BCH 405

Gene mapping
Gene mapping or genome mapping describes the methods used to identify the location of a gene
on a chromosome and the distances between genes. Gene mapping can also describe the
distances between different sites within a gene. The distances between various sites inside a gene
can also be described through gene mapping. Placing several molecular markers at specific
locations on the genome is the fundamental element of all genome mapping. There are many
types of molecular markers. When creating genome maps, genes can be observed as a particular
class of genetic markers mapped similarly to other markers.

Types of Gene Mapping

Genetic-linkage maps and physical maps are the two main categories of “Maps” used in gene
mapping. Both maps consist of genetic markers and gene loci. While physical maps involve
actual physical distances, often measured in number of base pairs, distances of genetic maps are
based on genetic linkage information. There are many gene mapping methods, including
comparative, physical, and genetic-linkage mapping. However, physical, and genetic-linkage
mapping are more common.
Physical mapping utilizes molecular biology techniques to inspect chromosomes. These
techniques consequently allow researchers to observe chromosomes directly so that a map may
be constructed with relative gene positions. Genetic mapping on the other hand uses genetic
techniques to indirectly find association between genes. Techniques can include cross-breeding
(hybrid) experiments and examining pedigrees. These techniques allow for maps to be
constructed so that relative positions of genes and other important sequences can be analyzed.
Genetic-linkage Mapping
Genetic-linkage maps show the location of each gene on a chromosome and their relative
distances from one another. Initially, these maps were created by tracing the inheritance of
several features, like eye colour and hair colour. Blood, saliva, or tissue samples from both
affected and unaffected family members are used to begin a genetic map. Saliva is the most often
used sample in genetic mapping, particularly personal genetic studies.
Genetic mapping is made possible by crossing over — a regular biological occurrence during
meiosis (cell division that produces sperm and egg cells). Chromosomes line up in pairs in the
centre of a cell during the first phase of meiosis, where they often “stick” to one another and
exchange similar fragments of themselves, also known as crossing over. Genetic mapping makes
it possible to determine which gene is present on each chromosome and where it is located
within that specific chromosome. Based on the distance between two genes, mapping can also
determine which gene is more likely to undergo recombination.
Physical Mapping
Physical maps always provide the actual DNA base pair distances between landmarks. It is one
gene mapping approach that has a high degree of accuracy in determining the sequence of DNA
base pairs.
A physical map provides the nucleotide numbers and the precise physical distance between
genetic markers. Radiation hybrid mapping, sequence mapping, and cytogenetic mapping are the
techniques used to produce a physical map. Physical mapping assembles larger DNA sections
using DNA markers and DNA fragments. Researchers can identify the locations of the DNA
bases from the overlapping sections of the fragments. Various physical mapping methods are
available to study genomes of multiple sizes and obtain different degrees of precision. Physical
mapping is a common approach used in genome sequencing to acquire an entire genome
sequence and determine whether there is any correlation between the specific DNA sequence and
phenotypic features.
Genetic Mapping Techniques
Recombination events are used in genetic mapping techniques to measure the distance between
genetic markers.
Random Fragment Length Polymorphism, or RFLP, measures the differences in homologous
DNA sequences to calculate the distance between two markers.
Currently, gene mapping analyses targeting single gene disorders use short tandem repeat
polymorphisms (STRP).
SNP (Single Nucleotide Polymorphism) is used in genome-wide association and linkage analysis
genetic research. Linkage analysis is studied using the inheritance of characteristic and genetic
signatures like SNPs and microsatellites.
Genome-Wide Association (GWA) studies the connections between traits and markers like SNPs
and microsatellites by treating the population as a single family. The method is used to map the
gene functions of common disorders.
Difference between genetic-linkage mapping and physical mapping

Genetic Mapping Physical Mapping

It is a technique which shows how genetic It is a technique which shows the physical
information is shuffled in a chromosome.. distance in a DNA sequence by working out
the number of base pairs.
It uses genetic markers to map the distance It uses restriction enzymes to cut the specific
between two genes. sequence of DNA.
It depicts the region of polymorphisms It depicts the actual distance of base pairs
(region where the DNA sequence differs) in along a stretch of DNA.
different individuals.
It gives mere insights for different regions of It depicts a more accurate representation of
chromosomes. the genome.

Applications:
Disease Mapping: Identifying genes associated with genetic disorders.
Marker-Assisted Breeding: Selecting for desirable traits in agriculture.
Evolutionary Studies: Understanding the genetic relationships between species.
Gene mapping has played a crucial role in advancing our understanding of genetics, inheritance
patterns, and the relationships between genes and various traits or diseases. Advances in
molecular biology and genomics continue to refine and expand our ability to map genes with
increasing precision.

Hybridomas applications of DNA technology in industries, agriculture,

medicine
Hybridoma cell lines are used to produce monoclonal antibodies for a wide range of applications
in research and therapeutics. Hybridomas are created by fusing two different cell types: B
lymphocytes (which produce antibodies) and myeloma cells (which can divide indefinitely).
Hybridomas, a technology developed by César Milstein and Georges Köhler in 1975, involve the
fusion of a specific type of antibody-producing cell (B cell) with a cancer cell (myeloma cell) to
create immortalized cells that produce monoclonal antibodies. This technology, central to DNA-
based methods, has found applications in various industries, agriculture, and medicine.
Industries
Biopharmaceutical Production: Hybridomas play a crucial role in the production of
monoclonal antibodies (mAbs) for therapeutic use. These mAbs are used in treating various
diseases, such as cancer and autoimmune disorders. They are employed in the development of
biopharmaceuticals, contributing to advancements in the pharmaceutical industry.
Protein Purification: Monoclonal antibodies produced by hybridomas are widely used in
protein purification processes. Affinity chromatography, utilizing these antibodies, helps isolate
and purify specific proteins of interest in industrial settings.
Diagnostic Assays: Hybridoma-derived monoclonal antibodies are used in diagnostic tests for
the detection of specific biomarkers, antigens, or pathogens. These tests are crucial in medical
diagnostics and quality control in various industries.
Bioprocessing: In bioprocessing, hybridomas are involved in the large-scale production of
therapeutic proteins, including monoclonal antibodies, through bioreactor systems.
Agriculture
Disease Detection in Crops: Monoclonal antibodies can be used to develop diagnostic tools for
identifying plant diseases. This aids in early detection and management of crop diseases in
agriculture.
Pest Control: Hybridomas can contribute to the development of methods for detecting and
controlling pests in crops by identifying specific antigens associated with harmful insects.
Genetic Engineering in Crops: DNA technology, including hybridoma-derived antibodies, can
be used in genetic engineering to develop crops with enhanced resistance to diseases or pests.
Medicine
Disease Diagnosis: Monoclonal antibodies derived from hybridomas are used in diagnostic tests
to detect specific markers associated with diseases, enabling early and accurate diagnosis.
Cancer Treatment: Monoclonal antibodies, such as those produced by hybridomas, are
employed in targeted cancer therapies. These antibodies can specifically target cancer cells,
minimizing damage to healthy tissues.
Autoimmune Diseases: Hybridoma-derived monoclonal antibodies are used in the treatment of
autoimmune diseases by modulating the immune response and reducing inflammation.
Vaccine Development: Monoclonal antibodies play a role in vaccine development, helping
researchers identify and characterize antigens that can be targeted to induce an immune response.
Research Tools: Hybridomas are essential tools in biomedical research, facilitating the study of
specific proteins, cells, and pathogens. They enable researchers to better understand the
molecular mechanisms underlying diseases.
The applications of hybridomas in industries, agriculture, and medicine showcase the versatility
and impact of this technology across different fields. From diagnostics to therapeutic
interventions, hybridomas and their derived monoclonal antibodies have become integral
components in various applications that benefit society.

Enzyme Technology
Enzyme technology is one of the corner stones of Industrial Biotechnology. The research in this
area involves both fundamental and applied enzymology, biocatalysis, molecular modelling,
structural biology and diagnostics. Enzyme technology involves the use of enzymes as
biocatalysts to perform specific functions in various industrial, medical, and research
applications. Enzymes are biological catalysts that accelerate chemical reactions without being
consumed in the process.
Applications of Enzyme Technology
Detergent Industry
Laundry Detergents: Enzymes like proteases and lipases are used in laundry detergents to break
down protein-based stains (such as blood and grass) and oily stains more effectively than
traditional chemical detergents.
Food and Beverage Industry
Brewing and Distilling: Enzymes, particularly amylases, are employed in the conversion of
starches into sugars during the production of beer and spirits.
Baking Industry Enzymes like amylases and lipases improve dough handling, texture, and shelf
life in baking processes.
Fruit Juice Production: Enzymes help in the clarification and extraction of juice from fruits,
improving the overall yield and quality.
Textile Industry
Biopolishing: Enzymes, such as cellulases, are used in biopolishing treatments to remove fuzz
and pills from fabric, resulting in a smoother and softer finish.
Biofuel Production
Ethanol Production: Enzymes, such as cellulases and amylases, are used in the conversion of
biomass into sugars, which can then be fermented to produce biofuels like ethanol.
Pharmaceuticals
Drug Manufacturing: Enzymes are used in pharmaceutical synthesis for the production of
specific drugs and intermediates in a more environmentally friendly manner.
Diagnostic Enzymes: Enzymes are employed in diagnostic assays for the detection of various
biomarkers, such as enzymes used in glucose monitoring for diabetes.
Environmental Applications
Waste Treatment: Enzymes play a role in breaking down organic matter in wastewater
treatment processes.
Bioremediation: Enzymes are utilized to degrade pollutants and contaminants in soil and water,
contributing to environmental cleanup.
Paper and Pulp Industry
Bleaching Processes: Enzymes, such as xylanases and ligninases, are used in the pulp and paper
industry to reduce the environmental impact of bleaching processes.
Genetic Engineering and Molecular Biology
DNA Manipulation: Enzymes like restriction endonucleases and DNA ligases are essential tools
in genetic engineering for manipulating DNA molecules.
Polymerase Chain Reaction (PCR): DNA polymerases, an enzyme crucial for PCR, are used in
the amplification of DNA for various applications in molecular biology.
Animal Feed Industry
Feed Digestibility: Enzymes like proteases and carbohydrases are added to animal feed to
enhance nutrient availability and improve digestibility.
Medical and Therapeutic Applications
Enzyme Replacement Therapy: Enzymes are used in medical treatments, such as enzyme
replacement therapy for individuals with genetic enzyme deficiencies.
Diagnostic Enzymes: Enzymes are used in clinical diagnostics, including assays for liver
function, cardiac markers, and more.
Advantages of Enzyme Technology
Specificity: Enzymes exhibit high substrate specificity, allowing for precise control over
reactions.
Mild Reaction Conditions: Enzymes often operate under mild conditions (e.g., moderate
temperatures and pH levels), reducing the need for harsh chemicals.
Biodegradability: Enzymes are generally biodegradable and environmentally friendly.
Catalytic Efficiency: Enzymes can catalyze reactions at impressive rates, increasing the overall
efficiency of processes.
Enzyme technology continues to evolve, offering sustainable and efficient solutions in a wide
range of industries, contributing to cleaner and more environmentally friendly processes.

Immobilized enzymes and application

Immobilization of enzymes (or cells) refers to the technique of confining/anchoring the enzymes
(or cells) in or on an inert support for their stability and functional reuse. By employing this
technique, enzymes are made more efficient and cost-effective for their industrial use. Some
workers regard immobilization as a goose with a golden egg in enzyme technology. Immobilized
enzymes retain their structural conformation necessary for catalysis.
Methods of Immobilization
Enzymes can be immobilized through physical or chemical methods, each with its own
advantages and considerations.

Physical Methods
Adsorption: Involves reversible immobilization by physically attaching enzymes onto a support
substance through non-specific forces like van der Waals, hydrogen bonds, and hydrophobic
interactions. Example: Adsorption on glass, alginate beads, or matrix involves enzyme
attachment to an inert material. This method is slow, and the active site may be blocked,
reducing enzyme activity.
Entrapment: An irreversible physical technique where enzymes are physically restricted in a
specified area or space. It enhances mechanical stability and reduces enzyme leaching, protecting
enzymes from denaturation. Example: Enzymes are trapped in insoluble beads or microspheres,
like calcium alginate beads. However, substrate arrival and product exit may be hindered.

Chemical Methods
Cross-linking: An irreversible method without support material, where enzyme molecules are
covalently bonded to create a matrix, maintaining enzyme activity. Spacer molecules like
poly(ethylene glycol) reduce steric hindrance. The covalent bonds lack self-healing properties
seen in chemo-adsorbed self-assembled monolayers.
Covalent Bonding: Enzymes are covalently bound to an insoluble support, providing strong
enzyme/support interaction and minimal protein leakage during catalysis. The activity depends
on carrier material shape, size, coupling method, and special conditions.
Affinity-Tag Binding: Combines physical and chemical methods, allowing enzymes to be
immobilized on a surface using non-covalent or covalent protein tags. This method is applicable
without prior enzyme purification. Example: Porous glass and derivatives are used, and the
porous surface can be adjusted for enzyme compatibility.

Diagram illustrating major enzyme immobilization methods. Reversible methods include

adsorption, ionic bonding, and affinity bonding. Irreversible methods include covalent binding,
entrapment, encapsulation, and cross-linking

Applications of Immobilized Enzymes:

Biocatalysis in Industry
Chemical Synthesis: Immobilized enzymes are used in the synthesis of various chemicals,
pharmaceuticals, and fine chemicals. For example, lipases are used in the synthesis of biodiesel.
Food and Beverage Industry
Brewing and Distilling: Immobilized enzymes, such as amylases, are used in beer production
for starch hydrolysis.
Fruit Juice Clarification: Pectinase immobilized on supports helps clarify fruit juices by
breaking down pectin.
Pharmaceuticals
Drug Synthesis: Immobilized enzymes are employed in pharmaceutical processes for the
synthesis of specific drugs and intermediates.
Waste Treatment
Bioremediation: Immobilized enzymes are used for the degradation of pollutants and the
treatment of wastewater.
Clinical Diagnostics
Enzyme Immobilization in Biosensors: Immobilized enzymes are used in biosensors for
clinical diagnostics, detecting specific analytes in blood or other bodily fluids.
Textile Industry
Biopolishing: Immobilized cellulases are used for biopolishing textiles, improving fabric feel
and appearance.
Analytical Chemistry
Enzyme Immobilization in Columns: Immobilized enzymes in columns are used for analytical
purposes, such as the detection and quantification of specific compounds.
DNA Technology
PCR (Polymerase Chain Reaction): Enzymes like DNA polymerase can be immobilized on
beads for use in PCR applications.
Medical Devices
Implantable Enzyme Systems: Immobilized enzymes are investigated for use in medical
devices, such as glucose sensors for diabetes management.
Biodegradable Materials
Enzyme Immobilization for Environmental Applications: Immobilized enzymes are used in
the production of biodegradable materials, contributing to sustainable practices.
There are several advantages of immobilized enzymes:
1. Stable and more efficient in function.
2. Can be reused again and again.
3. Products are enzyme-free.
4. Ideal for multi-enzyme reaction systems.
5. Control of enzyme function is easy.
6. Suitable for industrial and medical use.
7. Minimize effluent disposal problems.
8. high enzyme substrate ratio.
9. Minimum reaction time.
10. Continuous use of enzyme.

There are however, certain disadvantages also associated with immobilization.

1. The possibility of loss of biological activity of an enzyme during immobilization or
while it is in use.
2. Immobilization is an expensive affair often requiring sophisticated equipment.
3. Some enzyme become unstable after immobilization.
4. Sometimes enzymes become inactivated by the heat generated by the system.