Abstract

The goal of this experiment Is to study about-Recombinant DNA Technology In

Today's Medicine.

Genetic Engineering

Genetic Engineering plays a very important role, not only in scientific research,
but also in t~e diagnosis and treatment of disease. Recombinant DNA is a tool
in understanding the structure, function, and regulation of genes and their
products.
 0
RECOMBINANT DNA TECHNOLOGY
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Tbe ob]ecUves of Bt:combinont DNA tecbaotagy 1n,1ucte;
- Identifying genes

- Isolating genes

- Modifying genes

- Re-expressing genes In other hosts or organisms

➔ These steps permit scientists and clinicians to:

- Identify new genes and the proteins they encode

- To correct endogenous genetic defects

- To manufacture large quantities of specific gene products such as hormones,

vaccines, and other biologica l agents of medical Interest

➔ Genetic engineering produces proteins that offer advantages over proteins

isolated from other biologica l sources. These advantages include:

- High purity

- High specific activity

- Steady supply

- Batch-to-batch consistency

Steps in Synthesizine a Recombinant Protein

➔ Recombinant technology begins with the isolation of a gene of interest. The

gene is then Inserted Into a vector and cloned, A vector Is a piece of DNA that
is capable of independent growth; commonly used vectors are bacterial
plasmids and viral phages. The gene of Interest (foreign DNA) is integrated into
the plasmid or phage, and this is referred to as recombinant DNA.

➔ Before introducing the vector containing the foreign DNA into host cells to
express the protein, it must be cloned. Cloning Is necessary to produce
numerous copies of the DNA since the Initial supply is inadequate to insert into
host cells.

➔ Once the vector is isolated In large quantities, it can be introduced into the
desired host cells such as mammalian, yeast, or special bacterial cells. The host
cells will then synthesize the foreign protein from the recombinant DNA. When
the cells are grown in vast quantities, the foreign or recombinant protein can
be isolated and purified In large amounts.
➔ Recombinant DNA technology is not only an important tool in scientific
research, but has also resulted in enormous progress in the diagnosis and
treatment of certain diseases and genetic disorders in many areas of medicine.

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Genetic engineering has permitted

Jdent1ficat1gn of mutot1oas:

People may be tested for the presence of mutated proteins that may be
involved in the progression of breast cancer, retino-blastoma, and
neurofibromatosis

Diagnosis of affected and ,au/er states for hereditary diseases:

Tests exist to determine If people are carriers of the cystic fibrosis gene, the
Huntington's disease gene, the Tay-Sachs disease gene, or the Duchenne
muscular dystrophy gene.
Maaalna af human aeaes on cbromosomes:

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Scientists are able to link mutations and disease states to specific sites on
chromosomes.
Transfeufoo genes from one oraankm to oootber:

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People suffering from cystic fibrosis, rheumatoid arthritis, vascular disease, and
certain cancers may now benefit from the progress made in gene therapy.

tsolotfon ond ottecotfoo of genes;

Once gene modification becomes successful, alteration of genes to produce a
more functional protein than the endogenous protein may become possible,
opening up the route of gene therapy.

Performing structure and functfon analyses on proteins:

Researchers may now employ rational drug design to synthesize drug
compounds that will be efficacious and selective in treating disease.

lsolatton oftqrge auanttttes ofoure protein:

Insulin, growth hormone, follicle-stimulating hormone, as w ell as other
proteins, are now available as recombinant products. Physicians will no longer
have to rely on biological products of low purity and specific activity from
Inconsistent batch preparations to treat their patients

Recombinant DNA Technique

CRISPR

CRISPR technology or CRISPR-Cas9 utilizes a protein called Cas9, which acts like a pair of molecular
scissors and can cut DNA. CRISPRs are specialized stretches of DNA and are used in medical
biotechnology as a tool to edit genomes. This allows scientists to alter DNA and modify gene
functions, often called genetic engineering. There are many applications, like correcting genetic
defects, treating diseases, preventing the spread of diseases, improving crops, and more. But the
science of altering genomes has many ethical concerns surrounding it. From the ability to mutate
genes and the unknowns surrounding gene mutation, CRISPR is a controversial area of biomedical
science. Some new studies even show that perhaps CRISPR technology can create tumors and cancer
with DNA deletions that aren’t controlled or precise. Of course, pharmaceutical companies and other
scientific organizations that develop and utilize CRISPR technology are trying to downplay the
concerns and issues, so the reality of the benefits and damage of the technology is
somewhat unknown.

STEM CELL RESEARCH

Biotechnology plays a big part in supporting stem cell research, which supports the exploration of
growing stem cells in a lab setting or in vitro. This could help in situations where patients may be
suffering from a disease or disorder where implanting stem cells could help restore their vitality and
give them a new lease on life. How does it work? Because stem cells can repeatedly divide and
transform into other types of body cells, biotechnologists can learn how to work with their unique
profiles to encourage growth of specific types of cells. Though research is ongoing, it’s reported that
the results show hope for the future of this unique medical approach.
Genetically Engineered Insulin (Humulin)
Insulin is a peptide hormone produced
by beta cells in the pancreas of various
organisms including human beings. It
regulates
the metabolism of carbohydrates an
d fats by promoting the absorption
of glucose from the blood to skeletal
muscles and fat tissue and by causing
fat to be stored rather than used for energy. Insulin also inhibits the
production of glucose by the liver.

Except in the presence of the metabolic disorder diabetes

mellitus and metabolic syndrome, insulin is provided within the body in
a constant proportion to remove excess glucose from the blood, which
otherwise would be toxic. When blood glucose levels fall below a certain
level, the body begins to use stored glucose as an energy source
through glycogenolysis, which breaks down the glycogen stored in the
liver and muscles into glucose, which can then be utilized as an energy
source. As a central metabolic control mechanism, its status is also used
as a control signal to other body systems (such as amino acid uptake by
body cells). In addition, it has several other anabolic effects throughout
the body. When control of insulin levels fails, diabetes
mellitus can result.

Structure:

Insulin is composed of two

different types of peptide
chains. Chain A has 21 amino
acids and Chain B has 30 amino
acids. Both chains contain alpha
helices but no beta strands.
There are 3 conserved disulfide
bridges which help keep the
two chains together. Insulin can
also form dimers in solution due
to the hydrogen bonding between the B chains. The dimers can further
interact to form hexamers due to interaction between hydrophobic
surfaces. This scene highlights the hydrophobic and polar parts of an
insulin monomer at a pH of 7.

A number of insulin variants have been made to favor either the

monomeric or hexameric form. Deletion of the five C terminal residues
of the B chain creates a monomer only form. This portion of the B chain
is involved in hydrogen bonds between the B chain of one monomer and
the A and B chain of another monomer.

Need of Genetically Engineered Insulin:

The original form of the wonder cure for diabetes, these were once the
only type of insulin available, but are now rarely used. Animal insulin
was originally made
from ground-up
animal pancreas
tissue, and then later
was extracted from
healthy animals
(slaughtered pigs &
cows). The
metabolism of cows and pigs was close enough to human metabolism
that their animal insulin also worked well in human bodies. Beef insulin
has 3 differences from human; pork insulin has 1 difference from
human. The use of a mixture of beef and pork insulin was also possible.
It has been shown that human insulin is less immunogenic than animal
insulin. Porcine insulin is most similar to human insulin. The primary
amino acid sequences of bovine and porcine insulin differ from that of
human insulin by three and one amino acid, respectively. This greater
dissimilarity between human and bovine insulin has been postulated to
be the explanation for the greater antigenicity of bovine insulin as
compared with porcine insulin

One of the problems with animal insulin was antibody issues. The
body identifies them and tries to reject them. Pork insulin differs by 1
amino acid and beef insulin by 3 amino acids, so the body's immune
system can sometimes recognize them as foreign. Immunological
complications of insulin therapy have been evident since animal insulin
became available for the treatment of diabetes mellitus in 1922. In
insulin-allergic patients treated with conventional insulin preparations,
the insulin-specific IgE values are often 10- to 20-fold higher than in
patients without allergy. It has been shown that human insulin is less
immunogenic than animal insulin. Porcine
insulin is most similar to human insulin. Cross-
reactivity between human insulin and insulin
of animal origin has been reported. A major
problem is the cross-reactivity that occurs
between anti-insulin antibodies and the
various animal and human insulin preparations
in patients presenting with allergy to animal
insulin.

The usage of animal insulin has so greatly declined in modern

times that they have largely been withdrawn from the market. Newly
diagnosed diabetics are typically given synthesized or Genetically
Engineered human insulin.

What is “Proinsulin”?

Proinsulin is the prohormone precursor to insulin made in the beta

cells of the islets of Langerhans, specialized regions of the pancreas.
Proinsulin is synthesized on
membrane associated
ribosomes found on the rough
endoplasmic reticulum, where it
is folded and its disulfide
bonds are oxidized. It is then
transported to the Golgi
apparatus where it is packaged
into secretory vesicles, and
where it is processed by a series
of proteases to form
mature insulin. Mature insulin has 35 fewer amino acids; 4 are removed
altogether, and the remaining 31 form the C-peptide. The C-peptide is
abstracted from the center of the proinsulin sequence; the two other
ends (the B chain and A chain) remain connected by disulfide bonds.

When insulin was originally purified from bovine or porcine pancreata,

all the proinsulin was not fully removed. [3][4] When some people used
these insulins, the proinsulin may have caused the body to react with a
rash, to resist the insulin, or even to make dents or lumps in the skin at
the place where the insulin was injected. This can be described as
an iatrogenic injury due to slight differences between the proinsulin of
different species. Since the late 1970s, when highly
purified porcine insulin was introduced, and the level of insulin purity
reached 99%, this ceased to be a significant clinical issue. The main
challenge for production of insulin using rDNA techniques was
getting insulin assembled into mature form.

Humulin:

Humulin was the first medication produced using modern genetic

engineering techniques in which actual human DNA is inserted into a
host cell (E. coli in this case). Biosynthetic "human" insulin is now
manufactured for widespread clinical use using genetic engineering
techniques using recombinant DNA technology, which the
manufacturers claim reduces the presence of many impurities, although
there is no clinical evidence to substantiate this claim. Eli
Lilly marketed the first artificial insulin, Humulin, in 1982.

Humulin production method is as follows:

1. DNA coding for A and B polypeptide chains of insulin are

chemically synthesised a in the lab. Sixty three nucleotides are
sequenced to produce A chain of insulin and ninety nucleotide long
DNA designed to produce B chain of insulin, plus terminator codon
is added at the end of each chain sequence. Anti-codon for
methionine is added at the beginning of the sequence to
distinguish humulin from the other bacterial proteins.

2. Chemically synthesized A and B chain DNA sequence are inserted

into one of the marker gene which are present in the plasmid
vector. Genes are inserted into the plasmid with the help of
enzymes known as endonuclease and ligase.

3. The vector plasmids with the insulin gene are then introduced into
the E. coli bacterial cell. These cells are then allowed to replicate
by mitosis, along with the bacterial cell recombinant plasmid also
gets replicated producing the human insulin.

4. A and B polypeptide chains of insulin are then extracted and

purified from the fomenters in the lab. High-Performance Liquid
 Chromatography (HPLC) is used to get 100% pure humulin from
the mixture of proteins.

5. The A and B polypeptide chains of insulin are mixed together and

connected with each other by disulphide bond, forming the
Humulin or synthetic human insulin.

Advantages & Disadvantages of Humulin:

Humulin is the one and only human protein produced in the bacteria
with identical chemical structure to that of the natural human insulin.
Administration of humulin reduces the possibility of antibody production
and inflammatory response
in diabetic patients. Major
difficulty is the extraction of
humulin from a mixture of
host proteins present in the
fermentation broth.

Now days to overcome this

extraction problem synthetic
human insulin are produced
in the yeast cell instead of E. coli using the same procedure. As yeast is
Eukaryotes they secrete the whole humulin molecule with perfect three
dimensional structures, reducing the need for complex and time
consuming purification methods.

Now most of the diabetic patients are treated with synthetic human
insulin. Small group of patients claim that episodes of hyperglycaemic
complications have been increased after shifting from animal origin
insulin to humulin. No study till date shows the difference between the
frequency of hyperglycaemic complications in patient using humulin
(synthetic human insulin) and animal origin insulin.
Gene Therapy
Gene therapy is the therapeutic delivery of nucleic acid polymers into
a patient's cells as a drug to treat disease. Gene therapy is an
experimental technique that uses genes to treat or prevent disease. In
the future, this technique
may allow doctors to treat a
disorder by inserting a gene
into a patient’s cells instead
of using drugs or surgery.
Researchers are testing
several approaches to gene
therapy, including:

 Replacing a mutated
gene that causes
disease with a healthy
copy of the gene.

 Inactivating, or “knocking out,” a mutated gene that is functioning

improperly.

 Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of

diseases (including inherited disorders, some types of cancer, and
certain viral infections), the technique remains risky and is still under
study to make sure that it will be safe and effective. Gene therapy is
currently only being tested for the treatment of diseases that have no
other cures. It should be noted that not all medical procedures that
introduce alterations to a patient's genetic makeup can be considered
gene therapy. Bone marrow transplantation, and organ transplants in
general have been found to introduce foreign DNA into patients. Gene
therapy is defined by the precision of the procedure and the intention of
direct therapeutic effects.

Gene therapy was conceptualized in 1972, by authors who urged

caution before commencing human gene therapy studies.

The first attempt, albeit an unsuccessful one, at gene therapy (as well
as the first case of medical transfer of foreign genes into humans not
counting organ transplantation) was performed by Martin Cline on 10
July 1980. Cline claimed that one of the genes in his patients was active
six months later, though he never published this data or had it
verified and even if he is correct, it's unlikely it produced any significant
beneficial effects treating beta-thalassemia.

The first germ line gene therapy consisted of producing a genetically

engineered embryo in October 1996. The baby was born on July 21,
1997 and was produced by taking a donor's egg with healthy
mitochondria, removing its nuclear DNA and filling it with the nuclear
DNA of the biological mother - a procedure known as cytoplasmic
transfer.

This procedure was referred to sensationally and somewhat inaccurately

in the media as a "three parent baby", though mtDNA is not the primary
human genome and has little effect on an organism's individual
characteristics beyond powering their cells.

Gene therapy is a way to fix a genetic problem at its source. The

polymers are either expressed as proteins, interfere with protein
expression, or possibly correct genetic mutations.

The most common form uses DNA that encodes a functional,

therapeutic gene to replace a mutated gene. The polymer molecule is
packaged within a "vector", which carries the molecule inside cells.

The first commercial gene therapy, Gendicine, was approved in China in

2003 for the treatment of certain cancers. In 2011 Neovasculgen was
registered in Russia as the first-in-class gene-therapy drug for treatment
of peripheral artery disease, including critical limb ischemia. In
2012 Glybera, a treatment for a rare inherited disorder, became the first
treatment to be approved for clinical use in either Europe or the United
States after its endorsement by the European Commission.

ADA deficiency is one form of SCID (severe combined

immunodeficiency), a disorder that affects the immune system. ADA
deficiency is very rare, but very dangerous, because a malfunctioning
immune system leaves the body open to infection from bacteria and
viruses.
The disease is caused by a
mutation in a gene on
chromosome 20. ADA
deficiency is inherited in
an autosomal recessive
manner. The gene codes for
the enzyme adenosine
deaminase (ADA). Without
this enzyme, the body is
unable to break down a toxic
substance called
deoxyadenosine. The toxin
builds up and destroys
infection-fighting immune
cells called T and B
lymphocytes. Because ADA
deficiency affects the
immune system, people who have the disorder are more susceptible to
all kinds of infections, particularly those of the skin, respiratory system,
and gastrointestinal tract. They may also be shorter than normal. Sadly,
most babies who are born with the disorder die within a few months.

Treatments of ADA Deficiency includes:

 bone marrow transplant

 gene therapy

 ADA enzyme in PEG vehicle

On September 14, 1990, the first gene therapy to combat this

disease was performed by Dr. William French Anderson on a four-year-
old girl, Ashanti DeSilva, at the National Institutes of Health,
Bethesda, Maryland, U.S.A.

Conclusion
Biotechnology is the new wonder of science. It is truly multidisciplinary
in nature and it encompasses several disciplines of basic sciences and
engineering. The Science disciplines from which biotechnology draws
heavily are microbiology, chemistry, biochemistry, genetics, molecular
biology, immunology, cell and tissue culture and physiology. On the
engineering side it leans heavily on process chemical and biochemical
engineering since large scale cultivation of microorganisms and cells,
their downstream processing are based on them. It comes to us as a
great blessing...
Biotechnology utilizes the technique called genetic engineering or
recombinant DNA technology where a microorganism is isolated; its
genetic material is cut, manipulated, sealed, again inserted in an
organism and allowed to grow in a suitable environment under
controlled conditions to get the desired product. It looks easy but is a
very tedious job and it takes years for a research to achieve its goal.
Like every other thing, biotechnology too has some harmful
impacts:
1. Genetic engineering is a very vital part of biotechnology and the
cost of transferring genes from one species to another is very
expensive, which requires a huge amount of capital investment.
The cost of producing genetically- modified plants and
animals are sky- rocketing and the duration of return are also
not predictable.
2. Genetic engineering crosses boundaries of reproduction by
crossing genes of species that are completely unrelated; hence
giving rise to hazardous results as well as also increasing the risk
of harming multiple species.
3. When genetic material from certain viruses is used in the
production of transgenic crops, there are chances that these virus
genes will combine with crop genes to produce more destructive
viruses. The consumption of such crops is hazardous to human
health and can cause several life- threatening ailments. It can also
result in cancer, often malignant as well.
4. Biotechnology also poses a number of environmental threats.
Genetically modifies crops often infect monarch butteries and
other insect species.

The applications of biotechnology are so broad, and the advantages so

compelling, that virtually every industry is using this technology.
Developments are underway in areas as diverse as pharmaceuticals,
diagnostics, textiles, aquaculture, forestry, chemicals, household
products, environmental cleanup, food processing and forensics to name
a few. Biotechnology is enabling these industries to make new or better
products, often with greater speed, efficiency and flexibility.
Biotechnology must continue to be carefully regulated so that
the maximum benefits are received with the least risk.

Bibliography
http://en.wikipedia.org/biotechnology
http://en.wikipedia.org/insulin
http://www.genewatch.org/sub-568238
http://en.wikipedia.org/humulin
http://www.biotecharticles.com/Others-Article/Human-

Insulin-and-Recombinant-DNA-Technology-70.html
https://isaaa.org/resources/publications/pocketk/34/default.

asp
http://www.sciencedirect.com/
https://en.wikipedia.org/wiki/Gene_therapy
https://en.wikipedia.org/wiki/Adenosine_deaminase_deficie

ncy
http://www.diabetes.co.uk/insulin/animal-insulin.html
Biology textbook (N.C.E.R.T) Class 12th

Contents

 Introduction
 History
 Biotechnology in Agriculture
 Genetically Modified Crops
 RNA Interference (RNAi)