recent advances in recombinant dna technology

81 Pages·2015·1.54 MB·English

RECENT ADVANCES IN n
RECOMBINANT DNA TECHNOLOGY
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Project Report
For Elective Subject
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Submitted to the

Hemchandracharya North Guja.rat University,
Patan t
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IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR
THE DEGREE OF BACHELOR OF PHARMACY
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. SUBMITTED BY

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ALPESH P. PATEL

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CERTIFICATE

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This is to certify that the project report for elective subject
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entitled “Recent Advances in Recombinant DNA Technology”
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represents the bonafide work of Alpesh P. Patel carried ocut under my
guidance and supervision at the Department of Pharmacology, S. K.
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Patel College of Pharmaceutical Education and Research during
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academic year 2004-2005. He has collected the literature and completed
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the project work very sincerely and methodically. This work is up to my
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satisfaction.
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G u i d e : Principal (I/C):

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Mr. Rashwin J. Patel Dr. N. J. Patel

M.S. (Pharmn.) M.Pharm., Ph. D.

Lecturer Professor and Head,

D e p a r t m e n t o f P h a r m a c o l o g y , Department of Pharmacology
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S . K. Patel College of Pharm. S. K. Patel College of Pharm.
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E d u. & Research, Ganpat Edu. & Research, Ganpat
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Vidyanagar, Kherva-382711, Vidyanagar, Kherva-382711,

Dist: Mehsana. Dist: Mehsana.

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g Date:

Place:
ACKNOWLEDGEMENT
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First, I would like to express my salutation to God for giving me ithe
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strength,confidence and moral boost tosuccesful completion of this project.
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“You want to do the right thing &
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you want to do it for right reason
but if you don’t have right guid.ance
you can never hit the right ttarget.”
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Numerous people have been instrumental in enabling me to give a concrete
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shape to my thesis However, I must mention the names of few people who
have made catalytic impact on the development of this thesis.
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First and foremost, I would like to acknowledge the continuous encouragement
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and help extended to me by Mr.R . J. Patel for preparing this thesis. He has
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been my sole guide & philosopher throughout the period of my work. His
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extensive knowledge of the subject and the way he imparted the same to me
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has enabled me to develop the thesis in a cohesive manner and kindled within
me a passion for the subject.
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I also express my profound gratitude to Dr.N .J.PATEL our Principal, who has
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been a constant source of inspiration to steer me forward throughout the four
n years of my study. I wish to thank prof.Dr.M.M.PATEL for providing the
necessary facilities and constant inspiration through out my studies of
g B.Pharm. I take this opportunity to place on record my indebt ness to Mr.
J.L.PATEL for helping me when needed.

I am also thankful to my parents who lead me from darkness to light ,ignorance
to enlighten, confusion to clarity throughout my life
“Your soorow get divided and
Yourn happiness get multiplied
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Only with your friends.”
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A friend is a person who understand your filling, emotion, and help you to be what
you to be. I am specially thankful to my friends Rakesh , Jatin , Dacsharath , Pavan
,Nilesh & Bipin .
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I am also thankful to Bhargav, Jigar, Aashish ,Vijay, Ankit,Tarang,chintan,
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Mihir,Bhavesh ,Nilesh, Manish, Bhaumik , t& Tushar. I am thankful to my
classmates for their tremendous co-operatione throughout my studies.

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ALPESH P.PATEL
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INDEX

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SR.NO. CHAPTER PAGE
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1. INTRODUCTION 1
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2. RECOMBINANT DNA PREPARATION 4
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2.1 ISOLATION OF DNA FRAGMENTS 4
2.2 RESTRICTION ENDO NUCLEASES ENZYME 6
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2.3 DNA LIGASE - REJOINING OF DNA 8
2.4 CLONING VECTORS 9
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2.5 GENE EXPRESSION 14
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3. BIOMEDICAL IMPORTANCE 17
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4. MEDICINAL APPLICATION OF RECOMBINANT DNA 18
TECHNOLOGY n
4.1 HUMAN INSULIN 21
4.2 ANTI HEMOPHILbIC FACTOR 33
4.3 HUMAN GROWTH HORMONE 38
4.4 INTERLEUKINS 40
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4.5 INTElRFERON 42
4.6 HEfPATITIS –B VACCINE 46
4.7n ERYTHROPOETIN 47
4.8 TISSUE PLASMINOGEN ACTIVATOR (TPA) 49
i4.9 GRANULOCYTE COLONY STIMULATING FACTOR & 51
. GRANULOCYTE MACROPHAGE COLONY STIMULATING
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4.10 MONOCLONAL ANTIBODY 53
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5. RECENT ADVANCES IN RECOMBINANT DNA TECHNOLOGY 55
6. FUTURE PERSPECTIVE 59
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7. RECOMBINANT DNA PRODUCTS-UNDER CLINICAL TRIALS 60
8. STATUS OF BIOTECHNOLOGY IN THE WORLD AND IN INDIA 64
9. REFERENCES 76
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1. INTRODUCTION
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Biotechnology means the use of organisms or enzymes for production of useful
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materials . It‟s main application is in
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 Agriculture field
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 Food Technology
 Environmental Science
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 Major Impacts in area of Drugs and Vaccines

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The techniques utilized to produce biotechnological products are as follows:
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 Recombinant DNA technology
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 Monoclonal antibody technology
 Polymerase chain reaction
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 Gene therapy
 Nucleotide blockade or antisense nucleic acids
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 Peptide technology

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RECOMBINANT DNA TECHNOLOGY:
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The biochemical approach to understanding a complex biological process is to
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isolate and study the individual Components In vitro with goal of understanding the
overall process in whole organism. The most fertile source is DNA.
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Decades of advances in genetics, Biochemistry, Cell biology And Physical chemistry
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came together in laboratories of Paul Berg, Herbert Boyer and Stanley Cohen to
yield techniques for locating, isolating, preparing and studying small segments of
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DNA derived from larger chromosomes, which is known as DNA cloning.

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Revolutionary as it is, this technology is grounded in most fundamental biological and
biochemical principles. This techniques of DNA cloning made available by advances
in biotechnology that have provided medicinal agents fall into two broad areas . First
is r-DNA technology. which involve specific transplantation of functional unit of DNA
from one organism to another , forming a r-DNA.

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RECENT ADVANCES IN RECOMBINANT DNA TECHNOLOGY
The ability to specifically transfer to specific pieces of DNA and control its expression
was new and it‟s potential for revolutionizing the cell biology and biotechnology. The
potential result was shown in 1978, the human insulin, the first pharmaceutical
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product, produced by genetic engineering was available.

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u Fig.1: Biotechnology today

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This technology involves Specific transplantation of a functional unit of DNA (a gene)
from one organism to another, and form “Recombinant” organisms in order to
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achieve the aim and benefits of general recombination. It is a novel technique for
controlled recombination. It is a DNA that constitutes gene, allowing cells to
reproduces and maintain life.

DNA also plays major roll in production of proteins require for cellular maintenance
and function. The ability to selectively hydrolyze a population a population of DNA
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RECENT ADVANCES IN RECOMBINANT DNA TECHNOLOGY
molecules promoted a technique for joining two different DNA molecules. The two
different DNA molecules can be joined by using these two enzymes.
 Restriction endonuclease. n
 DNA Ligases.
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MILESTONES IN BIOTECHNOLOGY& RECOMBINANT DNA TECHNOLO.GY:
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1950- Discovery of interferon.
1972 - First recombinant DNA molecules generated a
1973 - Use of Plasmid vectors for gene cloning
1978 - Human genomic library constructed .
1979 - Insulin synthesized, First human viral antigetn
(Hepatitis-B) cloned e
1982 - Isolation, cloning, & characterization of human
Cancer gene n
1985 - Development of the Polymerase chain reaction
technique
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1988 - Development of gene gun.
1990 - Human genome project launched.
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1991 - First test of lgene therapy on human cancer patients
1994 - Fully humfan monoclonal antibodies produced in
genentically engineered mice
1997 - Cloning of sheep “ dolly” by E.Wilmat.
200i0 - Human genome mapped.
.2002 - Birth of first human clone “eve”.
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RECENT ADVANCES IN RECOMBINANT DNA TECHNOLOGY
2. RECOMBINANT DNA PREPARATION
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The various general steps involved in the preparation of Recombinant DNA can be
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briefly Summarized As.
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(1) Selection of DNA of interest
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(foreign on target or passenger DNA)
(2) A cloning vector (to carry inserted pieces of target DNA)
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(3) DNA ligases
(which joins pieces of two different DNA molecules together)
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(4) Restriction endonucleases enzymes
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(which makes internal cuts at specifics sites on DNA )
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(5) Host (to replicate the vector containing foreign
DNA: Prokaryotic / Eucaryotic cell)
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(6) Screening test for recombinants produced by insertion of
DNA.
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2.1: ISOLATION OF DNA FRAGMENTS:
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The first step in Recombinant DNA technology is selection of DNA of interest. To
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isolate the DNA of interest, GEL ELECTROPHORESIS method is used. Agarose or
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polyacrylamide gels usually are used to separate DNA fragments electrophoretiically.
Charged molecules are placed in electrical field and allowed to migrate toward the
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positive and negative poles. The molecules separate because they move at different
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rates due to their differences in charge and size.
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In practice, the fragment mixture is usually placed in wells molded with in a sheet of
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gel. The gel concentration varies with the size of DNA fragments to be separated.
Usually 1 to 3 % Agarose gels or 3 to 20 % Polyacrylamide gels are used. When an
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electrical field is generated in gel , the fragment move toward electrode. A simple
DNA molecule might yield only a few bands. The band or section containing the
desired fragment is located with the southern blotting technique and removed. The
location of pure fragment is determined by southern blotting and is extracted from the
gel.

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RECENT ADVANCES IN RECOMBINANT DNA TECHNOLOGY