacteriophage T2
is DNA
11

12. 1953
James Watson Francis Crick
(April 6 1928-) (Jun 8, 1916-Jul 28, 2004)
proposed
Double helical model for DNA
Nobel Prize In Physiology or Medicine in 1962 12

13. Watson and Crick with their model of DNA
13

14. 1958
Arthur Kornberg
discovered
DNA polymerase
March 3, 1918 – October 26, 2007
Nobel Prize in Physiology or Medicine 1959
14

15. 1966
Vin Thorne
introduced agar gels
to analyze DNA
separated superhelical , nicked and linear forms of
radiolabelled polyomavius DNA
15

16. 1967
Martin Gellert
and three other groups
independently isolated
DNA ligase enzyme
Martin Gellert
16

17. 1968
Werner Arber
(Jun 3, 1929 - )
Nobel Prize in Physiology or Medicine 1978
showed
in vitro restriction of
fd phage DNA
by E. coli extract
17

18. 1970
Isolated and characterized
the first restriction enzyme
Endonuclease R
( later renamed HindII )
Hamilton O. Smith
(Aug 23, 1931-)
Nobel Prize in Physiology or Medicine 1978
18

19. Howard Temin
(Dec 10, 1934 – Feb 9, 1994)
Nobel Prize in Physiology or Medicine 1975
David Baltimore
(Mar 7, 1938 - )
discovered
reverse transcriptase
1970
19

20. 1970
M. Mandel and A. Higa
developed
a transfection method for E. coli
20

21. 1971
Daniel Nathans
(Oct 30, 1928 – Nov 16, 1999)
Pioneered the application of
restriction enzymes :
Specific cleavage of SV 40 DNA
Nobel Prize in Physiology or Medicine 1978
Kathleen Janet Danna
21

22. 1972
Stanley Cohen
(Nov 17, 1922 - )
Cohen and coworkers
achieved
genetic transformation of
E. coli
by R-factor DNA (purified plasmid DNA)
22

23. 1972
constructed first
recombinant DNA
molecule in vitro
Paul Berg
(Jun 30, 1926 - )
Nobel Prize in Chemistry 1980
23

24. 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
Stanley Cohen
(Nov 17, 1922 - )
Herbert Boyer
(1936 - )
Boyer and Cohen were awarded Nobel Prize in Physiology or Medicine 1986
1973
24

25. 1974
Development of phage (viral) cloning vector
by three research groups
25

26. 1975
developed the
Southern blotting
technique
(1938 - )
Edward Mellor Southern
26

27. 1975
Identified
tumour inducing (Ti) plasmid in
Agrobacterium tumefaciens
Jeff Schell
(Jul 20, 1935-Apr 17, 2003)
Mark van Montagu
(Nov 10, 1933 - )
27

28. 1975
M. Grunstein and D.S. Hogness
developed
colony hybridization method
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29. F. Bolivar and co workers constructed the
plasmid vector pBR322
1977
29

30. Joachin Messing and co workers
reported
the construction of
bacteriophage M13 cloning vector (M13 mp1)
1977
30

31. 1977
devised methods for DNA sequencing
Walter Gilbert
(Mar 21, 1932 - )
Frederick Sanger
(Aug 13, 1918 –Nov 19, 2013 )
Nobel Prize in Chemistry 1980
31

32. 1977
Obtained the
complete nucleotide
sequence of a virus,
bacteriophage ΦX174
Frederick Sanger
(Aug 13, 1918 –Nov 19, 2013 )
32

33. 1977
Paul Berg and coworkers
introduced
nick translation technique
for radiolabelling of DNA
33

34. 1977
Somatostatin
becomes the first
human hormone
produced by
using
Recombinant
DNA technologyHerbert Boyer
(1936 - )
Arthur D. Riggs
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35. 1978
Construction of
Cosmid cloning
vectors
University of Basel
Switzerland
Barbara Lenore Hohn
35

36. 1978
Transformation of Yeast
Gerald R. Fink
36

37. 1980
Discovery of Fluorescent In Situ Hybridization (FISH)
(Direct method)
direct labeling of nucleic acid with fluorophores
37

38. 1980
Restriction Fragment Length Polymorphism
(RFLP)
Concept
D. Botstein, R. L. White, M. Skolnick and
R. W. Davis
gave
38

39. 1981
developed a general method for
site- directed mutagenesis in prokaryotes
Garry Ruvkun Frederick M Ausubel
39

40. 1982
Human insulin produced by recombinant
DNA methods goes on the market under
the trade name Humulin
1982
40

41. Genetic transformation of Drosophila
1982
Allen C. Spradling
41

42. 1982
Obtained the
complete nucleotide
sequence of
bacteriophage
lambda
Frederick Sanger
(Aug 13, 1918 - )
42

43. 1982
discovered self-splicing
of an intron RNA
Thomas Cech
(Dec 8, 1947 - )
Nobel Prize in Chemistry 1989 43

44. 1983
Sidney Altman
(May 7, 1939 - )
Showed enzymatic cleavage
of RNA by RNA
Discovery of ribozymes
Nobel Prize in Chemistry 1989 44

45. 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
a Ti plasmid derived vector
45

46. 1983
Construction of suicide plasmid
vectors
for
transposon mutagenesis
in Gram-negative bacteria
Alfred Pühler
46

47. 1984
D.C. Schwartz & C.R. Cantor
Introduced
Pulse Field Gradient Gel Electrophoresis
47