To give knowledge about application to r-DNA Technology.

1. Application of R -DNA technology
Prepared by:
Ms. Harshada R. Bafna.
M. Pharm (Quality Assurances)

2.  Recombinant DNA is a form of DNA constructed in the laboratory. It is generated by
transferring selected pieces of DNA from one organism to another.
 r-DNA technology has been exploited in order to provide selective improvements in
various specialties that include crop agriculture, pharmaceutics, gene therapy, vaccine
design and bioremediation.
 The technology used for producing artificial DNA through the combination of different
genetic materials (DNA) from different sources is referred to as Recombinant DNA
Technology. Recombinant DNA technology is popularly known as genetic
engineering.
 The recombinant DNA technology emerged with the discovery of restriction
enzymes in the year 1968 by Swiss microbiologist Werner Arber. 2

3. Application of R -DNAtechnologyin genetic engineering
Recombinant DNA technology has also proven important to the production of vaccines
and protein therapies such as human insulin, interferon and human growth hormone.
It is also used to produce clotting factors for treating hemophilia and in the development
of gene therapy.
The use of recombinant (r-)DNA technology to produce genetically engineered
organisms started in the early 1970s with the pioneering transfer of genes between
bacteria of the same Escherichia coli species.
DNA technology is also used to detect the presence of HIV in a person.
Gene Therapy – It is used as an attempt to correct the gene defects which give rise to
heredity diseases.
Clinical diagnosis – ELISA is an example where the application of recombinant.
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4. Interferon is an antiviral substance, and is the first line of defense against viral attacks.
The term interferon has originated from the interference of this molecule on virus
replication. It was originally discovered in 1957 by Alick Isaacs and Jean Lindemann
and was considered to be a single substance.
It is now known that interferon actually consists of a group of more than twenty
substances with molecular weights between 20,000-30,000 dalton. All the
interferons are proteins in nature and many of them are glycoproteins. They are
broadly categorized into three groups based on their structure and function:
1. Interferon-α (IFN-α)
2. Interferon-β (IFN-β)
3. Interferon-ƴ (IFN-ƴ)
1. Interferons
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5. Production of recombinant interferons:
 The complementary DNA (cDNA) was synthesized from the mRNA of a specific
interferon. This is inserted to a vector (say plasmid) which is introduced into E. coli
or other cells. The interferon can be isolated from the culture medium. This is the
basic mechanism of producing recombinant interferons.
 The production of interferons is relatively less in bacterial hosts, although E. coli
was the first to be used. This is mainly because most interferons are glycoproteins in
nature and bacteria do not possess the machinery for glycosylation of proteins.
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6. Production interferons by yeasts:
The yeast Saccharomyces cerevisiae is more suitable for the production of
recombinant interferons. This is mainly because the yeast possess the
mechanism to carry out glycosylation of proteins, similar to that occurs in
mammalian cells. The DNA sequence coding for specific human interferon
can be attached to the yeast alcohol dehydrogenase gene in a plasmid and
introduced into 4 yeast cells. The yield of interferons is several fold higher
compared to E. coli.
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7. Production of hybrid interferons:
Several attempts have been made to produce hybrid
interferons. This is advantageous since different
interferons with different antiviral activities can be
combined to produce a more efficient interferon. Further,
the glycosylation step can be bypassed, and bacteria can
be used to produce hybrid interferons. The hybrid
interferons are more reactive in performing their function.
The creation of hybrid genes from the genes of IFN-α2 and IFN-α3 is illustrated in Fig. These
genes are digested by restriction endonucleases. The resulting fragments are ligated to generate
hybrid genes. The appropriate hybrid genes can be selected and used for producing hybrid
interferons. E. coli can be employed for this purpose
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8. The Recombivax HB (Merck), a hepatitis B vaccine, is one of the most recent and
significant developments in the field of recombinant DNA technology, that essentially
comprise of highly specific antibodies which act like magic bullets.
It has been duly observed that hepatitis tends to cause a severe acute infection and may
ultimately lead to chronic infection and permanent liver damage. It is essentially caused
by hepatitis B virus (HBV) ; and recognized as an enveloped and double-stranded DNA
virus.
2. Hepatitis B Vaccine
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9. Importantly, the hepatitis B infection may be prevented through a vaccine created
using recombinant DNA technology. However, complete protection can be
accomplished via two vaccinations 1 month apart and a second dose 4 months later an
increased anti-HBs antibody titer value evidently shows successful vaccination.
The following categories of person(s) must be vaccinated :
 All health care staff members
 Patients with renal disease requiring hemodialysis
 Police personnel and other public safety workers
 Family members and sexual partners of those infected with HBV
 Persons who travel frequently and extensively abroad.
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10. 1. Genetic material (DNA) is extracted from the ensuing hepatitis virus. At this stage
the ‘surface proteins’ essentially provoke an immune response.
2. The ‘individual genes’ are adequately analyzed and identified.
3. The ‘specific gene’ which categorically directs production of surface protein is
located carefully.
4. In this most critical steps the gene is removed from the viral DNA and inserted into
the plasmid carefully.
5. The plasmids are accurately inserted into the corresponding yeast cells.
6. Yeast is allowed to grow via fermentation. In this manner the cells reproduce and
generate more quantum of surface protein.
Steps involves in hepatitis-B vaccine preparation:
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11. 7. After a duration of 48 hours the corresponding yeast cells are ruptured to free the
ensuring ‘surface protein’. The resulting mixture is duly processed so as to extract
the purify the surface protein.
8. A large amount of surface protein particles, in its purest form, are obtained which
ultimately provoke an immune response effectively.
9. The resulting surface proteins are adequately mixed with appropriate
preservations together with other ingredients to obtain the vaccine.
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13. Insulin is a hormone produced by β-cells of islets of Langerhans of pancreas. It was discovered by
sir Edward Sharpey Schafer (1916) while studying Islets of Langerhans. Pancreas is a mixed
gland situated transversely across the upper abdomen behind stomach and spleen. Insulin is a
peptide hormone produced by pancreas and is a central regulator of carbohydrates and fat
metabolism in the body.
Structure of Human Insulin:
 Chemically Human insulin is small, simple protein composed of 51 amino acids sequences and has a
molecular weight of 5808 Dalton.
 Insulin hormone is a dimer of a A- chain and a B-chain which are linked together by a disulphide bond.
 Fredrick Sanger et al (1954) gave the first complete description of insulin. Insulin consists of two
polypeptide:
Chain A- 21 amino acids long
Chain B-30 amino acids long Both chains are joined together by disulphide bond between two cysteine
residue
3. Insulin
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14. Insulin produced inside pancreas:
 At first Pancreatic β-cells synthesize pre-pro-
insulin, which is a 109 amino acids long
polypeptide
 Among 109 amino acids, 23 amino acids are
signal molecules which allows the pre-pro-
insulin to pass through cell membrane.
 Entering inside cell, it become 86 amino acids
long pro-insulin. It is still inactive.
 Some Proteolytic enzymes cut and expose the
active site of pro insulin converting it into
active form of insulin of 51 amino acids long.
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15. The basic step in recombinant DNA technology is similar for insulin production
also:
 At first suitable vector (plasmid) is isolated from E. coli and then it is cut open by
restriction endonuclease enzyme.
 The gene of interest (i.e. Insulin coding gene) is isolated from β-cell and inserted in
opened plasmid.
 Plasmid and gene of interest are recombined together by DNA ligase enzyme
 This recombined plasmid is inserted into suitable host cell (i.e. E. coli) and now this
recombined host cell starts producing insulin hormone.
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16. Technique for recombinant insulin
production:
The original technique of insulin synthesis in E.
coli has undergone several changes, for
improving the yield, e.g. addition of signal
peptide, synthesis of A and B chains separately
etc. The procedure employed for the synthesis
of two insulin chains A and B is illustrated in
Fig. 15.1. The genes for insulin A chain and B
chain are separately inserted to the plasmids of
two different E. coli cultures.
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