BIO TECHNOLOGY

Bio technology
• Techniques of using live organisms or enzymes –
produce products & processes – useful to humans
• Restricted sense – genetically modified organisms
• Eg: in vitro fertilization – test tube baby
• Synthesising gene and using
• Developing DNA vaccine
• Correcting defective gene
Principles of Bio-technology
• Genetic engineering – techniques – alter the
chemistry of genetic material (DNA and RNA) – to
introduce in host organisms – change the phenotype
of host organisms
• Phenotype - refers to an individual’s observable traits,
such as height, eye color and blood type
• It is determined by both their genomic makeup
(genotype) and environmental factors
• Bioprocess Engineering
• Maintenance of sterile ambience
• enable growth – desired microbe/eukaryotic
cell
• large quantities
• manufacture – antibiotics, vaccines, enzymes
etc.
Sexual Vs Asexual
Sexual Reproduction Asexual Reproduction
Provides opportunities for Preserves genetic information
variations and formulation of
unique combinations of genetic
set up beneficial to organisms as
well as population
Hybridisation procedure in plant
and animal breeding – lead to
inclusion & multiplication of
undesirable genes along with
desired genes
 Gene cloning
Techniques of
Recombinant
Genetic
DNA
Engineering
Gene transfer
Recombinant DNA (rDNA)
• Recombinant DNA (rDNA) is a form of artificial DNA that is created by
combining genetic material from different sources.
• The process involves cutting and splicing DNA fragments from various
organisms to create new genetic combinations that do not naturally
occur.
• This technology is a critical part of genetic engineering and has
numerous applications in medicine, agriculture, and biotechnology.
Applications of Recombinant DNA
• Medicine: Recombinant DNA technology is used to produce
therapeutic proteins like insulin, growth hormones, and vaccines. It's
also important for gene therapy and genetic diagnostics.
• Agriculture: Recombinant DNA is used to create genetically modified
crops that are resistant to pests, diseases, or environmental
conditions. Examples include “Bt crops” and drought-resistant plants.
• Research: Recombinant DNA is a fundamental tool in molecular
biology research, allowing scientists to study gene function, protein
synthesis, and gene regulation.
Gene cloning
• Gene cloning is the process of creating copies of a particular gene or
segment of DNA.
• It is a type of recombinant DNA technology used to isolate and make
multiple copies of a specific gene for further study or application.
• This process is fundamental in molecular biology, biotechnology, and
genetic engineering.
 Key Steps in Gene Cloning
Isolation of the Gene of Interest: The first step in gene cloning is to isolate
the gene of interest from the organism's genome. This can be done using
techniques like PCR (Polymerase Chain Reaction) or restriction enzymes to
cut out the specific gene from the chromosomal DNA.
Insertion into a Vector: Once the gene is isolated, it is inserted into a vector,
which is usually a plasmid (a small circular DNA molecule found in bacteria).
The plasmid serves as a carrier to transfer the gene into a host cell. The
insertion is typically carried out using restriction enzymes that create sticky
ends on both the plasmid and the gene, enabling them to join together. The
enzyme ligase is then used to seal the gene into the plasmid.
Transformation: The recombinant vector (plasmid + gene of interest) is
introduced into a host cell through a process known as transformation.
Bacteria (like E. coli) are commonly used as host cells because they are easy
to manipulate, replicate quickly, and can take up plasmids efficiently.
Selection of Transformed Cells: After transformation, only the cells that
have successfully incorporated the recombinant plasmid (and thus the
gene of interest) will grow. This is typically done using a selectable
marker, such as an antibiotic resistance gene, which allows researchers to
identify and isolate the cells that have successfully taken up the plasmid.
Gene Expression or Amplification: Once the gene is inside the host cell,
it can be expressed (producing the protein of interest) or simply
amplified (making many copies of the gene). In cases where protein
production is the goal, the transformed cells may be cultured, and the
protein can then be harvested and purified for further use, such as in
therapeutic applications (e.g., producing insulin or vaccines).
Applications of Gene Cloning
1.Protein Production: Gene cloning allows the production of proteins for therapeutic
uses. For example, bacteria or yeast cells can be used to produce human proteins such
as insulin, growth hormones, or clotting factors for patients with medical conditions.
2.Gene Therapy: In gene therapy, gene cloning is used to insert a normal, functional
gene into a patient’s cells to treat genetic disorders caused by defective genes. This
technique aims to correct genetic abnormalities at the molecular level.
3.Genetic Research: Cloning genes helps researchers understand the function of specific
genes, study genetic diseases, and explore how different genes interact with each
other. This is essential for advancing our knowledge of genetics and disease
mechanisms.
4.Agricultural Biotechnology: Gene cloning is used to produce genetically modified
organisms (GMOs) with desirable traits, such as pest resistance, drought tolerance, or
enhanced nutritional content in crops.
5.Diagnostics: Cloned genes can be used in diagnostics to detect the presence of
specific genes or genetic mutations associated with diseases.
Gene transfer
• Gene transfer is the process of moving genetic material
(DNA) from one organism to another.
• It is a key technique in genetic engineering and
biotechnology, allowing for the introduction of new genes
into an organism’s genome to confer desirable traits or study
gene function.
• Gene transfer can occur naturally in nature (horizontal gene
transfer) or artificially in laboratory settings (genetic
modification).
Why DNA cannot transferred easily?
• Piece of DNA – not able to multiply – in progeny cells of the organisms
• Progeny cells refer to the daughter cells that result from cell division.
• These cells are genetically related to the parent cell and inherit a copy
of its genetic material, either identical or, in the case of mutations or
specific processes like meiosis, slightly different.
• The process of cell division is crucial for growth, repair, reproduction,
and maintaining the functions of an organism.
When can get transferred? – Origin of
replication
• Gets integrated into genome of recipient
• Multiply and be inherited along with the host DNA
• The piece of DNA has become part of a chromosome – ability
to replicate
• In chromosome – specific DNA sequence – responsible for
initiating replication - origin of replication
• Can replicate and multiply itself in the host organism
• Also called cloning – making multiple identical copies of any
template DNA
Construction of an artificial
recombinant DNA
• Possibility of linking a gene encoding antibiotic resistance with a
native “plasmid” of Salmonella typhimurium
• Plasmid – autonomously replicating circular extra – chromosomal
DNA
• Stanley Cohen & Herbert Boyer – 1972 – isolating the antibiotic
resistance gene – piece of DNA - from plasmid
• Cutting of DNA - Molecular scissors – restriction of enzymes
• Cut – piece of DNA linked with plasmid DNA – plasmid DNA act as
“Vectors” – transfers – the piece of DNA
Cloning of antibiotic resistance gene
in E. Coli
• Link antibiotic resistance gene with the plasmid vector
• Using enzyme DNA ligase
• This act on cut DNA molecules and joins them
• Makes new combination of circular autonomously replicating DNA –
created in vitro – recombinant DNA
• Transferred to Escherichia coli, bacterium closely related to
Salmonella
• Replicate using – new host DNA polymerase enzyme – make multiple
copies – antibiotic resistance gene - Cloning
Basic steps in genetic modiifcation

• Identification of DNA with desirable genes

• Introduction of identified DNA into the host
• Maintenance of introduced DNA in the host
• Transfer of the DNA to its progeny
Tools of recombinant DNA
technology
• Restriction enzymes
• Polymerase enzymes
• Ligases
• Vectors and
• Host organism
Restriction enzymes
• Restriction enzymes, also known as restriction endonucleases, are
proteins that act as molecular scissors to cut DNA at specific
sequences.
• They are found naturally in bacteria and archaea, where they serve as
part of the organism's defense mechanism against viral DNA
(bacteriophages).
• The enzymes recognize a specific short sequence of DNA, typically 4-8
base pairs long, and cut the DNA at or near this site.
• Type I: These enzymes cut the DNA at a random position, far from their
recognition site, and also have methylase activity (they can methylate
DNA).
• Type II: The most commonly used in molecular biology, these enzymes
recognize specific sequences and cut the DNA at or near this site. They do
not have methylase activity. Type II enzymes are widely used in genetic
engineering, cloning, and sequencing. Examples include EcoRI and BamHI.
• Type III: These enzymes cut DNA a short distance from their recognition
site. Like type I enzymes, they also have methylase activity.
• Type IV: These enzymes recognize and cut modified (e.g., methylated) DNA,
primarily serving to protect the organism from viral DNA.
• The recognition sequence for most Type II restriction enzymes is a
palindromic sequence, meaning it reads the same forward and
backward.
• After binding to the recognition site, the enzyme cleaves the DNA,
creating either blunt ends or sticky (cohesive) ends, depending on the
enzyme.
Applications in Molecular Biology
• Cloning: Restriction enzymes are used to cut DNA into fragments that
can be inserted into plasmids or vectors for cloning.
• DNA Fingerprinting: By cutting DNA at specific sites, restriction
enzymes can help generate unique patterns for identification
purposes.
• Genetic Engineering: They are used to manipulate genes by cutting
and inserting specific pieces of DNA into organisms.
• Gene Mapping: Scientists use restriction enzymes to break down DNA
into smaller fragments and map out the genes or sequence.
4-8 base pairs long
• Due to a balance between specificity and frequency, which helps
optimize the enzyme's function.
• 4 base pairs are short enough to generate fragments of reasonable
sizes but long enough to avoid random, non-specific cuts.
• 8 base pairs are more specific, which is useful for targeting specific
regions of DNA, but they cut less often, creating fewer, larger
fragments.
Methylase activity
• Methylase activity refers to the ability of certain enzymes, known as
methyltransferases or DNA methylases, to transfer a methyl group (–CH₃)
from a donor molecule (typically S-adenosylmethionine, SAM) to a
specific nucleotide within the DNA.
• This process is called DNA methylation, and it plays an important role in
the regulation and protection of DNA.
• The methylation system is an important part of the restriction-
modification system that helps bacteria distinguish between their own
DNA and foreign DNA, especially viral DNA.
• Restriction-modification system that helps bacteria distinguish between
their own DNA and foreign DNA, especially viral DNA.
The first restriction endonuclease–
Hind II
• Hind II - Functioning depended on a specific DNA nucleotide
sequence - isolated and characterized five years later
• Always cut DNA molecules at a particular point by recognizing a
specific sequence of six base pairs
• This specific base sequence is known as the recognition sequence for
Hind I
• 900 restriction enzymes that have been isolated from over 230 strains
of bacteria each of which recognize different recognition sequences
Naming of enzymes
• First letter comes from the genus
• Second two letters – species of prokaryotic cell (from which they
isolated)
• Roman numbers – order in which the enzymes were isolated from –
strains of bacteria
• EcoRI
• Escherichia coli RY 13
• R – name of the strain
• I – roman number – order in which the enzymes were isolated from the
strain of bacteria