Biotechnology

Biotechnology is defined as application of scientific and engineering

principles of bio agents, system or organism for the production of useful
material and service industries for the benefit of living organisms including
human as well as to the universe such as baking, wine, beer production, dairy
products and single cell protein such as pruteen and mycoprotein, biogas,
biofuel, sewage treatment, biodegradation, bioremediation etc.
Biotechnology is the technologies applied to biology, molecular biology,
genetics, and many other subfields of biology. Biotechnology utilizes cellular
and biomolecular processes to create technologies and products that help
improve our lives and the nature. 
Depending on the tools and applications, it often overlaps with the
(related) fields of bio-engineering and other fields related to health sciences and
environment. The advent of biotechnology has expanded into diverse sciences
like immunology, virology and other subjects like health, agriculture, cell
biology, plant physiology, seed technology, etc.
In 2012, Kafarski develop a color code to differentiate the main area of
biotechnology.
1. Red biotechnology is the use of biotechnology in the medical
and pharmaceutical industries, and health preservation.
2. White biotechnology, also known as industrial biotechnology, is
biotechnology applied to industrial processes.
3. Green biotechnology is biotechnology applied to agricultural processes.
4. Blue biotechnology is based on the exploitation of sea resources to create
products and industrial applications.
5. Yellow biotechnology refers to the use of biotechnology in improvement in
food production and nutrition.
6. Gray biotechnology is dedicated to environmental applications and focused
on how to make pollution free environment.
7. Gold biotechnology refers to bioinformatics is an interdisciplinary field that
addresses biological problems using computational techniques.
8. Brown biotechnology is related to the management of arid lands
and deserts.
9. Violet biotechnology is related to law, ethical and philosophical issues
around biotechnology.
10. Dark biotechnology is the color associated with bioterrorism or biological
weapons and biowarfare which uses microorganisms, and toxins to cause
diseases and death in humans, livestock and crops.
Scope of Biotechnology
Biotechnology is a multidisciplinary pursuit that has emerged as a
demanding industry during the recent past. Besides being a branch of advance
biological sciences, it has attracted many multinational and international
companies including those are concerned with:
 The production of pharmaceutical products for the cure or control of many
human diseases such as antibiotics, vaccines, life-saving drugs and gene
therapy and tissue replacement.
 Improvement of clinical testing and diagnostic tools.
 Production of novel varieties of crop plants for product and yield production
and animals for better milk production and better breeding.

Plasmids
Recombinant DNA technology is an essential method for bringing about
desirable changes in the DNA of organisms. DNA fragments can be deleted or
added in other organisms, thereby creating variations not normally found in
nature. It can be used in cross-species genetics, in the curing of diseases,
making multiple copies of segments of genes, etc. which can be used in
industrial and medical technology.
A plasmid is a small DNA molecule within a cell that is physically
separated from chromosomal DNA and can replicate independently. They are
most commonly found as small circular, double-stranded DNA molecules in
bacteria; however, plasmids are sometimes present in archaea and eukaryotic
organisms. Plasmids can usually carry at least one gene which is beneficial for
their host. Plasmids base pair ranges from few thousands to hundred kilobases.
Although plasmids carry its separate genes from their host but are not
considered to be independent life. So, plasmids are considered the extra
chromosomal DNA that are stably inherited.
Plasmids usually carry one gene while in some cases a few genes.
Plasmids are replicated by the same machinery that replicates the bacterial
chromosome. Some plasmids are copied at about the same rate as the
chromosome, so a single cell is apt to have only a single copy of the plasmid. E.
coli and its plasmid considered the most variable type of host-vector system
known for DNA cloning.
Functions of Plasmids
Plasmids may contain genes that enhance the survival of an organism,
either by killing other organisms or by defending the host cell by producing
toxins. Some plasmids facilitate the process of replication in bacteria. Since
plasmids are so small, they usually only contain a few genes with a specific
function (as opposed to a large amount of noncoding DNA). Multiple plasmids
can coexist in the same cell, each with different functions.
Genes on the plasmids with high number of copies usually express at
high level. In nature, this gene often encodes proteins (various enzymes) that
protect the bacteria from antibiotics and other environmental factors.
Plasmids enter the bacterial cell with very ease. This may occur in nature
and may account for the rapid spread of antibiotic resistance in hospital and
elsewhere. Plasmids can be deliberately induced into the bacteria in the
laboratory thus transferring the cells, the incoming genes and their functions.
Plasmids can be from general to specific in function in the bacterial cell.
Conjugative Plasmid
Bacteria reproduce by sexual conjugation, which is the transfer of genetic
material from one bacterial cell to another, either through direct contact or a
bridge between the two cells.
Some plasmids contain genes called transfer genes that facilitate the
beginning of conjugation.
Non-Conjugative Plasmid
Non-conjugative plasmids cannot start the conjugation process, and they
can only be transferred through sexual conjugation with the help of conjugative
plasmids.
Types of Plasmids
There are five main types of plasmids: fertility F-plasmids, resistance plasmids,
virulence plasmids, degradative plasmids, and Col plasmids.
1. Fertility F-plasmids
Fertility plasmids, also known as F-plasmids, contain transfer genes that
allow genes to be transferred from one bacterium to another through
conjugation. These make up the broad category of conjugative plasmids.
F-plasmids are episomes, which are plasmids that can be inserted into
chromosomal DNA. Bacteria that have the F-plasmid are known as F positive
(F+), and bacteria without it are F negative (F –). When an F+ bacterium
conjugates with an F– bacterium, two F+ bacterium result. There can only be one
F-plasmid in each bacterium.
2. Resistance Plasmids
Resistance or R plasmids contain genes that help a bacterial cell defend
against environmental factors such as poisons or antibiotics. Some resistance
plasmids can transfer themselves through conjugation. When this happens, a
strain of bacteria can become resistant to antibiotics.
According to NPR (FDA), overuse of antibiotics to treat other infections,
like urinary tract infections, may lead to the proliferation of drug-resistant
strains.
3. Virulence Plasmids
 When a virulence plasmid is inside a bacterium, it turns that bacterium
into a pathogen, which is an agent of disease. Bacteria that causes disease can
be easily spread and replicated among affected individuals. The
bacterium Escherichia coli (E. coli) has several virulence plasmids. E. coli is
found naturally in the human gut and in other animals, but certain strains of E.
coli can cause severe diarrhea and vomiting. Some other bacterial species such
as Salmonella enterica also contains virulence plasmids and cause severe
infection in body.
4. Degradative Plasmids
Degradative plasmids help the host bacterium to digest compounds that
are not commonly found in nature, such as camphor, xylene, toluene, and
salicylic acid. These plasmids contain genes for special enzymes that break
down specific compounds. Degradative plasmids are conjugative.
5. Col Plasmids
Col plasmids contain genes that make bacteriocins (also known as
colicins), which are proteins that kill other bacteria and thus defend the host
bacterium. Bacteriocins are found in many types of bacteria particularly E. coli.
Transposons
Transposons ("jumping genes") are small pieces of DNA that encode
enzymes that enable the transposon to move from one DNA location to another,
either on the same molecule of DNA or on a different molecule. A transposable
element (TE, transposon, or jumping gene) is a DNA sequence that can change
its position within a genome, sometimes creating or reversing mutations and
altering the cell's genetic identity and genome size. Its size ranges from 1-
40kbp.

DNA Sequencing
DNA sequencing is the process of determining the nucleic acid sequence
– the order of nucleotides in DNA. It includes any method or technology that is
used to determine the order of the four bases: adenine, guanine, cytosine, and
thymine. The advent of rapid DNA sequencing methods has greatly accelerated
biological and medical research and discovery.
Sequencing DNA means determining the order of the four chemical
building blocks - called "bases" - that make up the DNA molecule. The
sequence tells scientists the kind of genetic information that is carried in a
particular DNA segment. For example, scientists can use sequence information
to determine which stretches of DNA contain genes and which stretches carry
regulatory instructions, turning genes on or off.
 Knowledge of DNA sequences has become indispensable for basic
biological research, and in numerous applied fields such as medical diagnosis,
biotechnology, forensic biology, virology and biological systematics.
The rapid speed of sequencing attained with modern DNA sequencing
technology has been instrumental in the sequencing of complete DNA
sequences, or genomes, of numerous types and species of life, including the
human genome and other complete DNA sequences of many microbial species.
The first DNA sequences were obtained in the 1970 by Ray Wu
academic researchers using laborious methods based on rectography (two-
dimensional chromatography). Following the development of fluorescence-
based sequencing methods with a DNA sequencer, DNA sequencing has
become easier and orders of magnitude faster.
Later, the first full DNA genome to be sequenced was that of in 1977.
Medical Research Council scientists deciphered the complete DNA sequence of
the Epstein-Barr virus in 1984.
DNA sequencing may be used to determine the sequence of individual
genes, larger genetic regions (i.e. clusters of genes or operons), full
chromosomes, or entire genomes of any organism.
The primary purpose of sequencing a genome is to obtain information of
medicinal value for future pursuits. Genomic sequences can provide information
on genetic variants that can lead through various diseases or can increase the
risk of disease development even in an asymptomatic people.
DNA sequencing is also the most efficient way to indirectly sequence
RNA or proteins (via their open reading frames). In fact, DNA sequencing has
become a key technology in many areas of biology and other sciences such as
medicine, forensics, anthropology, metagenomics, ecology, epidemiology and
microbiology.
RNA sequencing was one of the earliest forms of nucleotide sequencing.
The major landmark of RNA sequencing is the sequence of the first complete
gene and the complete genome of Bacteriophage identified and published by
Walter Fiers in 1992.
DNA sequencing shows the cost to sequence a genome diverging
drastically around 2008, falling from almost $10 million to close to $1,000
today. The first human genome took $2.7 billion and almost 15 years to
complete. Now, according to Cowen analyst Doug Schenkel, genome
sequencing and analysis cost around $1,400.
1. Sanger Sequencing Method
Sanger sequencing, also known as the “chain termination method”, is a
method for determining the nucleotide sequence of DNA. The method was
developed by two times Nobel Laureate Frederick Sanger and his colleagues in
1977, hence the name the Sanger Sequence.  It was the most widely used
sequencing method for approximately 40 years. More recently, higher volume
Sanger sequencing has been replaced by "Next-Gen" sequencing methods,
especially for large-scale, automated genome analyses. However, the Sanger
method remains in wide use, for smaller-scale projects, and for validation of
Next-Gen results.
Sanger sequencing is the process of selective incorporation of chain-
terminating dideoxynucleotides by DNA polymerase during in vitro DNA
replication; it is the most widely used method for the detection of SNVs.
The DNA sample is divided into four separate sequencing reactions,
containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and
dTTP) and the DNA polymerase.  Its ingredients are similar to those needed
for DNA replication in an organism, or for polymerase chain reaction (PCR),
which copies DNA in vitro.

2. Next Generation Sequencing

NGS method uses array-based sequencing which combines the techniques
developed in Sanger sequencing to process millions of reactions in parallel,
resulting in very high speed and throughput at a reduced cost.
NGS is also known as massively parallel and deep sequencing because
millions of fragments can be sequenced simultaneously per run. It describes a
DNA sequencing technology which has revolutionized genomic research.
Using NGS, an entire human genome can be sequenced within a single
day. NGS enables researchers to perform a wide variety of applications and
study biological systems at a level never before possible.
Next-generation sequencing refers to novel techniques of DNA
sequencing directly from DNA fragments without the need for cloning in
vectors, allowing the generation of enormous amounts of sequence data at high
speed and low cost from a single run. 
Next generation sequencing offers fast turnaround time and takes only
about 4 hours to complete a run. Now a days highly advanced methods can be
used to obtain DNA sequences.
1. Shotgun sequencing
Shotgun sequencing is a sequencing method designed for analysis of DNA
sequences longer than 1000 bp, up to and including entire chromosomes. This
method requires the target DNA to be broken into random fragments.
2. Bridge PCR
In this, fragments are amplified upon primers attached to a solid surface and
form DNA colonies or DNA clusters.
3. Polony sequencing
It was used to sequence a full E. coli genome in 2005. It combined an in
vitro paired-tag library with emulsion PCR, an automated microscope, and
ligation-based sequencing method.
4. Pyrosequencing
 Pyrosequencing is performed by detecting the nucleotide incorporated by
a DNA polymerase.
5. Illumina (Solexa) sequencing
This sequencing method based on reversible dye-terminators technology,
and engineered polymerases to sequence the nucleotides.

6. DNA nanoball sequencing 

DNA nanoball sequencing is used to determine the entire genomic
sequence of an organism including microbes.
7.Heliscope sequencing
It uses DNA fragments with added poly-A tail adapters which are attached to
the flow cell surface. It is used to read short sequences up to 35bp.
8. Sequencing by hybridization
It is a non-enzymatic method that uses a DNA microarray. 
9. RNAP sequencing
The sequence is deduced based on the four readouts with lowered
concentrations of each of the four nucleotide types, similarly to the Sanger
method.
10. Nanopore DNA sequencing
The DNA passing through the nanopore changes its ion current. This change
is dependent on the shape, size and length of the DNA sequence. It is also
known as 3rd Generation sequencing.
Second-generation sequencing that is currently the most commonly used
NGS technology consists of library preparation, amplification, and
sequencing steps while in third-generation sequencing, individual nucleic acids
are sequenced directly in order to avoid biases and have higher throughput.
Recently described fourth-generation sequencing aims conducting genomic
analysis directly in the cell. Classified to different generations, NGS has led to
overcome the limitations of conventional DNA sequencing methods and has
found usage in a wide range of molecular biology applications.

Social & Ethical Aspects of rDNA

technology
Although rDNA technology provides many benefits and advantages
including the improvement of medicines, crops, delivering safer medicines with
lower and cheaper cost, modifying fruits and vegetables colors and their taste,
agriculture and animal husbandry as well. However, several ethical and social
considerations and controversies are associated with genetic engineering. Many
people believe that altering human DNA is immoral and constitutes "playing
God". In addition to this, genetic engineering, fairly new technology, so there
are many questions about long term health effects of consuming genetically
altered plants and animals including humans.
Since recombinant DNA technology can contribute a gene to the host cells, a
concern is that some microbes can gain antibiotic resistance using rDNA
technology. Some environmental concerns can come from nearby plants,
pollinated from alternate plants which can change some properties of plants.
Some people also object insertion animal genes into plants and vice versa and
considered this unnatural. The point is that the evolution has created these
plants and animals to survive in this environment and don’t need to be altered
because the plants and animals are perfect as they are.
Wherever, the concern lies, a look at both pros and cons must be considered
to fully understand the social impact of genetic engineering, that may appear in
future.

Applications of rDNA technology

Besides providing valuable information about the nature and functions of
gene, it has several practical applications. Among these are the production of
various medicines, insulin, vaccines, hormones, interferon, genetically modified
food and crops.
Recombinant DNA technology is also used in agricultural products for
delayed ripening for longer duration, or to increase the sweetness of fruits, slow
down the process of spoiling of fruits, change in color, shape, size of them,
resistance against infections and plant viruses, enhancement of flavor, and
nutritional content.
Recombinant DNA technology also plays a vital role in animal husbandry as
well as different industries associated with human benefits.
1. Applications in Medicine: -
Recombinant DNA technology had made it possible to treat different
diseases by inserting new genes in place of damaged and diseased genes in the
human body. It has brought many revolutionary changes in the field of medicine
and introduced such methods of treating diseases and delivering the drug which
were just imaginary.
1. Insulin: -
Insulin is a hormone made up of protein. It is secreted in the pancreas by
some cells called as islet cells i.e. β-cells. This hormone is responsible for
controlling the glucose level in humans. If a person has decreased amount of
insulin in his body, he will suffer from a disease called diabetes.
 Recombinant DNA technology has allowed the scientists to develop human
insulin by using the bacteria as a host cell. It is believed that the drugs produced
through microbes are safer than the drugs produced traditionally.
2. Vaccines: -
Vaccine is a biological substance which is prepared from the suspension of
weak or dead pathogenic cells. It is injected in the body to enhance the
production of antibodies against antigen. Recombinant DNA technology
enables the scientists to develop vaccines by cloning the gene used for
protective antigen protein. Viral vaccines are most developed through this
technology for the treatment of various diseases for example, Herpes, Influenza,
Hepatitis and Foot and Mouth Disease, HIV and other viral infections.
3. 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. In recent
years, scientists have developed many growth hormones using recombinant
DNA technology. The disease of dwarfism is treated with this hormone.
4. Monoclonal Antibodies: -
When a foreign object enters the body, immune system of the body
releases a specific protein called as antibody, which fight against particular
foreign objects.
Hybridoma technology has made it possible to produce monoclonal
antibodies. In this technique, the lymphocytes or B cells are joined with
myeloma cells; the resulting substance is called as Hybridoma. This Hybridoma
produces unlimited antibodies in the culture. The antibody produced is called as
monoclonal antibody. These antibodies are used to produce vaccines against
different viral infections in the laboratory. They have ability to destroy bacteria
and other harmful pathogens which cause infection in the body by providing
body immune system.
5. Interferon: -
A glycoprotein which has the ability to block the multiplication or
division of viruses in the cells or in the nearby cells is called as interferon.
Interferon can be used to treat cancer like hairy cell leukemia. Recombinant
DNA technology produces this protein using E. coli. Interferon α and γ is used
to treat lymphoma and myelogenous leukemia.
6. Antibiotics: -
Antibiotics are the chemical substances which are used against bacterial
infections. They can be produced by microorganisms as well as in the
laboratory. They can destroy bacteria or other harmful microbes which cause
infections in the body. Alexander Fleming discovered penicillin for the first
time in 1928 using recombinant DNA technology. Other biotechnological
techniques are also being used to produce antibiotics.
7. Diagnosis of Infectious Diseases: -
Recombinant DNA technology has allowed the development of many
tests which are being used to diagnose diseases like TB, cancer, measles,
smallpox and hepatitis, allergy, HIV etc. If they are not diagnosed properly,
they can be a threat to human health.
In the diagnosis process, certain pathogens are isolated and identified, and
then diagnostic kits are produced when the genome of the specific pathogen is
known to kill it or block its pathogenic activity.
2. Applications in Agriculture Fields: -
Recombinant DNA technology is used in genetically modified plants by
adding or removing genes. Genes are often added to plant genomes to increase
plant resistance to viral, fungal and bacterial infections making herbicides less
necessary and to increase the sweetness of fruits. Genes can also be subtracted
to slow down the process by which fruit or vegetables spoil or to modify the
colors of flowers.
Although plants are more difficult to work with than bacteria, gene
insertions can be made into single plant cells. Then the cells can be cultivated to
form a mature plant.
The major method for inserting genes is through the plasmids of the
bacterium called Agrobacterium tumefaciens. This bacterium invades plant
cells, and its plasmids insert into plant chromosomes carrying the genes for
tumor induction. These tumor inducing bacteria are found in soil. Scientists
remove the tumor-inducing genes and obtain a plasmid that unites with the plant
cell without causing any harm.
Recombinant DNA and biotechnology have been used to increase the
efficiency of plant growth by increasing the efficiency of the plant’s ability to
fix nitrogen.
3. Applications in Genetically Modified Food
Genetically modified food is a product containing some quantity of any
genetically modified organism as an ingredient. rDNA technology involves
production of genetically modified food. Genes can be derived from plant or
even other organisms to give plants characteristics that are beneficial for both
producers and consumer of agricultural products. The changes or modifications
which are made into plant yield by genetic engineering are;
 Delayed food ripening for longer periods of time
 Resistance to insects and plant viruses
 Enhancement of flavor and level of nutritional content of crop
 Edible vaccines to prevent widespread diseases in developing countries e.g.
strawberry, tomato, potato etc.
GMF by rDNA technology is similar to the one used to produce human
insulin with an addition state after modifying genes of interest. The gene is then
introduced into plant cell so that plant will manufacture gene produced whether
its insecticide or vaccine or other plant substances.

4. Applications in Animal Husbandry

Another use of recombinant DNA technology is to add an outside gene to the
DNA of animal creating a transgenic animal. These genes are inserted into
animals before they born. DNA is introduced into a eukaryotic cell by a variety
of techniques, such as transformation, injection, viral infection, or bombardment
with DNA-coated tungsten particles to alters its protein contents.
For example, to produce a cow with low lactose milk or more lactose milk or
to dramatically increase milk production with cheaper and rapid desire products.
Genetic engineering will provide us genetically desire enzymes, various
proteins and increase production of meat, wool and other animal products
through common natural function of the animals.
Transgenic animals model advancements in DNA technology in their
development. The mechanism for creating one can be described in three steps:
1. Healthy egg cells are removed from a female of the host animal and
fertilized in the laboratory.
2. The desired gene from another species is identified, isolated, and cloned.
3. The cloned genes are injected directly into the eggs, which are then
surgically implanted in the host female, where the embryo undergoes a
normal development process.

5. Applications in Human Therapy

By using rDNA technology, many genes have been cloned in E. coli
particularly and yeast too. This has made it possible to produce unlimited
amount of human proteins and enzymes in vitro. Cultured cells, E. coli, yeast
cells and mammalian cells are being transferred with human genes are used to
manufacture more than 1100 products for human therapy. For example
1. TNF used for certain tumor cells.
2. IL-2 used in treatment of immune deficiency diseases particularly HIV.
3. Insulin can be used in treatment of diabetic patients.
4. Factor-VIII can be used for males suffering from Hemophilia A.
5. Factor-IX can be used for males suffering from Hemophilia B.
6. Human Growth Factor can be used to treat Dwarfs.
7. Erythropoietin is used for anemia patients.
8. Interferon is used for viral infections, in general, and particularly blood
cancer.
9. GM-CSF is used for stimulating the bone marrow after the transplantation
of bone marrow.
10. G-CSF is used for production of neutrophils movement from haemopoietic
stem cells.
11. Tissue Plasminogen activator is used for dissolving blood clots in blood
circulatory system.
12. Adenosine Deaminase Factor can be used to treat some form of SCID
symptoms.
13. Parathyroid hormone is used to treat cretenism and myxedema and goiter.
14. Monoclonal Abs can be used to treat various viral infections.
15. Hepatitis B Surface Antigen is used as a vaccine for Hepatitis B virus.
16. Chlorine Inhibitor is used to treat hereditary angioedema.
17. Prourokinase is used to treat heart diseases.
18. Taxol is used for cervical or ovarian cancer.

Gene Therapy
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 several 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 being tested only for
diseases that have no other cures.

Genetic screening tests

Some genetic tests are used even when symptoms of a disease are not seen,
but the genetic information may help in predicting if the person is at risk of
developing or are susceptible to a disease.
 Almost all genetic tests require a DNA sample from the patient, this is
usually obtained by either a blood sample or mouthwash (buccal swab). This is
then taken to a genetic testing lab for analysis. Researchers usually adopt
following for genetic screening tests:
1. Prenatal screening testing to screen for genetic diseases is offered to many
women during pregnancy. For example, the screening for Down syndrome in
women over 35 is usually carried out by amniocentesis or chorionic villus
sampling at 14 – 20 weeks of gestation.
2. Newborn screening is carried out routinely in most hospitals around the
world, screening newborns for several disorders including phenylketonuria,
cystic fibrosis (CF). A blood sample is taken from the newborn, this blood
sample is then sent to a laboratory for testing.
3. Carrier screening is used in people or populations to determine whether
they carry a mutative recessive gene that may not affect the individual’s
health but may affect the health of their future children. If a couple is carrier
for that genetic disease then before a genetic test is carried out, a doctor will
do a clinical examination and get a detailed family history. This will help the
doctor in working out which gene may be responsible for the disease.
Several techniques are used in the process of genetic testing, these include
PCR, Indirect gene tracking, Polymorphic repeat sequences.
DNA typing
DNA typing is a method in which our genetic material (DNA) is
converted into a barcode that, ultimately distinguishes each of us from nearly
everyone else on earth. DNA is easily recovered from many sources, so that
criminals often unwittingly leave their DNA at crime scenes, and the DNA of
victims is even sometimes carried away on the clothes of their assailants. By
using DNA, we are thus often able to place individuals at crime scenes, and in
the case of rape, can identify the man who "provided" the sperm.
DNA typing is or can be used for many different crimes and
circumstances:  rape, assault and murder, body identification, and establishing
parentage.  It is also useful in conservation (establishing that meat came from an
endangered species.  And a specific kind of DNA typing is used in molecular
epidemiology, to identify the source of infectious agents.

Gene Expression
Gene expression is the process by which information from a gene is used
in the synthesis of a functional gene product. These products are often proteins,
but in non-protein coding genes such as transfer RNA (tRNA) or small nuclear
RNA (snRNA) genes, the product is a functional RNA.
 The process of gene expression is used by all known life—eukaryotes
(including multicellular organisms), prokaryotes (bacteria and archaea), and
utilized by viruses—to generate the macromolecular machinery for life.
Several steps in the gene expression process may be modulated, including
the transcription, RNA splicing, translation, and post-translational modification
of a protein. Gene regulation gives the cell control over structure and function,
and is the basis for cellular differentiation, morphogenesis and the versatility
and adaptability of any organism. Gene regulation may also serve as a substrate
for evolutionary change, since control of the timing, location, and amount of
gene expression can have a profound effect on the functions (actions) of the
gene in a cell or in a multicellular organism.
In molecular genetics, gene expression is the most fundamental level at
which the genotype gives rise to the phenotype, i.e. observable trait. The genetic
code stored in DNA is "interpreted" by gene expression, and the properties of
the expression give rise to the organism's phenotype.
Background
In 1972, Walter Fiers became the first person to actually prove the
existence of the terminating enzyme.
Roger D. Kornberg won the 2006 Nobel Prize for his studies of the
molecular basis of eukaryotic transcription".
Transcription
Transcription is the process of creating a complementary RNA of a
sequence of DNA. During transcription, a DNA sequence is read by an RNA
polymerase, which produces a complementary, antiparallel RNA strand called
a primary transcript. As opposed to DNA replication, transcription results in an
RNA complement that includes the nucleotide uracil (U) in all instances
where thymine (T) would have occurred in a DNA complement.
Transcription can be explained as,
1. H-bonds breaks, DNA unwinds by helicase.
2. Free nucleotides of RNA are paired with complementary DNA bases.
3. RNA sugar-phosphate backbone is formed by RNA polymerase.
4. Hydrogen bonds of the untwisted RNA+DNA helix break and make free the
newly synthesized RNA strand.
5. RNA is further processed and then moves through the small nuclear pores to
the cytoplasm for translation.
Transcription is the first step leading to gene expression. The stretch of DNA
transcribed into an RNA molecule is called a transcription unit and encodes at
least one gene. If the gene transcribed encodes a protein, the result of
transcription is messenger RNA (mRNA), which will then be used to create that
protein via the process of translation. Alternatively, the transcribed gene may
encode for either ribosomal RNA (rRNA) or transfer RNA (tRNA), other
components of the protein-assembly process, or other ribozymes.

RNA splicing
RNA splicing, is modification of (pre-mRNA) transcript in which introns
are removed and exons are joined together. For nuclear-encoded genes, splicing
takes place within the nucleus either during or immediately after transcription.
Splicing is usually required in order to create an mRNA molecule that can be
translated into protein. For many eukaryotic introns, splicing is carried out in a
series of reactions which are catalyzed by the spliceosome, a complex of small
nuclear ribonucleoproteins (snRNPs). Self-splicing introns also exist.
Introns
The word intron is derived from the term intragenic region, a region
inside the gene where modification occurs and the corresponding sequence in
the unprocessed RNA transcript. As part of the RNA processing pathway,
introns are removed by RNA splicing either shortly after or concurrent with
transcription.
Introns are found in the genes of most organisms and many viruses. They
can be located in a wide range of genes, including those that generate proteins,
ribosomal RNA (rRNA), and transfer RNA (tRNA).
Spliceosomal introns often reside within the sequence of eukaryotic
protein encoding genes. Within introns, a donor site (5' end of the intron), a
branch site (near the 3' end of the intron) and an acceptor site (3' end of the
intron) are required for splicing.
Self-splicing
Self-splicing occurs for rare introns that form a ribozyme, performing the
functions of the spliceosome by RNA alone. There are three kinds of self-
splicing introns, Group I, Group II and Group III.
Group I and II introns perform splicing like the spliceosome without
requiring any protein. This similarity suggests that Group I and II introns may
be evolutionarily related to the spliceosome. Self-splicing may also be very
ancient, and may have existed in an RNA world present before protein.
tRNA splicing
tRNA (also tRNA-like) splicing is another rare form of splicing that
usually occurs in tRNA. The splicing reaction involves a different biochemistry
than the spliceosome and self-splicing pathways. For example, ribonucleases
cleave the RNA and the ligases join the exons together.
Exon
 An exon is any nucleotide sequence encoded by a gene that remains a
part of the final mature RNA produced.
The term exon refers to both the DNA sequence within a gene and to the
corresponding sequence in RNA transcripts. In RNA splicing, introns are
removed and exons are covalently joined to one another as part of generating
the mature messenger RNA.

Translation
Translation is the means by which a specific sequence of amino acids is
formed in accordance with the codons on the mRNA. A group of ribosomes
attached to the mRNA to form a structure called a polysome. The
complementary anticodon of a tRNA-amino acid complex is attracted to the
first codon on the mRNA. The second codon likewise attracts its
complementary anticodon.
The ribosome acts as a framework which holds the mRNA and tRNA-
amino acid complex together until the two amino acids form a peptide bond
between each other. Once they have combined, the ribosome will move along
the mRNA to hold the next codon—anticodon complex together until the third
amino acid is linked with the second. In this way a polypeptide chain is
assembled, by the addition of one amino acid at a time and subsequent
ribosomes may pass along the mRNA behind the first. In this way many
identical polypeptides are produced simultaneously.
Once each amino acid is linked, the tRNA, which carried it to the mRNA,
is released back into the cytoplasm. It is again free to combine with its specific
amino acid. The ribosome continues along the mRNA until it reaches one of the
nonsense at which point the polypeptide is cast off. The polypeptides so formed
must now assembled into proteins.
Proteins are polymers consisting of amino acids linked by peptide bonds
and the amino acid sequence of protein is its primary structure of protein.
The secondary structure is the shape, which a polypeptide chain formed
as a result of H-Bonding among amino acid residues in secondary polypeptide
backbone. In secondary structure the polypeptide full twist and most often spiral
as alpha helix chain although other configurations also occur.
Tertiary structure is due to the bonding and further twisting of
polypeptides into complex structures. All three bonds such as disulfide bonds,
H- bonds, ionic bonds contribute to the maintenance of tertiary structure.
Quaternary structure rise by combination of structure of 2 or more
polypeptide chains and associated with non-protein group (prosthetic groups)
into a large complex protein molecule.
The primary structure of protein is determined by the covalently linked
amino acids residue in the polypeptide backbone while secondary and other
higher order of structure of proteins are determined principally by non-covalent
forces such as H-bonding and ionic- bonds, Vander wall forces and hydrophobic
interactions.
Post Translation
The product of many genes is a protein whose actions produce the
----encoded by that genes. The resulted proteins are either enzymes, biological
catalyst that derive chemical reactions of cell. Structural components are
responsible for providing scaffolding and support for membrane, bones,
muscles, hair, nails in eukaryotes. Some proteins help in transporting substances
while other has regulatory communication and defense functions.

Biosensor
A biosensor is an analytical device, used for the detection of a chemical
substance, that combines a biological component with a physicochemical
detector. The sensitive biological element, e.g. tissue, microorganisms,
organelles, cell receptors, enzymes, Antibodies, nucleic acids, etc., is a
biologically derived material or biomimetic component that interacts with, binds
with, or recognizes the analyte under study.
History
The first ‘true’ biosensor was developed by Leland C. Clark, Jr in 1956
for oxygen detection. He is known as the ‘father of biosensors’ and his
invention of the oxygen electrode bears his name: ‘Clark electrode’. Later name
changed to biosensors.
These instruments have a wide range of applications ranging from clinical
through to environmental and agricultural. The devices are also used in the food
industry. Biosensor can also be used in:
 General healthcare monitoring
 Screening for disease
 Clinical analysis and diagnosis of disease
 Veterinary and agricultural applications
 Industrial processing and monitoring
 Environmental pollution control
Biosensors can provide cost-effective, easy-to-use, sensitive and highly
accurate detection devices in a variety of research and commercial applications. 
Applications in industry
Biosensors are used in the food industry to measure carbohydrates,
alcohols and acids, for example, during quality control processes. The devices
may also be used to check fermentation during the production of beer, yoghurt
and soft drinks. Another important application is their use in detecting
pathogens in fresh meat, poultry or fish.
Clinical and Diagnostic Applications
One well known example of a clinically applied biosensor is the glucose
monitor, which is used on a routine basis by diabetic individuals to check their
blood sugar level. These devices detect the amount of blood glucose in
undiluted blood samples allowing for the easy self-testing and monitoring that
has revolutionized diabetes management.
Ultra-Small Microelectrode Biosensors can be used for Brain Injury
Analysis. Bio–nano interactions help kill deadly microbes.
“Biosensors may be used in conjunction with enzyme-linked
immunosorbent assays (ELISA). Enzymes with high turnover numbers are used
in order to achieve rapid response.” The term “biosensor” is short for
“biological sensor.” The device is made up of a transducer and a biological
element that may be an enzyme, an antibody or a nucleic acid. The biological
element interacts with the analyte being tested and the biological response is
converted into an electrical signal by the transducer. 

Environmental applications
Biosensors are used to check the quality of air and water. The devices can
be used to pick up traces of organophosphates from pesticides or to check the
toxicity levels of wastewater, soil etc.
Major Components
1. Biocatalyst - Responsible to convert substrate into products.
2. Transducer – Responsible for determination of reaction.
3. Amplifying machine, Processing/ Interpretation Unit- completes
processing.
4. Display screen – For result.
Types
The most common types of bio-transducers used in biosensors are:

 Electrochemical biosensors
 Optical biosensors
 Electronic biosensors
 Piezoelectric biosensors
 Gravimetric biosensors
 Pyroelectric biosensors
Generations
 There are three so-called 'generations' of biosensors;
First generation biosensors where the normal product of the reaction diffuses
to the transducer and causes the electrical response.
Second generation biosensors which involve specific 'mediators' between the
reaction and the transducer in order to generate improved response.
Third generation biosensors where the reaction itself causes the response and
no product or mediator diffusion is directly involved.
Merits
A successful biosensor must possess the following beneficial features:
1. A biocatalyst must be highly specific for the purpose of the analyses be
stable under normal storage conditions. In the case of calorimetric enzyme
strips and Dipsticks show good stability over many assays.
2. The reaction should be as independent of physical parameters as like stirring,
pH and temperature is manageable. If the reaction involves cofactors or
coenzymes these should preferably be co-immobilized with the enzyme.
3. The response should be accurate, precise, reproducible and linear over the
useful analytical range without dilution or concentration. It should also be
free from electrical noise.
4. If the biosensor is to be used for invasive monitoring in clinical situations,
the probe must be tiny and biocompatible having no toxic or antigenic
effects. If it is to be used in fermenters it should be sterilizable.
5. The complete biosensor should be cheap, small, portable and capable of
being used by semi-skilled operators.
6. There should be a market for the biosensor by encourage the use of
traditional methods and discourage the decentralization of laboratory testing.
Drawbacks
1. pH and temperature parameter influence the performance of biosensor
activity.
2. Tedious measurements conditions to be developed.
3. Health and safety issues may be created because of nanoparticles in
biosensors that may cause serious illness in body.
4. Some social issues may develop.
5. Cells contain many enzymes and extreme care must be taken to ensure
selectivity of the unique response.
6. The time taken for cell-based sensors to return to baseline potential after
usage is lengthy.
7. Susceptibility to turbidity interfere with result used by biosensor.