Molecular pharmacology

1. BASIC PRINCIPLE OF RECOMBINANT DNA
TECHNOLOGY AND ITS APPLICATIONS
Presented by:
Prashanth M E
1ST Sem, M Pharm
Department of Pharmacology
Government College of Pharmacy,
Bengaluru
Guided by:
Dr. Rajesh M S
Assistant Professor
Department of Pharmacology
Government College of Pharmacy, Bengaluru

2. MOLECULAR BIOTECHNOLOGY
It is an scientific technique used to transfer specific units of genetic information from
one organism to another.
 The field of modern biotechnology started when recombinant human insulin
produced by bacteria was first marketed in the United States in 1982.
 The effort leading to this landmark began in the early 1970s when research
scientists developed protocols to construct new types of bacterial plasmids or
vectors, by cutting out and pasting pieces of DNA together to create a new piece
of DNA (recombinant DNA) that could be inserted into host bacterium such as
E.coli.

3. RECOMBINANT DNA TECHNOLOGY
It is the art of cutting and pasting genes.
Paul Berg, Herbert Boyer, Annie Change, and Stanley Cohen are the scientists made
the first recombinant DNA molecule in 1973.
 This technique uses a number of tools that to construct new combinations of
DNA (recombinant DNA or rDNA) in the laboratory for different purposes.
 The rDNA molecule thus constructed can be introduced into an appropriate host
cell, where it can be multiplied and generate many copies. This forms the basic
concept of the process known as gene cloning or DNA cloning
 Gene cloning can generate unlimited copies of a DNA molecule (e.g.,
recombinant DNA) by replication in a host cell.

4. The DNA from a donor organism is extracted,
enzymatically and joined (ligated) to another DNA
entity to form a new, recombined DNA molecule.
This cloning vector–insert DNA construct is
transferred and maintained within a host cell. It is
called transformation.
•Those host cells that take up the DNA construct are
identified and selected from those that do not.
•If required, a DNA construct can be created so that
the protein product encoded by the cloned DNA
sequence is produced in the host cell.

5. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloned DNA and a vehicle DNA to carry the DNA into a host cell and multiply.
The process of making a recombinant DNA requires the precise cutting and stitching
of DNA molecules,
This involves a number of molecular tools—the enzymes to cut and modify the DNA,
finally resulting in a recombinant DNA molecule.
The major enzymes required for the making of an rDNA molecule are:
 Type II restriction endonucleases
 DNA ligase
 Alkaline phosphatase.

6. 1. RESTRICTION ENDONUCLEASES
First isolated in 1968 by a group of three scientists—H.O. Smith, K.W. Wilcox, and
T.J. Kelley
These enzymes are also known as molecular scissors.
One of the first type II restriction endonucleases to be characterized was from the
bacterium Escherichia coli, and it was originally designated EcoRI.
It is a homodimeric protein that binds to a DNA region with a specific palindromic
sequence. The EcoRI recognition sequence consists of 6 base pairs (bp) and is cut
between the guanine and adenine residues on each strand.

7. Restriction Enzyme Nomenclature
The very name of the restriction enzymes consists of three parts:
• An abbreviation of the genus and the species of the organism to 3 letters, for
example- Eco for Escherichia coli identified by the first letter, E, of the genus and
the first two letters, co, of the species.
• It is followed by a letter, number or combination of both of them to signify the
strain of the species.
• A Roman numeral to indicate the order in which the different restriction-
modification systems were found in the same organism or strain

9. This enzyme is responsible for the formation of the phosphodiester linkage
between two adjacent nucleotides and thus joins two double-stranded DNA
fragments.
2. DNA LIGASE

10. 3. ALKALINE PHOSPHATASE
Self-ligation is the process of annealing the sticky ends
of the linearized vector without inserting the foreign
DNA fragment.
This can be prevented by using alkaline phosphatase.
It removes the phosphate group from the 5′ end of the
DNA molecule resulting in a 5′OH group.
bacterial alkaline phosphatase—BAP
calf intestine alkaline phosphatase—CAP

12. VECTORS: THE VEHICLE FOR CLONING
Vectors act as a vehicle for carrying foreign DNA into a host cell for multiplication.
 ORIGIN OF REPLICATION
 SELECTABLE MARKERS
o positive selection - antibiotic resistance genes
o negative selection - insertional inactivation.
 MULTIPLE CLONING SITES (MCS) OR POLYLINKER
 SMALL SIZE

14. a)PLASMID-CLONING VECTORS
Are derived from bacterial plasmids
 pBR322 Vectors
 pUC Series Vectors

15. b) BACTERIOPHAGE-CLONING VECTORS
The viruses that infect bacteria are called bacteriophages. viruses usually contain a
comparatively small DNA genome surrounded by a protein coat.
 Phage-cloning Vectors
DNA
replication
Packagin
g
Assembly
Infect E. coli

16.  M13-based Cloning Vectors
Derived from bacteriophage M13.
It is lysogenic filamentous phage with a circular
single-stranded DNA genome about 6,407 bp
(6.4 kb) in length
The advantages of M13-based vectors are that they contain the same polylinker and
alpha-peptide fragments as the pUC series and recombinants can be selected by the
blue → white color test.

17. c) COSMID-CLONING VECTORS
A cosmid can be defined as a plasmid that contains
a cos site from the lambda phage genome.
The simplest cosmid vector has a ColE1 origin of
replication, selectable markers (Ampr), and
suitable polylinker sites and lambda cos site.
Ligation of the cosmid vector and foreign DNA
fragments of sizes up to 45 kb is similar to
ligation into a lambda substitution vector.

18. d) YEAST ARTIFICIAL CHROMOSOMES (YACS)
The vectors that enable the formation of
artificial chromosomes with the foreign DNA
fragments and cloning into yeast. These are
used for the cloning of very large DNA
fragments in the range of 500 to 1,000 kb.

19. e) BACTERIAL ARTIFICIAL CHROMOSOMES (BACS)
These are the cloning vectors based on the extrachromosomal plasmids of E. coli
called F factor or fertility factor. These vectors enable the construction of artificial
chromosomes, which can be cloned in E. coli.
This vector is useful for cloning DNA fragments up to 350 kb, but can be handled
like regular bacterial plasmid vectors, and is very useful for sequencing large
stretches of chromosomal DNA. Like any other vector, BACs contain ori
sequences derived from E. coli plasmid F factor, multiple cloning sites (MCS)
having unique restriction sites, and suitable selectable markers.

20. f) ANIMAL AND PLANT VECTORS (SHUTTLE VECTORS)
There are some vectors developed for transforming plant and animal cells.
These are often called shuttle vectors as they replicate in both prokaryotic and
eukaryotic hosts.
The common features of such shuttle vectors
o Capable of replicating in two or more types of hosts including prokaryotic and
eukaryotic cells.
o Replicate autonomously, or integrate into the host genome and replicate when the
host cell multiplies.
o Commonly used for transporting genes from one organism to another (i.e.,
transforming animal and plant cells).

21. MAMMALIAN VECTORS
These are shuttle vectors developed for use in mammalian tissue culture.
These eukaryotic origins of replication are typically derived from well-
characterized mammalian viruses such as simian virus 40 (SV-40) with sv-40 ori
and large tantigen system or Epstein-Barr virus
In addition to the origin of replication, these shuttle vectors also carry antibiotic
resistance genes, which function in eukaryotic cells

22. PLANT VECTORS
These shuttle vectors are based on certain viruses and bacteria, which are
pathogenic to plants. Plant viruses such as the cauliflower mosaic virus (CMV),
tobacco mosaic virus (TMV), and Gemini viruses were used for developing vectors
for plant cell transformations, but with limited success.
The most successful shuttle vectors developed for the plant system are those that
are based on the Ti plasmid of agrobacterium tumifaciens, a bacterium that causes
tumor formation in plants.

23. HOST CELLS
The host cell can be bacteria, yeast, plant or animal cells.
E. coli is the most widely used organism in rDNA experiments.
• It is very simple, easy to handle, grows rapidly and is able to accept and maintain a
range of vectors.
• The doubling time of e. coli under ideal growth conditions is 20 minutes.
• As the cell undergoes multiplication, the rDNA within the cell also undergoes
multiplication independent of its genome.
If the recombinant protein that we want to produce is of eukaryotic origin, it is better
to opt for a eukaryotic host system such as yeast.
• Yeasts are the simplest of the eukaryotic systems, single-celled, genetically and
physiologically well-characterized, and easy to grow and manipulate.

24. MAKING RECOMBINANT DNA
• The isolation and purification of vectors
and the DNA fragments containing the
gene to be cloned.
o Digest the vector DNA with a suitable
restriction endonuclease and make it
linear with or without sticky ends
o Isolate the DNA fragment carrying the
gene by digesting the genome
o The sticky ends of the two DNA strands
come closer by the base
complementation and become hydrogen
bonded to each other.

25. TRANSGENICS—INTRODUCTION OF RECOMBINANT
DNA INTO HOST CELLS
Transformation
• Cells take up foreign
DNA from their
environment.
• Chemical treatments
can enhance the ability
of cells to take up the
foreign DNA
Transfection
• rDNA is mixed with
charged substances
such as calcium
phosphate, cationic
liposomes, and
overlayered on the
host cells.
• Uptake of the external
DNA by these host
cells.
Electroporation
• Electric current is used
to create transient
microscopic pores in
the cell membrane of
the host cell.
• Temporary openings
foreign DNA enters the
cell.
Microinjection
• DNA fragment or is
directly injected into
the nucleus of plant
and animal cells.
• Carried out without the
use of any specialized
vector.
• Help of a glass
microinjection tube or
syringe.
The biolistic method
• Introducing foreign
DNA into plant cells
with the help of a gene
gun.
• Microscopic particles
of gold or tungsten
coated with the DNA
of interest is
bombarded into the
cells at a high velocity.

26. IDENTIFICATION OF RECOMBINANTS
During the process of genetic transformation only a very low percentage of the total
cell population takes or receives the recombinant DNA.
In most of the cases there are two stages of selection.
First, one is the selection of transformed cells
The second one is to identify the transformed cells that have the recombinant
plasmid
The transformed cells contain a plasmid and can be identified by the
• Positive selection method - antibiotic-resistance genes
• Negative selection method – insertional activation

27. STEPS IN RECOMBINANT DNA TECHNOLOGY
(i) Selection and isolation of DNA insert
(ii)Selection of suitable cloning vector
(iii) Introduction of DNA-insert into vector
to form rDNA molecule
(iv) rDNA molecule is introduced into a
suitable host
(v) Selection of transformed host cells
(vi) Expression and Multiplication of DNA
insert in the host

28. APPLICATIONS
Production of Transgenic Plants:
For qualities like resistance to herbicides, insects or viruses or with expression of male sterility
etc.
Production of Transgenic Animals
• To increase the speed and range of selective breeding in case of animals
• For the production of better farm animals so as to ensure more commercial benefits.
• Production of certain proteins and pharmaceutical compounds.
• To study the gene functions in different animal species.

29. Production of Hormones:
• Bacterial cells like E.coli are utilized for the production of different fine chemicals like
insulin, somatostatin, somatotropin and p-endorphin.
• Human Insulin Hormone i.e., Humulin is the first therapeutic product which was produced
by the application of rec DNA technology.
Biosynthesis of Interferon:
• The gene of human fibroblasts is inserted into the bacterial plasmid.
• These genetically engineered bacteria are cloned and cultured so that the gene is expressed
and the interferons are produced in fairly high quantities.
• This interferon, so produced, is then extracted and purified.

30. Production of Vaccines:
• A number of vaccines have been synthesized through rDNA technology, which are effective
against numerous serious diseases caused by bacteria, viruses or protozoa.
• These include vaccines for polio, malaria, cholera, hepatitis, rabies, smallpox, etc.
• DNA-vaccine is the preparation that contains a gene encoding an immunogenic protein from
the concerned pathogen.
Production of Antibiotics:
• rDNA technology helps in increasing the production of antibiotics by improving the
microbial strains through modification of genetic characteristics.
• Some important antibiotics are tetracycline, penicillin, streptomycin, novobiocin,
bacitracin, etc.

31. Production of Commercially Important Chemicals:
• alcohols and alcoholic beverages obtained through fermentation
• organic acids like citric acid, acetic acid, etc. and vitamins produced by microorganisms.
Application in Enzyme Engineering:
• the enzymes are encoded by genes, and if there are changes in a gene then the enzyme
structure also changes.
• Enzyme engineering utilizes the same fact and can be explained as the modification of an
enzyme structure by inducing alterations in the genes which encode for that particular
enzyme.

32. Prevention and Diagnosis of Diseases:
• Monoclonal antibodies are useful tools for disease diagnosis.
• Monoclonal antibodies are produced by using the technique called hybridoma technology
• rDNA provides methods for the. prevention of a number of diseases like AIDS, cholera, etc.
Gene Therapy:
• Gene therapy is undoubtedly the most beneficial area of genetic engineering for human
beings.
• It involves delivery of specific genes into human body to correct the diseases. Thus, it is the
treatment of diseases by transfer and expression of a gene into the patients’ cells so as to
ensure the restoration of a normal cellular activity.

33. Applications in forensic science:
• The applications of rec DNA technology in forensic sciences depends on the technique
called DNA profiling or DNA fingerprinting.
• It enables us to identify any person by analysing his hair roots Wood stains, serum, etc.
• DNA fingerprinting also helps to solve the problems of parentage and to identify the
criminals.
Biofuel Production:
• Biofuels are derived from biomass and these are renewable and cost effective.
• Genetic engineering plays an essentially important role in a beneficial and largescale
production of biofuels like biogas. bio hydrogen biodiesel bio-ethanol., etc.
• Genetic engineering helps to improve organisms for obtaining higher product yields and
product tolerance.

34. Practical Applications of Genetic Engineering:
rDNA technology has an immense scope in Research and Experimental studies.
It is applied for:
a. Localizing specific genes.
b. Sequencing of DNA or genes.
c. Study of mechanism of gene regulation.
d. Molecular analysis of various diseases.
e. Study of mutations in DNA, etc.

35. REFERENCE
1. Glick BR, Pasternak JJ, Patten CL. Molecular biotechnology: Principles and
applications of recombinant DNA. 4th ed. Pasternak JJ, Glick BR, Patten CL, editors.
Washington, D.C., DC: American Society for Microbiology; 2009.
2. Nair AJ. Introduction to biotechnology and genetic engineering. New Delhi, India:
Laxmi Publications; 2008.
3. Satyanarayana U. Biochemistry - E-Book. 5th ed. New Delhi, India: Elsevier; 2017
4. Nagel KM. Principles of recombinant DNA technology. In: Introduction to Biologic
and Biosimilar Product Development and Analysis. Cham: Springer International
Publishing; 2018. p. 1–29.
5. S.A. shinde et.al, recombinant DNA technology and its applications: A review,
international journal of medipharm research p.79-88