BIOLOGY

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 INTRODUCTION
Recombinant DNA (rDNA) is a type of artificial DNA that is made by
joining together two or more DNA segment usually originating from
different organisms. More specially, a recombinant DNA molecule is a
vector, (most commonly ESCHERICHIA COLI) into which the desired
DNA regiment has been inserted to enable its cloning in an appropriate
host. This is achieved by using specific enzyme (restriction enzyme) for
cutting the DNA into suitable regiment and then for joining together the
appropriate regiments (ligation). Ligation plays a major role as it is the key
to proper results. In this manner gene may be produced which contain the
coding region from one organism joined to regulatory sequence from
another organism; such a gene is called chimeric gene. Multiple varieties of
proteins are created from recombinant DNA technology and it is used for
medications. Some can be extracts from humans, such as human growth
hormone (rHGH), human insulin, follicle-stimulating hormone (FSH) and
factor VIII. Other proteins, when used as medication, only have
recombinant DNA as a source, such as with erythropoietin. It has brought
many revolutionary changes in the field of medicine and introduced such
methods of treating diseases and delivering the drug. Genetic factors are of
great importance in adult-onset diseases such as atherosclerosis, cancer and
neuro-degenerative diseases. Advance in recombinant DNA technology has
made it almost easy to identify those persons who are at high-risk of
acquiring some of these diseases. Thus recombinant DNA technology has
lead to development in predictive medicine.

Application of Recombinant DNA

Technology
Recombinant DNA technology has made it possible to treat many diseases by
replacing damaged and diseased genes in the body with new genes.It has brought
revolutionary changes in the field of medicine and introduced such methods of treating
diseases and delivering drugs that were once just imaginary.

Applications of recombinant DNA technology

in medicine are:
(1) Diagnosis of Genetic Diseases

(2) DNA Typing (DNA Fingerprinting)

(3) Gene Therapy

(4) Recombinant DNA Technology in the Synthesis of Human Insulin

(5) Vaccine.

(6) Diagnosis of infection with HIV

(7) Human Growth Hormone

(8) Interferons

(9) Antibiotics

Diagnosis of Genetic Diseases:

Recessive mutant phenotypes that follow single-gene inheritance are
responsible for more than 500 human genetic diseases. A homozygous
person resulting from a marriage between two heterozygotes for the
same recessive allele will be affected by the disease. Cells derived from the
fetus can be screened at an early enough stage to allow the option of
abortion of afflicted fetuses. Many of the enzymes or other proteins that are
altered or missing in genetic diseases are now known.

Amniocentesis
To detect such genetic defects, fetal cells can be taken from the amniotic fluid,
separated from other components, and cultured to allow the analysis of chromosomes,
proteins, enzymatic reactions, and other biochemical properties. This
process, amniocentesis , can identify a number of known disorders.

Chorionic villus sampling (CVS)

It is a related technique in which a small sample of cells from the placenta is aspirated
out with a long syringe. CVS can be performed earlier in the pregnancy than
can amniocentesis, which must await the development of a large enough volume of
amniotic fluid.
Testing physiological properties or enzymatic activity in cultured fetal cells
limits the screening procedure to those disorders that affect characters or
proteins expressed in the cultured cells. However, recombinant
DNA technology improves the screening for genetic diseases in utero,
because the DNA can be analyzed directly. In principle, the gene being
tested could be cloned and its sequence compared with that of a cloned
normal gene. However, this procedure would be lengthy and impractical, so
shortcuts have to be devised to allow more rapid screening. Several of the
useful techniques that have been developed for this purpose are explained
in this section.

Alteration of restriction site by mutation

Sickle-cell anemia is a genetic disease that is caused by a well-
characterized mutational alteration. The disease results from an altered
hemoglobin, in which the amino acid valine substitutes for glutamic acid at
position 6 in the β-globin chain. The GAG-to-GTG change eliminates a
cleavage site for the restriction enzyme  MstII, which cuts the sequence
CCTNAGG (in which N represents any of the four bases). The change from
CCTGAGG to CCTGTGG can thus be recognized by Southern analysis
with the use of labeled β-globin cDNA as a probe, because
the DNA derived from persons with sickle-cell disease lacks one fragment
contained in the DNA of normal persons and contains a large (uncleaved)
fragment not seen in normal DNA.

Probing for altered sequences

When a genetic disorder can be attributed to a change in a
specific nucleotide, synthetic oligonucleotide probes can identify that
change. The best example is alpha1-antitrypsin deficiency, which leads to a
greatly increased probability of developing pulmonary emphysema. The
condition results from a single base change at a known nucleotide
position. A synthetic oligonucleotide probe that contains the wild-type
sequence in the relevant region of the gene can be used in a Southern
blot analysis to determine whether the DNA contains the wild-type or
the mutant sequence. At higher temperatures, a complementary sequence
will hybridize, whereas a sequence containing even a single mismatched
base will not.

PCR Tests
PCR test, also called a molecular test, detects genetic material of the virus using a lab
technique called polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) is used to make millions of copies of a target

piece of DNA. It is an indispensable tool in modern molecular biology and has
transformed scientific research and diagnostic medicine.
Genetic disorders are caused by mutations that range from simple changes in
the base sequence of the DNA double helix through to changes in large DNA
sequences and even whole chromosomes. PCR helps focus on the actual
segment of DNA that is of interest, rather than the whole genome. From a small
genetic sample, the genotypes can now be determined, and as a result, many
genetic disorders can be detected, diagnosed and monitored.
Pathogenic microorganisms, including some
viruses, bacteria, parasites and fungi, cause infectious diseases and can be
identified using PCR, aiding efficient diagnosis and treatment.
DNA Typing (DNA Fingerprinting):
DNA fingerprinting, also called DNA typing, DNA profiling, genetic
fingerprinting, genotyping, or identity testing, in genetics, method of isolating and
identifying variable elements within the base-pair sequence
of DNA (deoxyribonucleic acid).

1. DNA typing is a technique in which biological samples help in solving forensic

problems. This technique is used to establish that whether the suspected person has
committed a crime or not.

2. For DNA typing, biological samples like blood, skin, semen or hair are collected
from the site of crime. A portion of a sample is analyzed to confirm that there is
sufficient amount of intact DNA (un-degraded) for the further analysis.

3. If there is sufficient amount of intact DNA, it is digested with a restriction enzyme,

and the fragments are separated on an agarose gel and transferred by blotting onto a
nylon membrane.

4. This nylon membrane is then hybridized sequentially with four or five separate
radio-labeled probes that each recognizes a distinct DNA sequence. After each
hybridization reaction, the bands where the probe has bound to the digested. DNA
sample are visualized by autoradiography and the banding pattern for each sample is
noted.

5. Before the next probe is used, the first probe is completely removed from the
membrane. Since each hybridization and autoradiography step can take up to 10 to 14
days, the entire process may take many weeks and even several months.

6. A commonly used set of probes for this type of analysis consists of human mini-
satellite DNAs. These sequences occur throughout the human genome and consist of
tandemly repeated sequence.

7. The human mini-satellites DNA sequences are highly variable, and the chance of
finding two individuals in the population with the same DNA fingerprint is about one
in 105 to 108.
8. In addition to forensic applications of DNA typing, this technique may be used to
determine the paternity of a child. The DNA fingerprint banding pattern of the child
should be a composite of the patterns of its mother and father. Apart from these two
fields, it is also used in determining the frequency of a particular gene in a population
which gives rise to diversity. In case of the change in gene frequency or genetic drift,
Fingerprinting can be used to trace the role of this change in evolution.

9. In case when the amount of forensic sample collected is quite less but the DNA is
un-degraded then in that case, it is possible to amplify small portions of DNA of the
mini-satellite DNA by PCR.

In DNA fingerprinting, fragments of DNA are separated on a gel using a technique called electrophoresis.

Gene Therapy:

Gene therapy is a technique which involves the replacement of defective genes with

healthy ones in order to treat genetic disorders. It is an artificial method that introduces
DNA into the cells of the human body. The first gene therapy was successfully
accomplished in the year 1989.

The cell with the defective gene is injected with a normal gene which helps in the
normal functioning of the cell. This technique is employed mainly to fight against the
diseases in the human body and also to treat genetic disorders. The damaged proteins
are replaced in the cell by the insertion of DNA into that cell.

Types of Gene Therapy

Basically, there are two types of gene therapy

 Somatic Gene Therapy

This type usually occurs in the somatic cells of human body. This is related to a single
person and the only person who has the damaged cells will be replaced with healthy
cells. In this method, therapeutic genes are transferred into the somatic cells or the
stem cells of the human body. This technique is considered as the best and safest
method of gene therapy.

 Germline Gene Therapy

It occurs in the germline cells of the human body. Generally, this method is adopted to
treat the genetic, disease causing-variations of genes which are passed from the
parents to their children. The process involves introducing a healthy DNA into the
cells responsible for producing reproductive cells, eggs or sperms. Germline gene
therapy is not legal in many places as the risks outweigh the rewards.

Application of Gene Therapy

1. It is used in the replacement of genes that cause medical ill-health
2. The method generally destroys the problem causing genes
3. It helps the body to fight against diseases by adding genes to the human body
4. This method is employed to treat diseases such as cancer, ADA deficiency,
cystic fibrosis, etc.

Recombinant DNA Technology in the Synthesis of

Human Insulin:

Insulin is prepared by recombinant DNA (rDNA) technology for medicinal purposes

on a large scale. It was first produced in 1983 by an American Biotech company. The
trademark name is Humulin and it is licensed to Eli Lilly, the company which
manufactured it for the first time.
Genes, which code for functional A and B peptides of insulin, were inserted in the
plasmids of non-pathogenic E.coli strains. Both the chains are produced separately and
joined afterwards by disulphide linkages.
Insulin has been used for many years to treat diabetes. Diabetes is well managed by
taking insulin. Earlier insulin was extracted from the pancreas of killed cattle and pigs.
It had shortcomings. It used to stimulate allergic reactions and other immune
responses due to its foreign origin in some people. Another challenge was to cater to
the ever increasing demand and large scale production.
To overcome this, the production of insulin by recombinant DNA technology was
done and it has proved to be very beneficial. In fact, it was the first recombinant
medicine to be used in the USA.
Biosynthetic insulin produced by rDNA technology is purer than animal insulin. It
reduces the formation of antibodies against it.

Insulin is a protein hormone synthesised by the 𝛃 cells of the pancreas. It is

produced as a prohormone, i.e. Preproinsulin. The signal peptide cleaves to
give proinsulin. Proinsulin has to be further processed to become functional.
Proinsulin contains another peptide chain known as ‘C’ peptide in addition to
‘A’ and ‘B’ peptides, which are required for its functionality. From proinsulin,
the C peptide is removed at the time of maturation.
The genetically engineered insulin does not contain the C peptide.
VACCINE:

Vaccine is a biological substance that is prepared from the suspension of weak or dead
pathogenic cells. It is injected in the body to enhance the production of antibodies
against a particular antigen. Recombinant DNA technology has made it easier for
scientists to develop vaccines by cloning the gene used for protective antigen protein.
Viral vaccines are mostly developed from this technique, for example Herpes,
Influenza, Hepatitis, Foot and Mouth disease.

Hepatitis B Vaccine:
Hepatitis B (HepB) is a major public health problem worldwide. Approximately 30%
of the world’s population , have hepatitis B virus (HBV) infection. HBV is second
only to tobacco as a known human carcinogen. HepB vaccine is effective in
preventing HBV infections when it is given either before exposure or shortly after
exposure. At least 85-90% of HBV- associated deaths are vaccine-preventable.

Hepatitis B vaccine (rDNA) is a preparation of hepatitis B surface antigen (HBsAg), a

component protein of hepatitis B virus; the antigen may be adsorbed on a mineral
carrier such as aluminum hydroxide or hydrated aluminum phosphate. The antigen is
obtained by recombinant DNA technology.

Hepatitis B vaccine (rDNA) is produced by the expression of the viral gene coding for
HBsAg in yeast (Saccharomyces cerevisiae) or mammalian cells (Chinese hamster
ovary (CHO) cells or other suitable cell lines), purification of the resulting HBsAg and
the rendering of this antigen into an immunogenic preparation. The suitability and
safety of the cells are approved by the competent authority.

The antigen must comply following standard quality criteria: Total protein value,
Antigen content and identification using ELISA and SDS page electrophoresis,
Antigenic purity by reaction, Composition in terms of the content of proteins, lipids,
nucleic acids and carbohydrates, Host-cell- and vector-derived DNA presence,
Caesium content (used for purity of antigen) and lastly sterility.

Diagnosis of infection with HIV:

Each of the three widely used methods for diagnosing HIV infection has been
developed using recombinant DNA. The antibody test (ELISA or western blot) uses a
recombinant HIV protein to test for the presence of antibodies that the body has
produced in response to an HIV infection. The DNA test looks for the presence of
HIV genetic material using reverse transcriptase polymerase chain reaction (RTPCR).
Development of the RT-PCR test was made possible by the molecular cloning and
sequence analysis of HIV genomes.

Human Growth Hormones

Human growth hormone is a polypeptide hormone. It is responsible for growth,
reproduction of the cells and regeneration in humans as well as animals. It is secreted
by somatotroph cells present in the pituitary glands.

Before recombinant HGH became available, HGH for therapeutic use was obtained
from pituitary glands of cadavers. This unsafe practice led to some patients developing
CreutzfeldtJacob disease. Recombinant HGH eliminated this problem, and is now
used therapeutically. It has also been misused as a performance enhancing drug by
athletes and others. In recent days biotechnology has helped scientists to produce
many growth hormones. The dwarfism disease is successfully treated with this
hormone .

Interferon
A glycoprotein that has the ability to block the multiplication or division of viruses in
the cells or nearby cells are called interferons. It can be used to treat cancer like hairy
cell leukemia. Recombinant DNA technology produces this protein using E.coli.
Interferon alpha is used to treat lymphoma and myelogenous leukemia.

Antibiotics
Antibiotics are the chemical substances that are used against bacterial infections.
They can be produced by microorganisms as well as in the laboratory. They have the
ability to destroy microbes that cause harmful infections in the body. Alexander
Fleming discovered Penicillin in 1928 using recombinant DNA technology. Other
biotechnological techniques are also being used to produce antibiotics.

Conclusion

Recombinant proteins are widely used as reagents in

laboratory experiments and to generate antibody probes for
 examining protein synthesis within cells and organisms.
Thus the use of this advanced technology, Recombinant
DNA technology produces variety of products which are
used for medical purposes. It is a challenging field, and play
a key role in preventing genetic diseases, producing targeted
medicines, and providing patients with less toxic
pharmaceuticals. Hence it is gaining tremendous
significance in the field of medicine today.