Introduction to Biotechnology

 ‘Bio’ refers to life and ‘technology’ refers to the application of information for practical
use, i.e. the application of living organisms to create or improve a product.
 ‘Biotechnology means any scientific application that uses biological systems, living
organisms or derivatives thereof, to produce or alter products or processes for particular
use’
 ‘The utilization of living organisms, systems or processes constitutes biotechnology’
 Based on the Collins English Dictionary definition , biotechnology is the employment of
living organisms, their parts or processes, to develop active and useful products and to
provide services e.g. waste treatment. The term signifies a broad range of processes, from
the use of earthworms as a source of protein to the genetic modification of bacteria to
offer human gene products, e.g. growth hormones.
 According to the Golden Treasury of Science and Technology, biotechnology is a
discipline based on the harnessing of life processes which are controlled for the bulk
production of valuable substances.
 Biotechnology has been defined as ‘Janus-faced’. This means that there are two sides to
it. On one side, we know that the technology allows DNA to be modified so that genes
can be moved from one organism to another. On the other, it also entails comparatively
new techniques whose results are untested and should be met with care.
 Biotechnology is ‘the integrated use of microbiology, biochemistry and engineering
sciences in production or as service operation’.
 Biotechnology is the commercial employment of micro-organisms and living plant and
animal cells to create substances or effects beneficial to people. It includes the production
of antibiotics, vitamins, vaccines, plastics, etc.
 The term biotechnology is occasionally also applied to processes in which micro-
organisms such as yeasts and bacteria are cultured under strictly controlled environmental
conditions. For this reason, fermentation is occasionally called the oldest form of
biotechnology. Genetic engineering techniques are frequently, but not always, used in
biotechnology.
 The Oxford Dictionary of Biology [13] defines biotechnology as ‘the development of
techniques for the application of biological processes to the production of materials of
use in medicine and industry.’
 In summary, biotechnology is the ‘application of the theory of engineering and
biological science to generate new products from raw materials of biological origin,
e.g. vaccines or food’, or, in other words, it can also be defined as ‘the exploitation of
living organism/s or their product/s to change or improve human health and human
surroundings’.

Branches of biotechnology:
The definition of biotechnology can be further divided into different areas known as red,
green blue and white.
 Red biotechnology: This area includes medical procedures such as utilizing
organisms for the production of novel drugs or employing stem cells to
replace/regenerate injured tissues and possibly regenerate whole organs. It could
simply be called medical biotechnology.
  Green biotechnology: Green biotechnology applies to agriculture and involves such
processes as the development of pest-resistant grains and the accelerated evolution of
disease-resistant animals.
 Blue biotechnology: Blue biotechnology, rarely mentioned, encompasses processes
in the marine and aquatic environments, such as controlling the proliferation of
noxious water-borne organisms.
 White biotechnology: White (also called gray) biotechnology involves industrial
processes such as the production of new chemicals or the development of new fuels
for vehicles.

A distinction is made between ‘non-gene biotechnology’ and ‘gene biotechnology’:

 Non-gene biotechnology: Non-gene biotechnology works with whole cells, tissues or
even individual organisms. Non-gene biotechnology is the more popular practice,
involving plant tissue culture, hybrid seed production, microbial fermentation, production
of hybridoma antibodies and immunochemistry.
 Gene biotechnology: Gene biotechnology deals with genes, the transfer of genes from
one organism to another and genetic engineering.

Scope and importance of biotechnology:

Biotechnology is the science of the controlled application of biological agents for
beneficial use.
Since biotechnology is not an independent discipline, its well-known integration with
allied fields such as biochemistry, molecular biology and microbiology facilitates the
technological application of biological agents.
The major significance of this field of science in different fields will be evident from the
following examples.

Biotechnology in medicine:
 One of the major areas in biotechnology is the medical sector.
 The scope of biotechnology in medicine is to utilize techniques in living systems
to produce therapeutic proteins, which are usually called biopharmaceuticals or
recombinant proteins.
 Products such as monoclonal antibodies, DNA and RNA probes are produced for
the diagnosis of various diseases.
 Additionally, therapeutic protein-based drugs such as insulin and interferon have
been synthesized with bacteria for the treatment of human diseases.
 As described above, red biotechnology deals with production of medicinal drugs
that can be proteins (including antibodies that fight infection) or nucleic acids
(DNA or RNA). There is no involvement of chemicals in the synthesis process
since they are derived from micro-organisms which synthesize them naturally.
 The second major field of red biotechnology is gene therapy, which deals with the
diagnosis and treatment of genetic diseases and some other diseases such as
cancer. This therapy encompasses the manipulation of genes and the correction of
defective genes. During this process genes are inserted, deleted or modified.
 Pharmacogenomics and genetic testing both use techniques of red biotechnology
that are individual-specific.
  In pharmacogenomics the genetic information of the individual is derived, and
drugs are developed that can be inserted into that particular individual, whereas in
genetic testing different tests are conducted among family members to determine
genetic diseases, sex and carrier screening.
 Monoclonal antibodies, DNA and RNA probes are used for the diagnosis of
various diseases and valuable drugs such as insulin and interferon have been
synthesized by bacteria for the treatment of human diseases.
 DNA fingerprinting is utilized for the identification of parents and criminals.
 The development of recombinant vaccines for diseases such as human hepatitis B
using genetically engineered microbes is one of the list of notable achievements.
Industrial biotechnology:
 Industrial biotechnology was established for the large-scale production of alcohol
and antibiotics by micro-organisms.
 Currently, various pharmaceutical drugs and chemicals such as lactic acid,
glycerine, etc, are being produced by genetic engineering for better quality and
quantity.
 Biotechnology has provided us with a very efficient and economical technique for
the production of a variety of biochemicals, e.g. immobilized enzymes.
 Protein engineering is another important area where existing proteins and
enzymes are remodeled for a specific function or to increase the efficiency of
their function
Biotechnology and the environment:
 Environmental problems such as pollution control, the depletion of natural
resources for non-renewable energy, conservation of biodiversity, etc, are being
dealt with using biotechnology. For example, bacteria are being utilized for the
detoxification of industrial effluents, to combat oil spills, for treatment of sewage
and for biogas production.
 Biopesticides offer an environmentally safer alternative to chemical pesticides for
control of insect pests and diseases.
Biotechnology and agriculture:
 Currently the potential of plant tissue culture is widely utilized for the rapid and
economic clonal multiplication of fruit and forest trees, for the production of
virusfree genetic stock and planting material, as well as in the creation of novel
genetic variations through somaclonal variation.
 With the aid of rDNA technology, it has now become possible to produce
transgenic plants with desirable genes such as herbicide resistance, disease
resistance, increased shelf life, etc.
 Techniques such as molecular breeding have been employed to accelerate the
process of crop improvement.
 For instance, molecular markers, such as restriction fragment length
polymorphism (RFLP), and simple sequence repeats (SSRs) provide potential
tools for the indirect selection of both qualitative and quantitative traits, and also
for studying genotypic diversit.

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 Immobilization of Enzymes

Enzymes are biological catalysts that promote the transformation of chemical species in living
systems.
 These molecules, consisting of thousands of atoms in precise arrangements, are able to
catalyze the multitude of different chemical reactions occurring in biological cells.
 Their role in biological processes, in health and disease, has been extensively
investigated.
 They have also been a key component in many ancient human activities, especially food
processing, well before their nature or function was known.

 The term immobilized enzymes refers to “enzymes physically confined or localized in a

certain defi ned region of space with retention of their catalytic activities, and which can
be used repeatedly and continuously.”

Choice of Supports:
 The characteristics of the matrix are of paramount importance in determining the
performance of the immobilized enzyme system.
 Ideal support properties include physical resistance to compression, hydrophilicity,
inertness towards enzymes, ease of derivatization, bio-compatibility, resistance to
microbial attack, and availability at low cost.
 In particular, pore parameters and particle size determine the total surface area and thus
critically affect the capacity for binding of enzymes.
 Nonporous supports show few diffusional limitations but have a low loading capacity.
 Porous supports are in general preferred because the high surface area allows a higher
enzyme loading and the immobilized enzyme is more protected from the environment.
 Nanomaterials can serve as excellent support materials for enzyme immobilization,
offering ideal characteristics for balancing the key factors that determine the efficiency of
biocatalysts: surface area, mass transfer resistance and effective enzyme loading.
 Recently, enzymes immobilized to nanosized supports such as polymer microspheres,
fibers, tubes, as well as various metal and magnetic nanoparticles have been reported.
 Fig: Main features of support materials used for enzyme immobilization.

The classification of support can be done:

 According to Chemical composition of support (inorganic and organic) and
 According to material (classic and new materials)

Classification according to Chemical composition of support (inorganic and organic).

 The organic supports can be subdivided into natural and synthetic polymers.
 The physical characteristics of the matrices (such as mean particle diameter, swelling
behavior, mechanical strength, compression behavior) will be of major importance for the
performance of the immobilized systems and determine the type of reactor used under
technical conditions (i.e., stirred tank, fluidized, fixed beds).
 According to material (classic and new support materials)

Classic Materials:
 Materials have been sought which offer high stability, availability, relatively low price
and high affinity to the bound enzymes.
 A wide variety of materials of both inorganic and organic origin have been evaluated as
effective supports for biocatalyst and classified as classic material.

Fig: Classic materials of support

 Inorganic Materials:
 Silica and Inorganic Oxides: sol-gel silica, fumed silica, colloidal silica nanoparticles and
silica gel, inorganic oxides, titanium, aluminium and zirconium oxides
 Mineral Materials: The minerals used as supports for enzyme immobilization are mainly
clay materials such as bentonite, halloysite, kaolinite, montmorillonite and sepiolite.
 Carbon-based materials: Activated carbons and unmodified and modified charcoals
Organic Materials:
 Synthetic Polymers: Polymers containing carbonyl, carboxyl, hydroxyl, epoxy, amine
and diol groups,
 Biopolymers: Carbohydrates, albumin, gelatin, collagen, cellulose, keratins and
carrageenan as well as chitin, chitosan and alginate.

New Support Materials:

 These materials, of both organic and inorganic origin, are characterized by exceptional
thermal and chemical stability and very good mechanical properties.
 These materials possess remarkable quantities of various functional groups,
corresponding to the chemical groups of the proteins, which enhance enzyme binding and
surface modification.

Fig: New materials of inorganic, organic and hybrid origin, applied for enzyme immobilization .

Inorganic Materials
 Magnetic Particles:
 Mesoporous Materials: Materials such as zeolites, carbons and sol-gel matrices, as well
as precipitated and ordered mesoporous oxides are included in the mesoporous group.
 Nanoparticles: nanogold and grapheme
 Ceramic Materials: Alumina, zirconia, titania, silica, iron oxide and calcium phosphate
have been used as biomolecule carriers.
 Carbon Nanotubes:
 Graphene and Graphene Oxide:
Organic Materials
 Electrospun Materials: poly(vinyl alcohol), polystyrene, polyacrylamide and
polyurethane, as well as biopolymers such as chitin, chitosan, alginate and cellulose.
 Polymeric Membranes: Synthetic polymers can be used for the preparation of
membranes, for example poly(vinyl alcohol), polyurethane and poly(vinylidene fluoride).
Membranes made from biopolymers, for example from chitosan or cellulose, can also be
used.
Hybrid and Composite Materials
 Organic-Organic Hybrids: Organic-organic hybrids may be synthesized by connecting
together the following: (i) two synthetic materials, for example polyaniline and
polyacrylonitrile, polyethyleneimine, with epoxy-activated acrylate copolymer or
poly(acrylic acid) and polyvinyl alcohol (ii) a synthetic polymer with a biopolymer, such
as poly(acrylic acid) and cellulose, polyvinyl alcohol and chitosan, or polyaniline and
chitosan; (iii) two biopolymers, such as chitosan and alginate, chitin and lignin or
cellulose and dextra.
 Organic-Inorganic Hybrids: silica–dialdehyde starch (SiO2-DAS) mixed hybrid,
 Inorganic-Inorganic Hybrids: Calcium carbonate and gold nanoparticles (CaCO3-
AuNPs), sol-gel derived silica combined with multi-walled carbon nanotubes
(MWCNTs).

Methods of Immobilization:
One way of classifying the various approaches to immobilizing enzymes is in two broad
categories: irreversible and reversible methods.
In addition, immobilization methods are often classified by the type of chemical reaction used
for binding.

Fig: Methods of Immobilization

  Each immobilization method presents advantages and drawbacks.
 The choice of the most appropriate technique also depends on the nature of the enzyme
(biochemical and kinetics properties) and the carrier (chemical characteristics,
mechanical properties).
 The interaction between the enzyme and support provides an immobilized enzyme with
particular biochemical and physicochemical properties that determine their applicability
to specific processes.


Covalent Binding:
 Covalent binding is a conventional method for immobilization; it can be achieved by
direct attachment with the enzyme and the material through the covalent linkage.
 The covalent linkage is strong and stable and the support material of enzymes includes
polyacrylamide, porous glass, agarose and porous silica.
 Covalent method of immobilization is mainly used when a reaction process does not
require enzyme in the product, this is the criteria to choose covalent immobilization
method.
 The covalent binding is normally formed between the functional group in the support
matrix and the enzyme surface that contains the amino acid residues.
 The amino acid residues involved in the covalent binding are the sulfhydryl group of
cysteine, hydroxyl group of serine and threonin.
  The attachment between the enzyme and the support material can be achieved either
through direct linkage or through the spacer arm.
 The potentiality of using the spacer arm is that it provides the greater degree of the
mobility to the enzymes hence the enzymes show the higher activity when compared to
the direct attachment.
Entrapment:
 Enzymes are occluded in the synthetic or natural polymeric networks, it is a permeable
membrane which allowsthe substrates and the products to pass, but it retains the enzyme
inside the network, the entrapment can be achieved by the gel, fibre entrapping and
microencapsulation.
 The advantage of entrapment of enzyme immobilization is fast, cheap and mild
conditions required for reaction process.
 The disadvantage is that limitation in mass transfer.
 The support matrix protects the enzymes from microbial contamination, proteins and
enzymes in the micro Environment.
 Microencapsulation method is that the enzyme molecules are capsulated within spherical
semipermeable membranes with a selective controlled permeability.
 This method provides the large surface area between polymeric material and the enzyme.
 The drawback of this method is inactivation of enzyme during encapsulation.
Adsorption:
 This is a simple method of preparing an immobilized enzymes and the materials used for
adsorption are activated charcoal, Alumina, Ion exchange resins, this method is cheap
and easy for use and the disadvantage is a weak binding force between the carrier and the
enzyme.
 This method comes under carrier bound immobilization and the process of
immobilization is reversible. Adsorption is the easiest and oldest immobilization
techniques.
 The interaction between the enzyme and the surface of the matrix through weak forces by
salt linkage, hydrogen bonds, hydrophobic bonds, ionic bonds and van der waals forces.
 Based on the charges of the matrix and the protein arrangements the strongly bound, but
not distorted enzyme will be formed.
 The advantage of enzyme adsorption is minimum activation step and as a result of
minimum activation, no reagents required. It is cheap and easy way of immobilization.
Affinity Binding:
 The immobilization of enzyme linked to the matrix through the specific interactions.
 The Two methods are being followed in affinity immobilization.
 The first method is the activation of the support material which contains the coupled
affinity ligand, so that the enzyme will be added.
 The advantage of this method is the enzyme is not exposed to any harsh chemicals
conditions.
 The second method, the enzyme modified to another molecule which has the ability to
bind towards a matrix.
Metal Linked immobilization:
 In metal linked enzyme immobilization, the metal salts are precipitated over the surface
of the carriers and it has the potential to bind with the nucleophilic groups on the matrix.
  The precipitation of the ion on the carrier can be achieved by heating. This method is
simple and the activity of the immobilized enzymes is relatively high (30- 80%).
 The carrier and the enzyme can be separated by decreasing the pH, hence it is a reversible
process.
 The matrix and the enzyme can be regenerated, by the process.

APPLICATION OF THE IMMOBILIZED ENZYMES

Biomedical Application:
 Immobilized enzymes are used in medicine from 1990.
 Immobilized enzymes are used for diagnosis and treatment of diseases in the medical
field. The inborn metabolic deficiency can be overcome by replacing the encapsulated
enzymes (i.e, enzymes encapsulated by erythrocytes) instead of waste metabolites, the
RBC acts as a carrier for the exogenous enzyme drugs and the enzymes are
biocompatible in nature, hence there is no immune response.
 The enzyme encapsulation through the electroporation is a easiest way of immobilization
in the biomedical field and it is a reversible process for which enzyme can be regenerated
 The enzymes when combined with the biomaterials it provides biological and functional
systems.
Food industry Application:
 In food industry, the purified enzymes are used but during the purification the enzymes
will denature. Hence the immobilization technique makes the enzymes stable.
 The immobilized enzymes are used for the production of syrups.
 Immobilized beta-galactosidase used for lactose hydrolysis in whey for the production of
bakers yeast.
 The enzyme is linked to porous silica matrix through covalent linkage. This method is
not preferably used due to its cost and the other technique developed by Valio in 1980,
the enzyme galactosidase was linked to resin (food) through cross linking. This method
was used for the various purposes such as confectionaries and icecreams
.
Biodiesel Production:
 Biodiesel is monoalkyl esters of long chain fatty acids. Biodiesel is produced through
triglycerides (vegetable oil, animal fat) with esterification of alcohol (methanol, ethanol)
in the presence of the catalyst.
 The production of catalyst is a drawback of high energy requirements, recovery of
glycerol and side reaction which may affect the pollution. Hence the biological
production of liquid fuel with lipases nowadays has a great consideration with a rapid
improvement.
 Lipase catalyses the reaction with less energy requirements and mild conditions required.
But the production of lipase is of high cost, hence the immobilization of lipase which
results in repeated use and stability.
 The immobilization of lipase includes several methods entrapment, encapsulation, cross
linking, adsorption and covalent bonding.
 Adsortion method of immobilization is widely used in recent years when compared to
covalent bond, entrapment and cross linking.
 In the biological production of biodiesel the methanol inactivates the the lipase, hence the
immobilization method is an advantage for the biodiesel production.
  The low cost of lipase, candida sp as origin is of more industrial use.
Textile Industry:
 The enzymes derived from microbial origin are of great interest in textile industry.
 The enzymes such as cellulase, amylase, liccase, pectinase, cutinase etc and these are
used for various textile applications such asscouring,biopolishing, desizing, denim
finishing, treating wools etc.
 The textile industries now turned to enzyme process instead of using harsh chemical
which affects the pollution and cause damage to the fabrics.
 The processing of fabrics with enzymes requires high temperatures and increased pH, the
free enzymes does not able to withstand the extreme conditions.
 Hence, enzyme immobilization for this process able to withstand at extreme and able to
maintains its activity for more than 5-6 cycles.
 PolyMethyl Methacrylateis linked with cellulose covalently. In this method the
nanoparticle is synthesized with cellulase as core particle.
 Endoglucanase is a component of Cellulase enzyme, Endoglucanase is microencapsulated
with Arabic Gum is a natural polymer with the biodegradable property is used as a matrix
for encapsulation of endoglucanase.
 Encapsulation of endoglucanase prevented it to retain its activity in the presence of
detergents.

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BIOSENSORS:

 Biosensors are, sensing devices comprising a biological component (enzyme, antibody,

animal or plant cell, oligonucleotide, lipid, microorganisms, etc.) intimately connected to
a physical transducer (electrode, optical fiber, vibrating quartz, etc.). This dual
configuration permits a quantitative study of the interaction between a drug compound
and an immobilized biocomponent.
 A chemical sensor is a device that transforms chemical information from the
concentration of specific sample to total composition analysis into an analytical signal.
 Biosensors or Enzyme Electrodes invariably refer to such devices that sense and analyze
biological informations such as blood pressure (BP), temperature, heart rate,
sophisticated determination of chemical and enzymes in body fluids.
 The device may be used either in the laboratory or placed strategically within the body.
 Biosensor as a suitable probe, device or electrode, upon contact with an appropriate
sample, converts the desired analyte into a suitable corresponding measurable electrical
signal.
 Biosensors are unique and innovative tools that may provide complementary data and
information compared with more “classical” analytical instrumentation.
 Their use opens new investigation opportunities for the pharmaceutical research and
health care domains.
 Their uniqueness relies clearly on the presence of an immobilized biological component
closely connected to a physical transducer.
BASIC FEATURES OF BIOSENSORS
 Sensitivity: The value of the electrode response per substrate concentration.
 Linearity: Maximum linear value of the sensor calibration curve. Linearity of the sensor
must be high for the detection of high substrate concentration.
 Response Time: The necessary time for having 95% of the response.

BIOSENSOR TYPES:
 Biosensors started in the 1960s by the pioneers Clark and Lyons. Various types of
biosensors being used are enzymebased, tissue-based, immunosensors, DNA biosensors,
and thermal and piezoelectric biosensors.

Enzyme-based sensor:
 The first enzyme-based sensor was reported by Updike and Hicks in 1967.
 Enzyme biosensors have been devised on immobilization methods, i.e. adsorption of
enzymes by van der Waals forces, ionic bonding or covalent bonding.
 The commonly used enzymes for this purpose are oxidoreduc- tases, polyphenol
oxidases, peroxidases, and aminooxi-dases.
Microbe-based or cell-based sensor:
 The first microbe-based or cell-based sensor was actualized by Diviès.
 The tissues for tissue-based sensors arise from plant and animal sources. The analyte of
interest can be an inhibitor or a substrate of these processes.
 Rechnitz devel-oped the first tissue based sensor for the determination of amino acid
arginine.
Organelle-based sensors:
 Organelle-based sensors were made using membranes, chloroplasts, mitochondria, and
micro-somes.
 However, for this type of biosensor, the stability was high, but the detection time was
longer, and the specificity was reduced.
Immunosensors:
 Immunosensors were established on the fact that anti-bodies have high affinity towards
their respective antigens, i.e. the antibodies specifically bind to pathogens or toxins, or
interact with components of the host's immune system.
DNA biosensors:
 The DNA biosensors were devised on the property that single-strand nucleic acid
molecule is able to recognize and bind to its complementary strand in a sample.
 The interaction is due to the formation of stable hydrogen bonds between the two nucleic
acid strands.
Magnetic biosensors:
 Magnetic biosensors: miniaturized biosensors detecting magnetic micro- and
nanoparticles in microfluidic channels using the magnetoresistance effect have great
potential in terms of sensitivity and size.
Thermal biosensors or calorimetric biosensors:
 Thermal biosensors or calorimetric biosensors are devel-oped by assimilating biosensor
materials as mentioned before into a physical transducer.

Piezoelectric biosensors:
  Piezoelectric biosensors are of two types: the quartz crystal microbalance and the surface
acoustic wave device.
 They are based on the measurement of changes in resonance frequency of a piezoelectric
crystal due to mass changes on the crystal structure.
Optical biosensors:
 Optical biosensors consist of a light source, as well as numerous optical components to
generate a light beam with specific characteristics and to beeline this light to a
modulating agent, a modified sensing head along with a photodetector.
Genetically-encoded biosensors:
 Green fluorescent protein and the subsequent autofluor-escent protein (AFP) variants and
genetic fusion reporters have aided the development of genetically-encoded biosensors.
 This type of biosensor is user-friendly, easy to engineer, manipulate and transfer into
cells.
 Single-chain FRET biosen-sor is another example. They consist of a pair of AFPs, which
are able to transfer fluorescence resonance energy between them when brought close
together.
 Different methods may be used to regulate changes in Förster resonance energy transfer
(FRET) signals based on intensity, ratio or lifetime of AFPs.
Peptide and protein biosensors:
 Peptide and protein biosensors are easily manufactured through synthetic chemistry
followed by enzymatic labelling with synthetic fluorophores.
 Due to their independence of genetically-encoded AFPs, they are readily utilized to
control target activity and constitute attractive alternatives and have an added advantage
of being able to enhance signal-to-noise ratio and sensitivity of response through
introduction of chemical quenchers and photoactivatable groups.

TYPICAL SENSING TECHNIQUES FOR BIOSENSORS

1. Electrochemical
2. DNA Microarray
3. Fluorescence
4. Impedance spectroscopy
5. SERS (Surface Enhanced Raman Spectroscopy)
6. SPM (Scanning probe microscopy)
7. QCM (Quartz crystal microbalance).

APPLICATIONS OF BIOSENSORS

Biosensors have been applied in many fields namely food industry, medical field, marine sector
etc., and they provide better stability and sensitivity as compared with the tradition-al methods.

In food processing, monitoring, food authenticity, quality and safety

 An arduous quandary in food processing industry is of quality and safety, maintenance of
food products and processing.
 Traditional techniques performing chemical experiments and spectroscopy have
shortcomings due to human fatigue, are expensive and time consuming.
  Alternatives for food authen-tication and monitoring with objective and consistent mea-
surement of food products, in a cost effective manner, are desirable for the food industry.
 Thus development of biosen-sors in response to the demand for simple, real-time,
selective and inexpensive techniques is seemingly propitious Biosensors are used for the
detection of pathogens in food.
 Presence of Escherichia coli in vegetables, is a bioindicator of faecal contamination in
food.23,24 E. coli has been measured by detecting variation in pH caused by ammonia
(produced by urease–E. coli antibody conjugate) using potentiometric alter-nating
biosensing systems.
 Washing the vegetables such as sliced carrots and lettuce with peptone water provides us
with the liquid phase. It is then separated by amalgamating it in a sonicator, to disaffiliate
bacterial cells from food items.
 Enzymatic biosensors are also employed in the dairy industry. A biosensor, based on a
screen-printed carbon electrode, was integrated into a flow cell.26 Enzymes were
immobilized on electrodes by engulfment in a photocrosslink- able polymer. The
automated flow-based biosensor could quantify the three organophosphate pesticides in
milk.
 One of the popular food additives extensively used today are sweeteners, which are
adversely causing undesirable diseases including dental caries, cardiovascular diseases,
obesity and type-2 diabetes. It is believed that artificial sweeteners are addictive and coax
us to eat more high-energy food unconsciously, inadvertently causing weight gain. Thus
their detection and quantification are of prime importance.
 Traditional methods to distinguish the two types of sweeteners are ion chromatographic
methods, which are complicated and laborious.
In fermentation processes
 In fermentation industries, process safety and product quality are crucial. Thus effective
monitoring of the fermentation process is imperative to develop, optimize and maintain
biological reactors at maximum efficacy.
 Biosensors can be utilized to monitor the presence of products, biomass, enzyme,
antibody or by-products of the process to indirectly measure the process conditions.
 Biosensors precisely control the fermentation industry and produce reproducible results
due to their simple instrumentation, formidable selectivity, low prices and easy
automation.
Biosensing technology for sustainable food safety
 The term food quality refers to the appearance, taste, smell, nutritional value, freshness,
flavour, texture and chemicals.
 Smart monitoring of nutrients and fast screening of biological and chemical
contaminants are of paramount importance, when it comes to food quality and safety.
 Material science, nanotechnology, electromechanical and microfluidic systems are
striding in to make sensing technology imminent for use in market.
 Biosensors are being employed to perceive general toxicity and specific toxic metals, due
to their capability to react with only the hazardous fractions of metal ions.
 Pesticides pose grave threats to the environment. The common pesticides used are
organophosphates and carbamic insecticide species.
In medical field
 In the discipline of medical science, the applications of biosensors are growing rapidly.
  Glucose biosensors are widely used in clinical applications for diagnosis of diabetes
mellitus, which requires precise control over blood-glucose levels.
 Blood-glucose biosensors usage at home accounts for 85% of the gigantic world market.
 Biosensors are being used pervasively in the medical field to diagnose infectious
diseases.
 A promising biosensor technol-ogy for urinary tract infection (UTI) diagnosis along with
pathogen identification and anti-microbial susceptibility is under study.
 The various other biosensors applications include:
 quanti-tative measurement of cardiac markers in undiluted serum, microfluidic
impedance assay for controlling endothelin- induced cardiac hypertrophy,
immunosensor array for clinical immunophenotyping of acute leukemias,
 effect of oxazabor- olidines on immobilized fructosyltransferase in dental dis-
eases;
 histone deacylase (HDAC) inhibitor assay from resonance energy transfer,
biochip for a quick and accurate detection of multiple cancer markers and
neurochemical detection by diamond microneedle electrodes.
Fluorescent biosensors
 Fluorescent biosensors are imaging agents, for use in cancer and drug discovery.
 They have enabled insights into the role and regulation of enzymes at cellular level.
GFP-based and genetically encoded FRET biosensors play a vital role.
 Fluorescent biosensors are used in drug discovery pro-grammes for the identification of
drugs by high throughput, high content screening approaches, for postscreening analysis
of hits and optimization of leads.
Biodefense biosensing applications
 Biosensors can be used for military purposes at times of biological attacks.
 The main motive of such biosensors is be to sensitively and selectively identify
organisms posing threat in virtually real time called biowarfare agents (BWAs) namely,
bacteria (vegetative and spores), toxins and viruses.
Biosensors in plant biology
 Revolutionary new technologies in the areas of DNA sequenc-ing and molecular
imaging, have lead to advancements in plant science.
-----------X-----------

Protein engineering

Protein engineering is the design of new enzymes or proteins with new or desirable functions. It
is based on the use of recombinant DNA technology to change amino acid sequences.

Method in protein engineering

The most classical method in protein engineering is the so-called “rational design” approach
which involves “site-directed mutagenesis” of proteins.
 Site-directed mutagenesis allows introduction of specific amino acids into a target gene.
 There are two common methods for site-directed mutagenesis. One is called the “overlap
extension” method.
  This method involves two primer pairs, where one primer of each primer pair contains
the mutant codon with a mismatched sequence.
 These four primers are used in the first polymerase chain reaction (PCR), where two
PCRs take place, and two double-stranded DNA products are obtained.
 Upon denaturation and annealing of them, two heteroduplexes are formed, and each
strand of the heteroduplex involves the desired mutagenic codon.
 DNA polymerase is then used to fill in the overlapping 3’ and 5’ ends of each
heteroduplex and the second PCR takes place using the nonmutated primer set to amplify
the mutagenic DNA.
 The other site-directed mutagenesis method is called “whole plasmid single round PCR”.
 This method forms the basis of the commercial “QuikChange Site-Directed Mutagenesis
Kit” from Stratagene.
 It requires two oligonucleotide primers with the desired mutation(s) which are
complementary to the opposite strands of a double-stranded DNA plasmid template.
 Using DNA polymerase PCR takes place, and both strands of the template are replicated
without displacing the primers and a mutated plasmid is obtained with breaks that do not
overlap.
 DpnI methylase is then used for selective digestion to obtain a circular, nicked vector
with the mutant gene.
 Upon transformation of the nicked vector into competent cells, the nick in the DNA is
repaired, and a circular, mutated plasmid is obtained.

Protein engineering applications

A variety of protein engineering applications have been reported in the literature. These
applications range from biocatalysis for food and industry to environmental, medical and
nanobiotechnology applications.

Food and detergent industry applications:

 Early reports on the importance of protein engineering methods to design new enzymes
for enzyme biotechnological industries.
 The enzymes used in food industry were emphasized as an important group of enzymes,
the industrially important properties of which could be further improved by protein
engineering.
 Those properties include thermostability, specificity and catalytic efficiency.
Environmental applications
 Environmental applications of enzyme and protein engineering are also another important
field.
 Early reports on enzyme and cell applications in industry and in environmental
monitoring, such as environmental biosensors.
 Protein engineering of oxygenases, an important group of enzymes with high selectivity
and specificity, which enable the microbial utilization and biodegradation of organic,
toxic compounds.
 Protein engineering, isolation and study of new extremophilic microorganisms, genetic
engineering developments are all promising advances to develop new biocatalysts for
petroleum refining.
  Petroleum biorefining applications such as fuel biodesulfurization, denitrogenation of
fuels, heavy metal removal, depolymerisation of asphaltenes.
Medical applications:
 Medical applications of protein engineering are also diverse.
 The use of protein engineering for cancer treatment studies is a major area of interest.
 Pretargeted radioimmunotherapy has been discussed as a potential cancer treatment.
 By pretargeting, radiation toxicity is minimized by separating the rapidly cleared
radionuclide and the long-circulating antibody.
 Advances in protein engineering and recombinant DNA technology were expected to
increase the use of pretargeted radioimmunotherapy.
 Protein engineering applications for therapeutic protein production is an important area,
particularly for medicine.
 Protein engineering applications with antibodies are also diverse. Owing to advances in
recombinant DNA technology, “antibody engineering” is possible.
 A variety of examples include antibody fragments which have been conjugated to
bioluminescence, fluorescence, quantum dots for optical imaging, as well as iron oxide
nanoparticles for magnetic resonance imaging.
 It is obvious that molecular imaging tools based on antibodies will find more applications
in the future regarding diagnosis and treatment of cancer and other complex diseases
-----------------X-------------

AMYLASE
 Enzymes that break down starch or glycogen present in the saliva of humans and some
other mammals, where it begins the chemical process of digestion.
 All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds, produced by a
variety of living organisms, ranging from bacteria to plants and humans.
1. Classification
 Based on how they break down starch molecules, amylase are classified into:
1. α-Amylase
2. β-Amylase
3. γ-Amylase

α-Amylase
 α-Amylase is a hydrolase enzyme that catalyzes hydrolysis of internal α-
1, 4- glycosidic linkages in starch to yield products like glucose and maltose.
 It is a calcium metalloenzyme i.e. it depends on the presence of a metal co-factor for its
activity.
 The optimum pH for activity is found to be 7.0.
 The substrate that α-amylase acts upon is starch.
 Starch is a polysaccharide composed of two types of polymers – amylose and
amylopectin.
 Amylose constitutes 20-25% of the starch molecule. It is a linear chain consisting of
repetitive glucose units linked by α-1,4-glycosidic linkage.
 Amylopectin constitutes 75-80% of starch and is characterized by branched chains of
glucose units.
  The linear successive glucose units are linked by α-1, 4-glycosidic linkage, while
branching occurs every 15-45 glucose units where α-1, 6 glycosidic bonds are present.

β-Amylase

 β-Amylase is an exo-hydrolase enzyme that acts from the non-reducing end of a

polysaccharide chain by hydrolysis of α-1, 4-glucan linkages to yield successive
maltose units.
 Since, it is unable to cleave branched linkages in branched polysaccharides such as
glycogen or amylopectin, the hydrolysis is incomplete and dextrin units remain.
 Primary sources of β-Amylase are the seeds of higher plants and sweet potatoes.
 The optimal pH of the enzyme ranges from 4.0 to 5.5.
 β-Amylase can be used for different applications on the research as well as industrial
front.
 It can be used for structural studies of starch and glycogen molecules produced by
various methods.

γ-Amylase

 γ-Amylase cleaves α(1-6) glycosidic linkages, in addition to cleaving the last α(1-4)
glycosidic linkages at the non-reducing end of amylose and amylopectin, unlike the other
forms of amylase, yielding glucose.
 γ-amylase is most efficient in acidic environments and has an optimum pH of 3.

Production Methods of Amylase:

 There are mainly two method whichare use for production of α-Amylase on a
commercial scale. These are:
1) Submerged fermentation (SMF)
2) Solid State fermentation (SSF)

1) Submergedfermentation (SMF):
 Submerged fermentation (SmF) employs free flowing liquid substrates, as
molasses and broths.
 The products yieldedin fermentation are secreted into the fermentation broth.
 This fermentation technique is suitable for microorganisms such as bacteria that
require high moisture content for their growth.
 SMF is primarily used for the extraction of secondary metabolites that need to be
used in liquid form.
 This method has several advantages.
 SmF allows the utilization of genetically modified organisms to a
greater extent than SSF.
 The sterilization of the medium and purification process of the end
products can be done easily.
 Also the control of process parameters like temperature, pH, aeration,
oxygen transfer and moisture can be done conveniently

Purification of Amylaze:
 Purification methods commonly employed are precipitation, chromatography and liquid
liquid extraction depending on the properties of the enzyme desired.
 A combination of the above methods is used in a series of steps to achieve high purity.
 The crude extracellular enzyme sample can be obtained from the fermented mass by
filtration and centrifugation.
 The crude amylase enzyme can be precipitated and concentrated using ammonium
sulphate precipitation or organic solvents.
 The precipitated sample can be subjected to dialysis against water or a buffer for further
concentration.
 This can be followed by any of the chromato-graphic techniques like ion exchange, Gel
filtration and affinity chromatography for further separation and purification of the
enzyme.
 In a method of purification of the enzyme produced by Aspergillus spp. the enzyme was
precipitated followed by dialysis and then, column chromato-graphy.

CATALASE:
 Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. It
catalyzes the decomposition of hydrogen peroxide to water and oxygen.
 It is a very important enzyme in reproductive reactions.
 Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long.
 It contains four porphyrin heme (iron) groups that allow the enzyme to react with the
hydrogen peroxide.
 The optimum pH for human catalase is approximately 7.
 Catalase is used in the food industry for removing hydrogen peroxide from milk prior to
cheese production.
 Another use is in food wrappers where it prevents food from oxidizing.
 Catalase is also used in the textile industry, removing hydrogen peroxide from fabrics to
make sure the material is peroxide-free.

PEROXIDASE
 Peroxidases are hydrogen peroxide (H2 O2 ) decomposing enzymes concomitant with the
oxidation of wide range of phenolic as well as non- phenolic substrates (RH)

2RH + H2 O2 → 2R° + 2H2 O

 They are ubiquitous in nature being found in bacteria, fungi, algae, plants and animals.
 The plant peroxidases, belonging to Class III peroxidase, are implicated in various vital
processes of plant growth and development throughout the plant life cycle including cell
wall metabolism, lignification, suberization, reactive oxygen species (ROS) metabolism,
auxin metabolism, fruit growth and ripening, defense against pathogens etc.
 On the basis of presence or absence of heme, the peroxidases have been classified into
heme and non-heme peroxidases.
 Heme peroxidases have further been assigned to two superfamilies, namely peroxidase-
cyclooxygenase superfamily (PCOXS) and the peroxidase-catalase superfamily (PCATS)
  Due to versatility in reaction catalyzed by peroxidases, and their ubiquitous nature, they
have immense potential to be an industrial enzyme with application in various medicinal,
immunological, biotechnological and industrial sectors.
 The peroxidases find applications in bioremediation, textile synthetic dye decolorization,
polymer synthesis, paper and pulp industry, in development of biosensor, diagnosis kits
etc.
 In view of various applications, the identification of newer sources of novel peroxidase
offering resistance towards temperature, pH, salts, heavy metals, organic solvents etc. is
highly desirable.
Applications of peroxidases:
Application as biosensor
 Biosensors have significance in medicine, quality control, food and environmental
monitoring as well as in research. Enzyme based biosensors have advantageous over
other analytical techniques with regards to high selectivity and high sensitivity.
 Horse radish peroxidase (HRP) has widely been used in development of biosensors. HRP
biosensors are made by applying various detection methods including an amperometric
immunosensor, mass balance, potentiometric methods, photovoltaic spectroscopy, optical
and chemiluminescent methods etc.
 Soybean peroxidase (SBP) has been reported to be advantageousover HRP for the
biosensor manufacturing.
Application in analytical and diagnostic kits
 Among peroxidases, horseradish peroxidases are most commonly used for the analytical
purposes.
 In combination with cholesterol oxidase and cholesterol esterase, peroxidases have also
been exploited for developing cholesterol detection kits that help in quantification and
monitoring of human serum cholesterol.
 Peroxidases have been used in developing kits for the diagnosis of bladder and prostate
cancers through the detection of 8-hydroxydeoxyguanosine and its analogs in urine.
Application in de-colorization of industrial dyes
 Enzymes such as lignin peroxidases (LiPs) and manganese peroxidases (MnPs), are
involved in the decolorization of synthetic azo dyes such as orange II.
 Horseradish peroxidase (HRP) is reported to degrade phenol and substituted phenols via a
free radical polymerization mechanism and can be effective in degrading and
precipitating industrially important azo dyes, such as Remazol .
 A purified peroxidase produced by Geotrichum candidum dec1, was involved in
decolorization of dyes.
 Peroxidases have been shown to have great potential in the decolorization process to
decrease textile industry pollutant residue.
 Uses of peroxidases, such as horseradish, Brassica campestres turnip, tomato, bitter
gourd, soybean, Ipomea palmata and Saccharum spontaneum peroxidases, for degrading
and detoxifying polyaromatic hydrocarbons, polychlorinated biphenyls, and other
synthetic industrial dyes have been reported.
Application in pulp and paper industry
 Lignin, a phenolic heteropolymer, shows negative impact on the proper exploitation of
plant biomass for the pulp and paper industry.
Analytical applications
 Enzyme Linked Immunosorbent Assays (ELISA) has advantages over other
immunoassays in simplicity, low cost, reliability, simple equipment requirement etc.
 ELISA is usually developed to detect antigens or antibodies by producing an enzyme
such as peroxidase catalyzed color changes.
 The HRP-based or associated microarrays (biochips) have been used in various
applications such as expression analysis, recombination and gene mapping, mutation
analysis etc.

LIPASE

 Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are carboxylesterases that catalyze the

hydrolysis of triglycerides at the water-liquid interface.
 In non-aqueous conditions they catalyze the reverse reaction (such as esterification,
interesterification and transesterification) producing glycerides from glycerol and fatty
acids.
 Lipases can be obtained from animals mainly from forestomach tissue of calves or lambs
and pancreatic tissues of hogs and pigs.
 Plant lipases are also available but not exploited commercially because of the yield and
the processes involved.
 Generally, bacteria, fungi, yeast and actinomycetes are recognized as preferred sources of
extracellular lipases, facilitating the enzyme recovery from the culture broth: although
Candida, Pseudomonas, Mucor, Rhizopus and Geotrichum sp. stand out as the major
commercially viable strains.
APPLICATION OF LIPASES:
Lipases are forecasted to be among the fastest growing enzymes based on their
applications in organic synthesis, pharmaceuticals, and by expanded penetration into the
detergent industry and biofuel production.
Lipase in detergency
 lipase from Humicola strain capable of dissolving fatty stains.
 Bacillus sp. B207 and Pseudomonas paucimobilis produce alkaline lipases that can be
used as additives in the formulation of detergent.
Food industry applications:
 Modification of fats and oil based on their structure and composition is of great
importance in the food industries.
 The products obtained from lipase catalyzed reactions are considered to have wide
application in flavour synthesis, wines, baked foods, emulsifiers, supplements and
dairy products.
Flavour development:
 Low molecular weight esters such as ethyl, isobutyl, amyl and isoamyl acetates have
been widely used for flavour development in the food industry.
 Flavour substances such as S-methyl butanethioate and S-methyl 3-methyl
butanethioate are important components of the dairy aromas, especially cheese aroma
and of fruit aromas like strawberry and banana.
Lipases in oleochemical industry
Lipases in fatty acids unsaturation
 Biodiesel production
Biodegradation of plastics

PROTEASE
 Proteases are the group of enzymes that have been found in several microorganisms
like bacteria and fungi which are involved in breakdown of complex protein
molecules into simple polypeptide chains.
 The induction of protease requires a substrate like peptone, casein and other proteins.
 The ammonia as final product of enzymatic reaction of substrate hydrolysis,
responses enzyme synthesis by a well known mechanism of catabolite repression.
 This extracellular protease has also been commercially exploited to assist protein
degradation in various industrial processes.

Application of Alkaline Protease

Alkaline proteases are one of the most important groups of industrial enzymes widely used in
detergent, food and leather tanning industries.
Alkaline proteases can also be used on the hydrolysis of fibrous proteins such as horn, feather
and hair for converting them into useful biomass other potential industrial application of alkaline
protease include its utilization in peptide synthesis, resolution of racemic mixture of amino acids,
hydrolysis of gelatin laws of X-ray films and also in the recovery of silver.
Food Processing Industries
 In food industry, protease helps in processing and production of food products such as
meat, milk products and beverage.
 Proteases are used as tonic for proper digestion for children.
 Protease also plays an important role in processing tea, coffee and coco by oxidizing for
producing complete product.
 Microbe helps in sugar fermentation for ethanol production, along with other enzymes,
alkaline protease also aids in fermentation.
Leather Making
 Both fungal and bacterial proteases are used for leather processing, protease helps in
hydrolysis of non collagenous part of the skin non fibrillar protein.
 The leather sample processed by using alkaline protease was found to have maximum
softness.
Textile Industry
 Silk production is the back bone of textile industry, quality of silk determines the quality
of a fabric.
 Alkaline protease plays a major role in production of quality silk by removing gum and
other impurities produced along with silk’s native form, even synthetic fabric also treated
with protease for complete smooth finish.
Detergent Additives
 Protease helps removal of blood and other proteinaceous compounds.
 Protease has a great role in industries as detergent for sterilization since chemical
steriliants fails to remove minute trapped dirts.
 Hence, microbial protease commercially produced are used for cleaning large industrial
boilers, surgical instruments and also for various domestic purposes.
Medical applications of Alkaline protease
 Alkaline proteases shows a large variety of functions in medical field, which includes
from basic molecular level to whole organism therapeutic use such as haemostasis and
inflammation.
 Alkaline proteases are used extensively in the pharmaceutical industry for preparation of
medicines such as ointments for debridement of wounds.
 Additionally, a group of serine protease from Indian Earthworm has been studied for its
anti-inflammatory potential.
 Enzymes like caspase primarily involves in killing of abnormal cells, caspase is alkaline
protease enzyme which aids in proper immune system.
 A serine protease from Indian earthworm was evaluated for its antitumor activity against
breast cancer cell lines and result shown tremendous scope for protease in development
of anti-cancer therapeutics.
 Silver recovery from photographic films and x-ray films involves burning the films
directly oxidation of metallic silver followed by electrolysis stripping the silver-gelatin
layer using microbial enzymes especially protease which breaks the gelatin layer
embedded with silver in films approximately 1.5 % to 2.0 % (by weight) silver in its
gelatin layers. By using this method, pollution free stripping can be done.
 Alkaline proteases are like proteolytic enzyme aid in obtaining protein free nucleic acid
proportion.
 The most widely used proteolytic enzyme in nucleic acid purification is Proteinase K.
The Proteinase K also quickly inactivates the nucleases which might degrade the nucleic
acids present in the sample. It also helps in preventing degradation of DNA or RNA and
hence, high yield can be achieved.
----------------X---------------

PENICILLINASE

Penicillinase (penicillin amido-fl-Iactamhydrolase, EC 3.5.2.6) has been investigated since the

enzyme was first detected in a strain of E. coli/' and has been known to be produced by a wide
variety of microorganisms.
It has been reported that alkalophilic bacteria which grow in alkaline media produce alkaline
enzymes.
Production of Penicillinase
Media:
Isolation medium consisted of (per liter)
Glucose, :10 g;
Polypepton, :5 g;
Yeast extract, :5 g;
K2HPO4, :1 g;
MgSO.· :7g;
HzO, :0.2 g;
Agar, :20 g and
Andrade's indicator,(O) :40 ml.
Culture medium consisted of (per liter):
Glucose or glycerol, :2 g;
Polypepton, :5 g;
Yeast extract, :5 g;
K2HPO., :1 g and
MgS04'7HzO, : 0.2 g.

Before use, pH of the medium was adjusted to 9.0 with 10% sodium bicarbonate which was
sterilized separately.
Isolation of the organism
 The penicillinase-producing bacteria were isolated from soil according to the method.
 A bacterial strain No. 170 was selected as one of the most potential penicillinase
producer, and used for the present study.
Culture.
 Using 300 ml-flask containing 100 ml of medium, the strain No. 170 was cultivated at
30Q C for 23 hr on a rotary shaker; 19 hr after inoculation 10,000 units of benzyl
penicillin was added as an inducer of penicillinase, and cultivation was further continued
for 4 hr.
 The cell growth was followed by measuring the optical density at 610 nm.
Assay of the enzyme activity
 The penicillinase activity in the culture filtrate was determined by iodometry.'O) One unit
of enzyme activity was defined as the amount of enzyme which hydrolyzes 1 IIl110le of
benzyl penicillin per hr at 30°e.

Use:
 Some bacteria produce the enzyme penicillinase that destroys the beta-lactam ring of the
antibiotic, making the penicillin ineffective.
 Penicillinase resistant penicillins are used to treat resistant strains of staphylococci and
other infections.
--------------X-------------

GENETIC ENGINEERING TECHNOLOGY

Genetic Engineering Technology has made possible the manipulation of genetic information and
has been described as the new revolution in science. It is responsible for the development of new
industrial applications and areas of basic research. In the pharmaceutical field, alone, it has
applications to the production of natural and synthetic drugs (proteins/enzymes) at levels that can
not be achieved by conventional methods.
Through the application of recornbinant DNA and genetic engineering technologies the
approaches for detecting disease, for developing new drugs for treating disease and the methods
for drug manufacturing have been advanced. The purpose of this review is to highlight the
principles of these technologies and to describe some of the methods employed.
GENES AND GENE EXPRESSION
A fundarnental knowledge of heredity and the expression of genetic traits is necessary in order to
appreciate the principles of genetic engineering. DNA (deoxyribonucleic acid) is the molecule
that encodes all of the genetic characteristics of a cell and of an organism. DNA is composed of a
polymer of nucleotides attached to a ribose sugar-phosphate backbone (Figure I). There are four
different nucleotides, deoxyadenylate, deoxyguanylate, deoxycytidylate and deoxythymidylate.
DNA is normally in a double polynucleotide strand (double-stranded) configuration.

Gene Cloning:
APPLICATION
 In the pharmaceutical industry the cloned gene can be used directly in a diagnostic test.
 The utilization of recombinant DNA techniques for the isolation and expression of genes
that encode human interferon proteins has made production of large amounts of
interferon more economical than conventional production methods.
 A disease state that is caused by a dysfunction in a gene or the expression of that gene
may be alleviated by gene therapy.
 The strategy is to introduce and express a normal gene analog in the appropriate tissue of
the diseased organism using genetic engineering technology