Animal Biotechnology

Cloning vector
Certainly! Let's dive into more detailed characteristics of each cloning vector:
1. *Plasmids:*
- *Structure:* Circular, double-stranded DNA typically found in bacteria.
- *Replication:* Replicates independently of the host genome, often with high copy
numbers.
- *Selectable Markers:* Often carries antibiotic resistance genes for easy selection
in host cells.
- *Applications:* Commonly used for gene cloning, protein expression, and
creating genetically modified organisms.

2. *Cosmids:*
- *Structure:* Hybrid vectors with plasmid and phage characteristics.
- *Insert Size:* Can carry larger DNA fragments (up to 45-50 kb).
- *Packaging:* Utilizes phage packaging mechanisms for DNA transfer.
- *Applications:* Valuable for constructing genomic libraries due to their ability to
accommodate larger inserts.

3. *Phagemids:*
- *Structure:* Hybrid vectors containing plasmid and phage elements.
- *Advantages:* Combines the stability of plasmids with the efficient packaging of
phages.
- *Applications:* Often used in phage display systems for the expression and
selection of proteins.

4. *Lambda Bacteriophage:*
- *Structure:* Linear double-stranded DNA virus.
- *Insert Size:* Efficiently carries large DNA fragments (up to 20-25 kb).
- *Integration:* Can integrate into the host genome or replicate independently.
- *Applications:* Useful for constructing genomic libraries and cloning large DNA
fragments.

5. *M13:*
- *Structure:* Single-stranded DNA phage.
- *Applications:* Primarily used for DNA sequencing and cloning small DNA
fragments (up to about 10 kb).
- *Phagemid Usage:* Incorporated into phagemids for efficient single-stranded
DNA cloning.

6. *BAC (Bacterial Artificial Chromosome):*

- *Structure:* Large, low-copy-number vectors.
- *Insert Size:* Designed for stable maintenance of large DNA inserts (up to 300
kb).
- *Applications:* Suitable for cloning entire genes, large genomic fragments, and
artificial chromosomes.

7. *YAC (Yeast Artificial Chromosome):*

- *Structure:* Designed for use in yeast cells.
 - *Insert Size:* Can accommodate very large DNA fragments (up to several
megabases).
- *Applications:* Useful for cloning large eukaryotic genes and studying gene
regulation.

8. *MAC (Mammalian Artificial Chromosome):*

- *Structure:* Similar to YAC but designed for mammalian cells.
- *Applications:* Allows the study of large genomic regions in mammalian systems,
including potential gene therapy applications.

9. *Expression Vectors:*
- *Characteristics:* Engineered to facilitate efficient expression of inserted genes.
- *Components:* Include strong promoters, enhancers, and other regulatory
elements.
- *Applications:* Used for producing proteins of interest, studying gene function,
and creating recombinant proteins.

These vectors collectively provide a diverse toolkit for genetic manipulation and
exploration across various organisms and research objectives.

Restriction enzymes

Introduction

Watson and Crick's description, in 1953, of the double helical structure of the DNA
molecule opened the door to a new era in biological understanding and research.
Scientists, now knowing the molecular structure of the hereditary molecule, could
begin both to elucidate and to manipulate its function. These new studies were,
however, dependent on the discovery and use of the many enzymes that are able to
modify or join existing DNA molecules, or to aid in the synthesis of new DNA
molecules. One such discovery was restriction endonucleases.

Restriction Endonucleases/Enzymes
Restriction enzymes are bacterial proteins that recognize specific DNA sequences and
cut DNA at or near the recognition site. These enzymes are widely used in molecular
genetics for analyzing DNA and creating recombinant DNA molecules.

Biological Function and Historical Background

Restriction enzymes apparently evolved as a primitive immune system in bacteria. If
viruses infects a bacterial cell containing restriction enzymes, the viral DNA is
fragmented, which prevents destruction of the bacterial cell by the virus. The term
"restriction" derives from the phenomenon in which bacterial viruses are restricted
from replicating in certain strains of bacteria by enzymes that cleave the viral DNA,
but leave the bacterial DNA untouched. In bacteria, restriction enzymes form a system
with modification enzymes that methylate the bacterial DNA. Methylation of DNA at
the recognition sequence typically protects the bacteria from cleaving its own DNA.
Since the 1970s, restriction enzymes have had a very important role in recombinant
DNA techniques, both in the creation and analysis of recombinant DNA molecules.
The first restriction enzyme was isolated and characterized in 1968, and over 3,400
restriction enzymes have been discovered since. Of these enzymes, over 540 are
commercially available.

Nomenclature and Classification

Restriction enzymes are named based on the organism in which they were discovered.
For example, the enzyme Hind III was isolated from Haemophilus influenzae, strain
Rd. The first three letters of the name are italicized because they abbreviate the genus
and species of the organism. The fourth letter typically comes from the bacterial strain
designation. The Roman numerals are used to identify specific enzymes from bacteria
that contain multiple restriction enzymes. Typically, the Roman numeral indicates the
order in which restriction enzymes were discovered in a particular strain. There are
three classes of restriction enzymes: types I, II, and III.

Type I restriction systems consist of a single enzyme that performs both modification
(methylation) and restriction activities. These enzymes recognize specific DNA
sequences, but cleave the DNA strand randomly, at least 1,000 base pairs (bp) away
from the recognition site.

Type III restriction systems have separate enzymes for restriction and methylation,
but these enzymes share a common subunit. These enzymes recognize specific DNA
sequences, but cleave DNA at random sequences approximately twenty-five bp from
the recognition sequence. Neither type I nor type III restriction systems have
found much application in recombinant DNA techniques.

Type II restriction enzymes, in contrast, are regularly used in recombinant DNA

techniques. In type II, restriction and modification are catalysed by separate enzymes.
One enzyme recognizes and cuts DNA, the other enzyme recognizes and methylates
the DNA. Type II restriction enzymes cleave the DNA sequence at the same site at
which they recognize it. The only exception are type IIs (shifted) restriction enzymes,
which cleave DNA on one side of the recognition sequence, within twenty nucleotides
of the recognition site. Type II restriction enzymes discovered to date collectively
recognize over 200 different DNA sequences.

The substrates for restriction enzymes are more-or-less specific sequences of double-
stranded DNA called recognition sequences. Examining the following table will
illustrate some important points (recognition sites are shown as double stranded
DNA).

Type II restriction enzymes are enzymes that recognize specific DNA sequences and
cleave the DNA at or near these recognition sites. Here's a more detailed study of
Type II restriction enzymes:

1. *Recognition Sequence:*
- *Definition:* Type II restriction enzymes recognize specific DNA sequences,
which are usually palindromic, meaning they read the same backward and forward.
- *Example:* EcoRI recognizes the palindromic sequence GAATTC.

2. *Cutting Site:*
- *Location:* Type II restriction enzymes cleave DNA at specific positions within
or near their recognition sequences.
 - *Result:* The cleavage results in fragments with either blunt ends or overhanging
ends (sticky ends), depending on the enzyme.

3. *Enzyme Structure:*
- *Domains:* Type II restriction enzymes typically have distinct domains for DNA
binding and catalysis.
- *Multimeric Nature:* Some enzymes are homodimers (two identical subunits),
while others are homotetramers (four identical subunits).

4. *Catalytic Mechanism:*
- *Hydrolysis:* Cleavage involves hydrolysis of the phosphodiester bond in the
DNA backbone.
- *Metal Ions:* Many Type II restriction enzymes use metal ions, such as Mg²⁺, as
cofactors for catalysis.

5. *Overhangs (Sticky Ends) vs. Blunt Ends:*

- *Sticky Ends:* Some enzymes create overhanging ends, which can base-pair with
complementary overhangs on other DNA fragments.
- *Blunt Ends:* Others produce blunt ends, which have no overhangs.

6. *Methylation Sensitivity:*
- *Protection Mechanism:* Many Type II restriction enzymes are sensitive to the
methylation state of their recognition sites.
- *Host DNA Protection:* The host organism's DNA is often methylated at these
sites, protecting it from cleavage by its own restriction enzymes.

7. *Applications:*
- *Molecular Cloning:* Type II restriction enzymes are widely used in molecular
biology for DNA cloning. They allow the insertion of specific DNA fragments into
plasmids or other vectors.
- *DNA Fragment Analysis:* These enzymes are also used in DNA fingerprinting
and other genetic analyses.

8. *Commercial Availability:*
- *Variety:* There is a vast array of commercially available Type II restriction
enzymes with different recognition sequences and cutting patterns.
- *Nomenclature:* The enzymes are often named based on the organism they are
derived from (e.g., EcoRI from Escherichia coli).

9. *Evolutionary Significance:*
- *Defense Mechanism:* In bacteria, restriction enzymes act as a defense
mechanism against invading phages by cleaving their DNA.
- *Methylase Pairing:* The restriction enzymes work in conjunction with DNA
methylases, which methylate the host DNA to protect it from self-cleavage.

Understanding the properties and functions of Type II restriction enzymes is

fundamental for their successful use in various molecular biology techniques,
contributing significantly to genetic research and biotechnology.
DNA modification enzymes

1. *DNA Methyltransferases:*
- *Function:* These enzymes add methyl groups to cytosine bases in DNA, a
process known as DNA methylation.
- *Role:* DNA methylation is involved in gene regulation, genomic imprinting, and
maintaining chromosomal stability.

2. *DNA Glycosylases:*
- *Function:* DNA glycosylases recognize and remove damaged or mismatched
bases by cleaving the glycosidic bond between the base and the sugar in the DNA
backbone.
- *Role:* Repair of DNA damage, ensuring genomic integrity. Base excision repair
(BER) is a common mechanism involving DNA glycosylases.

3. *Restriction Enzymes:*
- *Function:* These enzymes recognize specific DNA sequences and cleave the
DNA at or near these sequences.
- *Role:* Used in molecular biology for genetic engineering (e.g., cloning), as they
enable the precise cutting of DNA at desired locations.

4. *DNA Ligases:*
- *Function:* DNA ligases join broken DNA strands by catalyzing the formation of
phosphodiester bonds between adjacent nucleotides.
- *Role:* Essential in DNA repair processes, particularly in sealing nicks and gaps.

5. *Topoisomerases:*
- *Function:* These enzymes alter the topological state of DNA by introducing or
removing supercoils.
- *Role:* Important for DNA replication, transcription, and repair, helping to
relieve torsional stress.

6. *Helicases:*
- *Function:* Helicases unwind the DNA double helix by breaking hydrogen bonds
between complementary bases.
- *Role:* Critical for processes like DNA replication and repair, where access to
single-stranded DNA is necessary.

7. *DNA Polymerases:*
- *Function:* DNA polymerases synthesize new DNA strands by adding
nucleotides complementary to a template DNA strand.
- *Role:* Essential in DNA replication and repair, ensuring accurate duplication of
genetic information.

Understanding the functions of these enzymes is fundamental to grasp the intricate

processes that govern DNA modification and maintenance within cells.
Certainly, let's go into more detailed explanations of the function and working of each
DNA modification enzyme:

1. *Alkaline Phosphatase:*
- *Function:* Alkaline phosphatase removes phosphate groups from the 5' ends of
DNA molecules.
- *Working:* During cloning procedures, DNA is often cut with restriction enzymes.
Alkaline phosphatase is then used to dephosphorylate the 5' ends of the linearized
DNA, preventing the ends from rejoining (self-ligation) and promoting the insertion
of a foreign DNA fragment.

2. *Terminal Transferase:*
- *Function:* Terminal transferase adds nucleotides to the 3' ends of DNA
molecules.
- *Working:* In DNA labeling or tailing procedures, terminal transferase catalyzes
the addition of nucleotides to the 3' overhangs of DNA, facilitating various
applications such as labeling DNA for hybridization studies or modifying DNA ends
for cloning.

3. *Reverse Transcriptase:*
- *Function:* Reverse transcriptase catalyzes the synthesis of complementary DNA
(cDNA) from an RNA template.
- *Working:* In reverse transcription reactions, reverse transcriptase synthesizes a
complementary DNA strand from an RNA template. This process is fundamental in
converting RNA into a form (cDNA) that can be further amplified and studied,
commonly used in techniques like RT-PCR.

4. *T4 DNA Kinase:*

- *Function:* T4 DNA kinase phosphorylates the 5' termini of DNA and RNA.
- *Working:* T4 DNA kinase adds phosphate groups to the 5' ends of DNA or RNA
molecules. This enzymatic activity is crucial for preparing DNA fragments for
subsequent ligation reactions or for labeling nucleic acids for various applications.

5. *Ligases:*
- *Function:* Ligases catalyze the formation of phosphodiester bonds between the
3' hydroxyl and 5' phosphate ends of DNA fragments.
- *Working:* DNA ligases are essential in DNA cloning. After restriction enzymes
cut DNA, ligases seal the nicks in the DNA backbone by joining the 3' and 5' ends of
the fragments, creating a continuous DNA strand.

6. *CRISPR Cas-9:* (Clustered Regularly Interspaced Short Palindromic

Repeats and CRISPR-associated protein 9)
- *Function:* CRISPR Cas-9 is an endonuclease that induces site-specific double-
strand breaks in DNA.
- *Working:* Cas-9 is guided to a specific DNA sequence by a guide RNA (gRNA).
Once at the target site, Cas-9 introduces breaks in the DNA. Cellular repair
mechanisms then kick in, leading to the addition, deletion, or replacement of genetic
material, enabling precise genome editing.
These enzymes, with their specific functions, play critical roles in molecular biology
techniques, allowing scientists to manipulate DNA for various research and practical
applications.

Transformation techniques

Transformation is the introduction of DNA into bacterial cells. For transformation,

cells should be in early logarithmic phase. Since DNA is a very hydrophilic molecule,
it won't normally pass through a bacterial cell's membrane. In order to make bacteria
take in the plasmid, they must first be made "competent" to take up DNA. This is
done by creating small holes in the bacterial cells by suspending them in a solution
with a high concentration of calcium. Bacterial cell wall is permeable to chloride ions
but not calcium ions. As the chloride ions enter the cell, water molecules accompany
the charged particles. This leads to swelling which is necessary for uptake of DNA.
DNA can then be forced into the cells by incubating the cells and the DNA together
on ice, followed by placing them briefly at 42oC (heat shock), and then putting them
back on ice. The heat shock treatment, leads to expression of heat shock genes, which
increases the ability of bacterial cells to survive and also enhances the uptake of DNA.
Exact temperature is important to obtain the highest transformation efficiency. If the
temperature is too cold the bacteria will not take up the DNA, while if it is too hot the
bacteria will die. The surviving cells are then allowed to revive from the heat shock
and multiply by suspending them in a medium such as Luria Bertani (LB) broth and
incubating at a 37ºC temperature for about an hour. The cells are then plated on an
agar medium containing antibiotic. The plasmid DNA often contains a gene whose
encoded protein renders the bacteria immune to the antibiotic. Therefore, only
bacteria that have been transformed will be able to survive.

Transformation is a genetic engineering technique used to introduce foreign DNA into

host cells, allowing the cells to express new traits or functions. Here's a detailed
explanation of transformation techniques:

1. *Natural Transformation:*
- *Process:* Some bacteria are naturally competent, meaning they can take up
foreign DNA from their environment.
- *Mechanism:* During natural transformation, the bacterial cell incorporates
external DNA fragments into its genome through a series of complex molecular
processes.

2. *Chemical Transformation:*
- *Process:* Chemical transformation involves treating bacterial cells with
chemicals that increase their permeability to DNA.
- *Common Method:* Calcium chloride (CaCl₂) treatment is often used, making the
cell membranes more porous and allowing foreign DNA to enter.

3. *Electroporation:*
- *Process:* In electroporation, an electric field is applied to cells, creating
temporary pores in the cell membrane.
- *Mechanism:* The electric field facilitates the entry of DNA into the cells, as the
pores permit the passage of foreign genetic material.
4. *Heat Shock Transformation:*
- *Process:* Heat shock transformation involves subjecting cells to a sudden
increase in temperature.
- *Purpose:* This transiently increases the permeability of the cell membrane,
allowing DNA to enter.

5. *Biological (Biolistic) Transformation:*

- *Process:* Also known as biolistic or gene gun transformation, it involves the use
of microscopic particles coated with DNA.
- *Mechanism:* The particles are propelled into the target cells, and upon impact,
the DNA-coated particles can be integrated into the host genome.

6. *Agrobacterium-Mediated Transformation:*
- *Applicability:* Commonly used in plant genetic engineering.
- *Bacterium:* Agrobacterium tumefaciens, a soil bacterium, is modified to carry
the desired DNA and transfer it to plant cells.

7. *Liposome-Mediated Transformation:*
- *Process:* Liposomes, which are lipid vesicles, can encapsulate and deliver DNA
into cells.
- *Mechanism:* The liposomes fuse with the cell membrane, releasing the DNA
into the cell.

8. *Viral Vector-Mediated Transformation:*

- *Application:* Often used in eukaryotic cells, especially for gene therapy.
- *Mechanism:* Viral vectors, modified to be non-pathogenic, are used to deliver
therapeutic genes into target cells.

9. *Microinjection:*
- *Process:* A micropipette is used to inject DNA directly into the nucleus of a
target cell.
- *Application:* Commonly used in animal cells and embryos for genetic
manipulation.

10. *Pulsed-Field Gel Electrophoresis (PFGE):*

- *Application:* Primarily used in yeast and certain bacteria.
- *Process:* High-voltage electric fields are applied in a specific direction,
allowing the movement and separation of large DNA fragments.

Transformation techniques play a pivotal role in genetic engineering, enabling the

introduction of specific genetic material into various organisms for research,
industrial, and medical purposes. The choice of the method depends on the
characteristics of the host organism and the desired outcome of the genetic
modification.
Certainly, let's delve into detailed explanations of Calcium Chloride Transformation
and Electroporation, two common methods used in genetic engineering for
introducing foreign DNA into cells:

1. *Calcium Chloride Transformation:*

- *Process:*
- Cells are treated with a solution containing calcium chloride (CaCl₂).
- The presence of CaCl₂ increases the permeability of the cell membrane.
- This treatment allows the cell to take up foreign DNA from the surrounding
environment.

- *Mechanism:*
- Calcium ions (Ca²⁺) interact with the negatively charged phosphate groups on the
cell membrane and DNA, neutralizing the charges.
- The neutralization reduces the electrostatic repulsion between the cell membrane
and DNA, facilitating the entry of DNA into the cell.

- *Protocol:*
- Cells are incubated in a CaCl₂ solution containing the DNA of interest.
- Following this treatment, the cells are often subjected to a heat shock, which
further aids in DNA uptake.
- The foreign DNA may integrate into the host genome or exist as an
extrachromosomal element (e.g., plasmid).

- *Applications:*
- Commonly used in bacterial transformation, especially with Escherichia coli.
- Efficient for introducing plasmids carrying specific genes into bacterial cells.

2. *Electroporation:*
- *Process:*
- Cells are exposed to an electric field of high voltage for a short duration.
- This electric field induces temporary pores in the cell membrane.
- These pores allow for the entry of foreign DNA into the cells.

- *Mechanism:*
- The electric field disrupts the cell membrane, creating temporary gaps or pores.
- The pores allow DNA molecules to move into the cell through electrophoresis,
driven by the electric field.

- *Protocol:*
- Cells are mixed with the DNA of interest.
- The cell-DNA mixture is then subjected to a brief pulse of high-voltage
electricity.
- The electroporated cells can then recover in a culture medium, and the introduced
DNA may become integrated into the cellular genome.

- *Applications:*
- Widely used in various cell types, including bacteria, yeast, and mammalian cells.
- Effective for introducing both plasmid DNA and linear DNA fragments into cells.
- Commonly employed in molecular biology and genetic engineering experiments.
Both Calcium Chloride Transformation and Electroporation are versatile methods
with applications in different organisms. The choice between these methods depends
on factors such as the type of host organism, the efficiency of transformation required,
and the nature of the genetic modification being introduced.

Construction of genomic and CDNA libraries

Certainly, let's delve into more detail for each step of constructing genomic and
cDNA libraries:

### Genomic Library Construction:

1. *Isolation of DNA:*
- Begin by obtaining high-quality genomic DNA from the organism of interest using
methods like phenol-chloroform extraction or commercial DNA extraction kits.

2. *Fragmentation:*
- Use restriction enzymes to cleave the genomic DNA into fragments. These
enzymes recognize specific DNA sequences, generating fragments with cohesive
(sticky) ends.

3. *Cloning:*
- Choose a vector (commonly a plasmid or bacteriophage) with compatible cohesive
ends. Ligase is then used to join the DNA fragments with the vector, creating
recombinant DNA.

4. *Transformation:*
- Introduce the recombinant DNA into a host organism, often bacteria. This is done
through a process like heat shock, allowing the bacteria to take up the recombinant
vectors.

5. *Screening:*
- Identify transformed bacterial colonies by selective markers (e.g., antibiotic
resistance) on the vector. Screening involves techniques like hybridization or
polymerase chain reaction (PCR) using specific probes or primers to identify clones
containing the desired genomic sequences.

### cDNA Library Construction:

1. *mRNA Extraction:*
- Isolate mRNA from the cells of interest. This can be achieved using techniques
like poly(A) tail selection or oligo(dT) priming.

2. *Reverse Transcription:*
- Use reverse transcriptase to synthesize complementary DNA (cDNA) from the
mRNA template. This results in a single-stranded cDNA molecule.

3. *Second Strand Synthesis:*

 - Convert the single-stranded cDNA into double-stranded DNA. This is typically
achieved through the use of DNA polymerase.

4. *Cloning:*
- Similar to genomic libraries, insert the double-stranded cDNA into a vector,
creating recombinant DNA. Vectors used for cDNA libraries often lack introns to
ensure that the cloned DNA represents only exonic regions.

5. *Transformation:*
- Introduce the recombinant DNA into a host organism, commonly bacteria, through
methods like electroporation or heat shock.

6. *Screening:*
- Identify clones containing cDNA sequences of interest. This often involves using
probes or primers specific to the cDNA of interest, followed by screening techniques
like PCR or hybridization.

These libraries are valuable resources for studying the genetic information of
organisms and understanding gene expression patterns in different tissues or under
various conditions.

Screening by colony and plaque hybridization

Screening by colony and plaque hybridization is a technique used to identify clones

containing specific DNA sequences in a genomic or cDNA library.

### Colony Hybridization (for Genomic Libraries):

1. *Replica Plating:*
- Duplicate the bacterial colonies onto a nitrocellulose or nylon membrane. This
creates a replica of the colony arrangement.

2. *Cell Lysis:*
- Lyse the cells on the membrane, transferring cellular contents, including DNA, to
the membrane.

3. *DNA Denaturation:*
- Denature the DNA on the membrane, making it accessible for hybridization.

4. *Hybridization:*
- Incubate the membrane with a labeled probe specific to the target DNA sequence.
The probe will hybridize (bind) to complementary sequences on the membrane.

5. *Washing:*
- Wash the membrane to remove unbound probe, leaving only the specifically
bound probe on the membrane.

6. *Detection:*
- Visualize the hybridized colonies using autoradiography or other detection
methods. Positive colonies will indicate the presence of the target DNA sequence.
### Plaque Hybridization (for cDNA Libraries in Bacteriophages):

1. *Plaque Lift:*
- Transfer phage plaques from the agarose gel onto a nitrocellulose or nylon
membrane.

2. *Denaturation and Neutralization:*

- Denature and neutralize the DNA on the membrane, making it accessible for
hybridization.

3. *Hybridization:*
- Incubate the membrane with a labeled probe specific to the cDNA of interest.

4. *Washing:*
- Wash the membrane to remove unbound probe, leaving only the specifically
bound probe on the membrane.

5. *Detection:*
- Visualize the hybridized plaques using autoradiography or other detection methods.
Positive plaques indicate the presence of the desired cDNA sequence.

These hybridization techniques are powerful tools for identifying clones with specific
genes or sequences in large libraries. They allow researchers to isolate and study
individual DNA fragments of interest from a diverse pool of genomic or cDNA
sequences.

Blue and white colony hybridization is a technique commonly used in molecular

biology, particularly in screening recombinant plasmids. This method utilizes a lacZ
(beta-galactosidase) gene and its interaction with the X-gal (5-bromo-4-chloro-3-
indolyl-beta-D-galactopyranoside) substrate to distinguish between recombinant and
non-recombinant colonies.

### Blue and White Colony Screening:

1. *Introduction of Recombinant Plasmids:*

- Transform host cells (e.g., E. coli) with recombinant plasmids containing an insert
of interest. These plasmids typically have a lacZ gene within the multiple cloning site
(MCS).

2. *Selection:*
- Plate transformed cells on a medium containing a selective agent (e.g., antibiotic)
to ensure only cells with the plasmid survive.

3. *X-gal and IPTG Addition:*

- Add X-gal and IPTG (Isopropyl β-D-1-thiogalactopyranoside) to the agar medium.
IPTG induces lacZ expression.

4. *Beta-Galactosidase Activity:*
 - Cells with non-recombinant plasmids express active beta-galactosidase, turning
the colonies blue when X-gal is cleaved.

5. *Recombinant Plasmids:*
- Cells with recombinant plasmids have disrupted lacZ due to the insertion, leading
to inactive beta-galactosidase. These colonies remain white or paler in color.

6. *Screening:*
- Identify white (or paler) colonies as potential clones with the desired insert.

This method allows for a rapid visual identification of recombinant colonies, making
the screening process more efficient. The blue-white screening technique is widely
used in cloning experiments, where researchers aim to identify bacterial colonies
containing plasmids with the inserted DNA fragment.

cDNA library screening by immunological methods involves using antibodies to

identify clones containing specific gene products. Here's a general overview of the
process:

1. *Expression of cDNA in a Fusion Protein:*

- Clone the cDNA into an expression vector that fuses the cDNA with a reporter
gene or protein tag. Common tags include epitope tags like FLAG or His6.

2. *Protein Expression:*
- Introduce the recombinant vectors into host cells for protein expression. This
results in the production of fusion proteins that incorporate the encoded cDNA.

3. *Protein Extraction:*
- Extract proteins from the host cells, including the expressed fusion proteins.

4. *Immobilization of Proteins:*
- Immobilize the extracted proteins onto a solid support, such as a membrane or
microplate.

5. *Blocking:*
- Block nonspecific binding sites on the solid support to prevent unwanted
interactions.

6. *Incubation with Antibodies:*

- Incubate the solid support with specific antibodies that recognize the epitope tag or
the protein product of interest. These antibodies can be monoclonal or polyclonal.

7. *Washing:*
- Wash away unbound antibodies to reduce background noise.

8. *Detection:*
- Add a secondary antibody linked to a detection molecule (e.g., enzyme,
fluorophore) that can visualize the presence of the primary antibody.
9. *Visualization:*
- Visualize the signal either by color development (for enzyme-linked antibodies) or
fluorescence (for fluorophore-linked antibodies).

10. *Identification of Positive Clones:*

- Positive clones in the cDNA library are those that express the protein recognized
by the specific antibody.

This method allows for the identification of clones in the cDNA library that produce
proteins with specific immunological characteristics. It is particularly useful when the
goal is to identify clones expressing proteins with particular functions or properties.

All Techniques

STUDYING FOLLOWING TECHNIQUES THROUGH PHOTOGRAPHS

Southern blotting

(1) Blotting describes the immobilization of sample macromolecules i.e. nucleic

acid and proteins on to a solid support, generally nylon or nitrocellulose
membranes. The blotted macromolecules are then used as targets in
subsequent hybridization experiments.
(2) The original method of blotting is named after Edwin Southern in 1975 for
detecting DNA fragments in an agarose gel that are complementary to a given
RNA and DNA sequence. The method is named after its inventor, the British
biologist Edwin Southern. Other blotting methods (i.e. western blot, northern
blot, southwestern blot) that employ similar principles, but using RNA or
protein, have later been named in reference to Southern's name.
(3) Southern blotting is a technique for transferring DNA molecules from agarose
gel to a solid support such as nitrocellulose filter or nylon membrane. Gel
embedded DNA is depurinated in the presence of HCl.
(4) Depurination, followed by alkaline hydrolysis (in denaturation solution)
breaks the DNA fragments into smaller pieces at the depurinated site, thus
allowing more efficient transfer from the gel to membrane. It also denatures
the fragment into single DNA strands for later hybridization to the probe and
destroys any residual RNA that may still be present in the DNA.
(5) Denatured molecules move upward by capillary action of buffer and on
coming in contact with the nylon membrane they get bound to it. Buffer
transfer by capillary action from a region of high water potential to a region of
low water potential (usually filter paper and paper tissues) is then used to
move the DNA from the gel on to the membrane; ion exchange interactions
bind the DNA to the membrane due to the negative charge of the DNA and
positive charge of the membrane.
(6) After transfer membrane is exposed to UV that cross-links DNA to the
membrane. It is based on the formation of cross-links between a small fraction
of thymine residues in DNA and positively charged amino groups on the
surface of nylon membrane.
(7) In the procedure referred to as Southern blotting, the agarose gel is mounted
on a filter paper wick which dips into reservoir containing transfer buffer. The
nylon membrane is sandwiched between the gel and a stack of paper towels
 which serve to draw the transfer buffer through the gel by capillary action. The
DNA molecules are carried out of the gel by the buffer flow and immobilized
on the membrane.
(8) The membrane is then incubated with a probe which is single-stranded DNA.
This probe will form base pairs with its complementary DNA sequence and
bind to form a double-stranded DNA molecule. The probe is labeled before
hybridization either radioactively or enzymatically (e.g. alkaline phosphatase
or horseradish peroxidase).
(9) Finally, the location of the probe is detected by directly exposing the
membrane to X-ray film or chemiluminescent methods.

APPLICATIONS OF SOUTHERN BLOTTING:

1. Southern blotting technique is used to detect DNA in given sample.

2. DNA finger printing is an example of southern blotting.
3. Used for paternity testing, criminal identification, victim identification
4. To isolate and identify desired gene of interest.
5. Used in restriction fragment length polymorphism
6. To identify mutation or gene rearrangement in the sequence of DNA
7. Used in diagnosis of disease caused by genetic defects
8. Used to identify infectious agents

Western Blotting

(1) Western Blotting is a technique that involves transfer of the resolved proteins
on the gel to the surface of a polymer sheet. The method of transferring
proteins from gel to membrane originated in the laboratory of George Stark at
Stanford. The name Western Blot was given to the technique by W. Neal
Burnette. The Western Blot (alternately, immunoblot) is a method of detecting
specific proteins in a given sample of tissue homogenate or extract separated
by gel electrophoresis. This method involves transfer of electrophoresed
protein bands from a polyacrylmide onto membrane of nitrocellulse to which
they bind strongly. The bound proteins are then available for specific protein-
ligand interaction.An antibody that is specific for the protein of interest is
 added to the sheet and reacts with the antigen. The antibody-antigen complex
on the sheet then can be detected by rinsing the sheet with a second antibody
specific for the first (e.g., goat antibody that recognizes mouse antibody). A
radioactive label on the second antibody produces a dark band on x-ray film
(an autoradiogram). Alternatively, an enzyme on the second antibody
generates a colored product, as in the ELISA method. Western blotting makes
it possible to find a protein in a complex mixture, the proverbial needle in a
haystack.
(2) The protein sample is subjected to electrophoresis on an SDS polyacrylamide
gel. Polyacrylamide Gel Electrophoresis (PAGE) is usually used for proteins.
In PAGE, a polyacrylamide gel is formed from a mixture of acrylamide and a
bis-acrylamide cross linker. Proteins are loaded onto the gel and move through
the gel under the influence of an electric field. All things being equal, the
protein will move according to its charge density, but in actual fact in most
PAGE techniques, it is the size of the pores in the gel that have more
profound effect on the rate of protein migration.
(3) Electrophoresis is somewhat similar to chromatography with electricity.
Compounds with charges on them will migrate in an electric field at a rate
proportional to their charge density, i.e. their charge divided by their mass
(small, highly charged things move quicker than heft, slightly charged things).
Electrophoresis is widely used to separate proteins and DNA.
(4) In SDS PAGE, proteins are denatured in such a way as to make them migrate
in a predictable fashion. SDS-PAGE is a denaturing technique. First proteins
are unravelled by boiling, then disulfide bonds are broken with
mercaptoethanol. Hence we add subunits and not the original protein to the gel.
We then add the negatively charged detergent sodium dodecyl sulfate (SDS),
which linearises the protein chains and coats the protein with negative charge.
This swamps the natural charge on the protein, so all proteins will have the
same charge density. Consequently, when the proteins are loaded, they move
according to how easily they can navigate through the pores in the gel alone:
this is proportional to (the logarithm of) their size alone.
(5) Detergents, salts and buffers are used in the lysis of cells and to solubilize
proteins. Protease and phosphatase inhibitors are added to prevent the
digestion of the sample by its own enzymes. SDS-PAGE (SDS-
Polyacrylamide Gel Electrophoresis) maintains polypeptides in a denatured
state once they have been treated with strong reducing agents to remove
secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl
groups [SH and SH]) and thus allows separation of proteins by their molecular
weight. Sampled proteins become covered in the negatively charged SDS and
move to the positively charged electrode through the acrylamide mesh of the
gel. Smaller proteins migrate faster through this mesh and the proteins are thus
separated according to size (usually measured in kilo Daltons, kDa). The
concentration of acrylamide determines the resolution of the gel. The greater
the acrylamide concentration the better the resolution of lower molecular
weight proteins. The lower the acrylamide concentration the better the
resolution of higher molecular weight proteins.
(6) When voltage is applied along the gel, proteins migrate into it at different
speeds. These different rates of advancement (different electrophoretic
mobilities) separate into bands within each lane.
 (7) Western blotting involves passing of current through transfer buffer, which
leads to transfer of proteins from gel onto membrane. Due to flow of buffer
ions towards oppositely charged electrode, negatively charged protein
molecules tend to move towards anode. But due to presence of membrane in
their path, these molecules having size bigger than pore size in membrane fail
to reach the respective electrode and get bound to the membrane, which is
placed towards anode. The membrane with its transferred proteins is referred
to as a blot. Once transferred onto membrane, the separated proteins can be
examined further. This involves probing the blot, usually using an antibody
(primary) to detect a specific protein.

Application
1. To determine the size and amount of protein in given sample.
2. Disease diagnosis: detects antibody against virus or bacteria in serum.
3. Western blotting technique is the confirmatory test for HIV. It detects anti
HIV antibody in patient’s serum.
4. Useful to detect defective proteins. For eg Prions disease.
5. Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and
Herpes.

POLYMERASE CHAIN REACTION

(1) The polymerase chain reaction (PCR) is a technique for exponentially

amplifying a specific fragment of DNA, via enzymatic replication, without
using a living organism.
(2) PCR can be used for amplification of a single or few copies of a piece of DNA
across several orders of magnitude, generating millions or more copies of the
DNA piece. Developed in 1983 by Kary Mullis, who won the Nobel Prize for
his work, PCR is now a common technique with wide applications in medical
 and biological research labs, such as in gene manipulation, prenatal diagnosis,
DNA profiling, archaeology, paleontology, diagnosis of hereditary and
infectious diseases etc.
(3) PCR is carried out by adding the following components to a solution
containing the target sequence: (1) a pair of primers that hybridize with the
flanking sequences of the target, (2) all four deoxyribonucleosidetriphosphates
(dNTPs), and (3) a heat-stable DNA polymerase.
(4) Two primers anneal to denatured DNA template at opposite sides of the target
region, and are extended by DNA polymerase to give new strands.
Denaturation of DNA template, annealing of primers to the template and
extension of the annealed primer constitute one cycle of reaction. After one
cycle, new DNA strands of variable length are produced. In cycle 2, the
original template strands and the new strands from cycle 1 are separated,
yielding a total of four primer sites with which primers anneal. The primers
that are hybridized to the new strands from cycle 1 are extended by the
polymerase as far as the end of the template, leading to a precise copy of the
target region. In cycle 3, double stranded DNA molecules are synthesized, that
are precisely identical to the target region. Further cycles lead to exponential
doubling of the target region.
(5) PCR primers are short fragments of single stranded DNA (17-30 nucleotides
in length) that are complementary to DNA sequences that flank the target
region of interest. Primer provides 3’OH group to which the Taq DNA
polymerase and synthesize a new strand, complimentary to the template strand.
(6) The PCR is carried out in small reaction tubes (0.2-0.5 ml volumes),
containing a reaction volume typically of 20-50 μl that are inserted into a
THERMAL CYCLER. This is a machine that heats and cools the reaction
tubes within it to the precise temperature required for each step of the reaction.
(7) The PCR usually consists of a series of 25 to 35 cycles. Most commonly, PCR
is carried out in three steps, often preceded by one temperature hold at the start
and followed by one hold at the end:
(i) Primary Denaturation: Prior to the first cycle, during an initialization
step, the PCR reaction is often heated to a temperature of 94°C, and
this temperature is then held for 5 minutes. This first hold is employed
to ensure that the DNA template is completely denatured.
(ii) Secondary Denaturation:Following this first hold of primary
denaturation, cycles of amplification begin, with one step at 94°C for
one minute.
(iii) Annealing:The denaturation is followed by the annealing step. In this
step the reaction temperature is lowered so that the primers can anneal
to the single-stranded DNA template. Brownian motion causes the
primers to move around, and hydrogen bonds are constantly formed
and broken between primer and template. Stable bonds are only
formed when the primer sequence very closely matches the template
sequence, and to this short section of double-stranded DNA the
polymerase attaches. The annealing temperature (Ta) is calculated as
discussed above.
(iv) Extension: The annealing step is followed by an extension/elongation
step during which the DNA polymerase synthesizes new DNA strands
complementary to the DNA template strands. The temperature at this
step depends on the DNA polymerase used. Taq polymerase has a
 temperature optimum of 70-74°C; thus, in most cases a temperature of
72°C is used. The hydrogen bonds between the extended primer and
the DNA template are now strong enough to withstand the higher
temperature. Primers that have annealed to DNA regions with
mismatching bases dissociate from the template and are not extended.
(v) Final Extension:A final elongation step of 5-15 minutes after the last
cycle may be used to ensure that any remaining single-stranded DNA
is fully extended.
(vi) Final Hold:A final hold at 4°C for an indefinite time may be
employed for storage of the reaction, e.g., if reactions are run overnight.

(8) Several features of this remarkable method for amplifying DNA are
noteworthy. First, the sequence of the target need not be known. All that is
required is knowledge of the flanking sequences. Second, the target can be
much larger than the primers. Targets larger than 10 kb have been amplified
by PCR. Third, primers do not have to be perfectly matched to flanking
sequences to amplify targets. With the use of primers derived from a gene of
known sequence, it is possible to search for variations on the theme. In this
way, families of genes are being discovered by PCR. Fourth, PCR is highly
specific because of the stringency of hybridization at high temperature (54°C).
Stringency is the required closeness of the match between primer and target,
which can be controlled by temperature and salt. At high temperatures, the
only DNA that is amplified is that situated between primers that have
hybridized. A gene constituting less than a millionth of the total DNA of a
higher organism is accessible by PCR. Fifth, PCR is exquisitely sensitive. A
single DNA molecule can be amplified and detected.

Real-Time Polymerase Chain Reaction (Real-Time PCR or qPCR) is a molecular

biology technique that allows for the quantification of DNA during the polymerase
chain reaction process. Here's a brief overview:

1. *Principle:* Real-time PCR monitors the amplification of a targeted DNA

molecule in real-time as the reaction progresses, as opposed to traditional PCR, where
analysis occurs after the reaction is complete.

2. *Fluorescent Probes or Dyes:* Fluorescent dyes or probes are used to detect the
amplification of the target DNA during each cycle of the PCR reaction.

3. *Amplification Monitoring:* The fluorescence signal increases proportionally to

the amount of DNA being amplified. The real-time monitoring allows for the
determination of the threshold cycle (Ct), which is the cycle number at which the
fluorescence signal crosses a predetermined threshold.

4. *Quantification:* The Ct values are used for quantification. A lower Ct value

indicates a higher starting amount of the target DNA in the sample.

5. *Applications:* Real-time PCR is widely used for gene expression analysis,

quantification of viral load, detection of pathogens, and various other applications
where precise quantification of DNA is essential.
Real-time PCR provides accurate and quantitative results, making it a valuable tool in
molecular biology and diagnostics.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a molecular

biology technique used to amplify and analyze RNA. Here's a brief explanation:

1. *Reverse Transcription:* In the first step, RNA is converted into complementary

DNA (cDNA) using an enzyme called reverse transcriptase. This process is known as
reverse transcription.

2. *cDNA Amplification:* The obtained cDNA is then subjected to PCR, where

specific regions of interest are selectively replicated. This involves using DNA
primers that are complementary to the target sequences.

3. *Amplification Monitoring:* The progress of the PCR reaction is monitored in

real-time using fluorescent dyes or probes. This allows for the quantification of the
starting amount of RNA based on the amount of amplified DNA.

4. *Analysis:* After PCR, the results can be analyzed using techniques like gel
electrophoresis or, more commonly, by measuring the fluorescence emitted during
each cycle in real-time PCR.

RT-PCR is widely used in gene expression studies, viral load quantification, and the
detection of RNA viruses. It provides a powerful tool to study RNA expression levels
and dynamics in various biological samples.

APPLICATIONS OF PCR
PCR Is a Powerful Technique in Medical Diagnostics, Forensics, and Molecular
Evolution
(1) PCR can provide valuable diagnostic information in medicine. Bacteria and
viruses can be readily detected with the use of specific primers. For example,
PCR can reveal the presence of human immunodeficiency virus in people who
have not mounted an immune response to this pathogen and would therefore
be missed with an antibody assay. Finding Mycobacterium tuberculosis bacilli
in tissue specimens is slow and laborious. With PCR, as few as 10 tubercle
bacilli per million human cells can be readily detected. PCR is a promising
method for the early detection of certain cancers.
(2) This technique can identify mutations of certain growth-control genes, such as
the ras genes. The capacity to greatly amplify selected regions of DNA can
also be highly informative in monitoring cancer chemotherapy. Tests using
PCR can detect when cancerous cells have been eliminated and treatment can
be stopped; they can also detect a relapse and the need to immediately resume
treatment. PCR is ideal for detecting leukemias caused by chromosomal
rearrangements.
(3) PCR is also having an effect in forensics and legal medicine. An individual
DNA profile is highly distinctive because many genetic loci are highly
variable within a population. For example, variations at a specific one of these
locations determines a person's HLA type (human leukocyte antigen type);
organ transplants are rejected when the HLA types of the donor and recipient
are not sufficiently matched. PCR amplification of multiple genes is being
 used to establish biological parentage in disputed paternity and immigration
cases.
(4) Analyses of blood stains and semen samples by PCR have implicated guilt or
innocence in numerous assault and rape cases.The root of a single shed hair
found at a crime scene contains enough DNA for typing by PCR (Figure
6.10).DNA is a remarkably stable molecule, particularly when relatively
shielded from air, light, and water. Under such circumstances, large fragments
of DNA can remain intact for thousands of years or longer. PCR provides an
ideal method for amplifying such ancient DNA molecules so that they can be
detected and characterized.
(5) PCR can also be used to amplify DNA from microorganisms that have not yet
been isolated and cultured.
(6) The sequences from these PCR products can be sources of considerable
insight into evolutionary relationships between organisms.

DNA sequencing

(1) DNA sequencing is the fundamental need for modern gene manipulation. The
power of DNA sequencing is its ability to reduce genes and genomes to
chemical entities of defined structure. DNA fragment can also be analyzed to
determine the nucleotide sequence of the DNA and to determine the
distribution and location of restriction sites.
(2) DNA Is Usually Sequenced by Controlled Termination of Replication (Sanger
Dideoxy Method).
(3) This procedure capitalizes on two properties of DNA polymerase: (1) its
ability to synthesize faithfully a complementary copy of a single stranded
DNA template and (2) its ability to use 2’, 3’- dideoxynucleoside
triphosphates as substrates. The process involves the extension of a short
primer by DNA polymerase. Both linear DNA and circular DNA can be
sequenced by this method.
(4) In dideoxy DNA sequencing, the DNA is first denatured to single strands and
a short oligonucleotide primer is annealed to one of the strands. The
oligonucleotide is designed so that its 3’ end is next to the DNA sequence of
interest. The oligonucleotide acts as a primer for DNA synthesis, and the 5’-
 to-3’ orientation chosen ensure that the DNA made is a complementary copy
of the DNA sequence of interest.
(5) The key to DNA sequencing is the generation of DNA fragments whose length
depends on the last base in the sequence. Collections of such fragments can be
generated through the controlled interruption of enzymatic replication. The
same procedure is performed on four reaction mixtures at the same time. In all
these mixtures, a DNA polymerase is used to make the complement of a
particular sequence within a single-stranded DNA molecule. In practice, the
klenow fragments of DNA polymerase I, which lacks the 5’ 3’ exonuclease
activity of the intact enzyme, is used to synthesize a complementary copy of
the single stranded target sequence. The synthesis is primed by a fragment,
usually obtained by chemical synthetic methods that is complementary to a
part of the sequence known from other studies.
(6) DNA synthesis is carried out in the presence of the four deoxynucleoside
triphosphates, one or more of which is labeled in four separate incubation
mixes, each reaction mixture contains a small amount of the 2 ,3 -dideoxy
analog of one of the nucleotides. Therefore, in each reaction there is a
population of partially synthesized radioactive DNA molecules, each having a
common 5’ end but each varying in length to a base specific 3’end since
incorporation of this analog blocks further growth of the new chain because it
lacks the 3 -hydroxyl terminus needed to form the next phosphodiester bond.
(7) The concentration of the dideoxy analog is low enough that chain termination
will take place only occasionally. The polymerase will sometimes insert the
correct nucleotide and other times the dideoxy analog, stopping the reaction.
For instance, if the dideoxy analog of dATP is present, fragments of various
lengths are produced, but all will be terminated by the dideoxy analog (Figure
6.4). Importantly, this dideoxy analog of dATP will be inserted only where a T
was located in the DNA being sequenced. Thus, the fragments of different
length will correspond to the positions of T. Four such sets of chain-
terminated fragments (one for each dideoxy analog) then undergo
electrophoresis, and the base sequence of the new DNA is read from the
autoradiogram of the four lanes.
(8) Fluorescence detection is a highly effective alternative to autoradiography. A
fluorescent tag is attached to an oligonucleotide priming fragment a differently
colored one in each of the four chain-terminating reaction mixtures (e.g., a
blue emitter for termination at A and a red one for termination at C). The
reaction mixtures are combined and subjected to electrophoresis together. The
separated bands of DNA are then detected by their fluorescence as they
emerge from the gel; the sequence of their colors directly gives the base
sequence. Sequences of as many as 500 bases can be determined in this way.
Alternatively, the dideoxy analogs can be labeled, each with a specific
fluorescent label. When this method is used, all four terminators can be placed
in a single tube, and only one reaction is necessary. Fluorescence detection is
attractive because it eliminates the use of radioactive reagents and can be
readily automated.

Application
(1) Knowledge of the sequence of a DNA region may be important in
understanding inherited human disorders. It is also important for planning any
substantial manipulation of the DNA.
(2) The information on sequencing is also useful, for example, in computer
database analyses for identifying gene sequences and regulatory sequences
within the fragment and for comparing the sequences of homologous genes
from different organisms. Furthermore, the DNA sequences of a protein-
coding gene can be translated by computer to provide information about the
properties of the protein for which it codes. Such information can be helpful to
an investigator who wants to isolate and study an unknown protein product of
a gene for which a clone is available. Therefore, this method is now favoured
for large-scale DNA sequence determination. This technique has superseded
alternative methods because of its simplicity
(3) Sanger and coworkers determined the complete sequence of the 5386 bases in
the DNA of the X174 DNA virus in1977, just a quarter century after
Sanger's pioneering elucidation of the amino acid sequence of a protein. This
accomplishment is a landmark in molecular biology because it revealed the
total information content of a DNA genome.This tour de force was followed
several years later by the determination of the sequence of human
mitochondrial DNA, adouble-stranded circular DNA molecule containing
16,569 base pairs. It encodes 2 ribosomal RNAs, 22 transfer RNAs,and 13
proteins. In recent years, the complete genomes of free-living organisms have
been sequenced. The first such sequence to be completed was that of the
bacterium Haemophilus influenzae. Its genome comprises 1,830,137 base
pairs and encodes approximately 1740 proteins.Many other bacterial and
archaeal genomes have since been sequenced. The first eukaryotic genome to
be completely sequenced was that of baker's yeast, Saccharomyces cerevisiae,
which comprises approximately 12 million base pairs, distributed on 16
chromosomes, and encodes more than 6000 proteins.

(4) The ability to determine complete genome sequences has revolutionized

biochemistry and biology.

Sequencing of DNA by the Sanger method

Sanger sequencing, also known as chain termination sequencing, is a widely used
method for determining the nucleotide sequence of DNA. This technique was
developed by Frederick Sanger and his colleagues in the late 1970s and has since
played a crucial role in many areas of molecular biology, genetics, and genomics.

### Sanger Sequencing Method:

1. **Principle:**
- Sanger sequencing relies on the synthesis of DNA strands in the presence of chain-
terminating dideoxynucleotides (ddNTPs). These ddNTPs lack the 3' hydroxyl group
necessary for the formation of the phosphodiester bond, resulting in chain termination.

2. **Components:**
- **Template DNA:** The DNA sequence to be determined.
- **DNA Primer:** Short oligonucleotide complementary to a specific region of the
template DNA.
- **DNA Polymerase:** Enzyme responsible for synthesizing the complementary
strand.
- **dNTPs (Deoxynucleotide Triphosphates):** Regular nucleotides (A, T, C, G)
needed for DNA synthesis.
- **ddNTPs (Dideoxynucleotide Triphosphates):** Modified nucleotides that
terminate DNA synthesis.

3. **Procedure:**
- A reaction mixture is prepared with the template DNA, primer, DNA polymerase,
regular dNTPs, and a small amount of one of the four ddNTPs (A, T, C, or G).
- The mixture is divided into four separate tubes, each containing a different ddNTP.
This results in four reactions, each terminated at a specific nucleotide.
- DNA synthesis begins when the primer binds to the template DNA, and as the
polymerase incorporates nucleotides, it may occasionally incorporate a ddNTP instead
of a regular dNTP, causing chain termination.

4. **Fragment Separation:**
- After the synthesis reactions, the fragments are separated by size through gel
electrophoresis. This is typically done in a polyacrylamide gel.
- The gel is exposed to X-rays or a laser, revealing the positions of the terminated
fragments.

5. **Visualization and Analysis:**

- The gel electrophoresis results are visualized on an autoradiogram or using
fluorescent dyes.
- The sequence is read from the bottom (3' end) to the top (5' end) of the gel,
corresponding to the synthesized DNA fragments.
- The position of each terminated fragment indicates the identity of the nucleotide at
that position in the sequence.

6. **Output:**
- The result is a chromatogram that represents the sequence of the template DNA.
Peaks or bands correspond to the incorporation of specific nucleotides.
### Advantages and Limitations:

- **Advantages:**
- High accuracy and reliability.
- Suitable for sequencing relatively short DNA fragments (up to a few hundred
nucleotides).
- Widely used for validating and confirming DNA sequences.

- **Limitations:**
- Labor-intensive and time-consuming.
- Limited throughput compared to modern high-throughput sequencing methods.
- Practical constraints on read length.

Sanger sequencing played a pivotal role in the Human Genome Project and remains
an important tool for targeted sequencing and validation of genetic information.
However, for large-scale genome sequencing, newer technologies such as next-
generation sequencing have largely replaced Sanger sequencing due to their higher
throughput and efficiency.

Next-Generation Sequencing (NGS) is a high-throughput DNA sequencing

technology that has revolutionized genomic research and various applications in the
life sciences. Here's a concise explanation:

1. *DNA Fragmentation:* The DNA sample is broken into small fragments.

2. *Library Preparation:* Adapters are added to the fragmented DNA, creating a

library of DNA fragments with known sequences at both ends.

3. *Amplification:* The DNA library is amplified through polymerase chain reaction

(PCR) to generate sufficient material for sequencing.

4. *Sequencing:* The prepared library is loaded onto a sequencer, where massively

parallel sequencing occurs. This means many fragments are sequenced simultaneously.

5. *Base Calling:* The sequencer reads the fluorescence signals generated during
sequencing, converting them into base calls (A, T, C, G).

6. *Data Analysis:* The generated sequences are then aligned to a reference genome
or assembled de novo, and bioinformatics tools are used to analyze the data.

NGS enables the rapid and cost-effective sequencing of entire genomes, exomes, or
specific genomic regions. It has applications in genomics, transcriptomics,
epigenomics, and metagenomics, providing insights into genetic variation, gene
expression, and more.

DNA Fingerprinting

(1) A fingerprint is an imprint made by the pattern of ridges on the pad of a

human finger. These ridges are commonly believed to provide traction for
grasping objects. Fingerprints are usually considered to be unique, with no
 fingers having the exact same friction ridge characteristics. A persons’s
fingerprint can be used as a biometric method to identify human individuals.
(2) DNA Fingerprinting is DNA based individual identification. The inventor of
DNA fingerprinting was Sir Alec Jeffreys at the University of Leicester in
1985.
(3) To identify individuals, forensic scientists scan 13 DNA regions, or loci, that
vary from person to person and use the data to create a DNA profile of that
individual (sometimes called a DNA fingerprint). There is an extremely small
chance that another person has the same DNA profile for a particular set of 13
regions. Scientists can use these variable regions to generate a DNA profile of
an individual, using samples from blood, bone, hair, and other body tissues
and products. In criminal cases, this generally involves obtaining samples
from crime-scene evidence and a suspect, extracting the DNA, and analyzing
it for the presence of a set of specific DNA regions (markers).
(4) Three types of DNA variability are commonly used in DNA fingerprinting
(i) Single Nucleotide Polymorphism (SNP) is a single nucleotide change-
a point mutation- at one position in genome (SNP locus). They are the
most common type of DNA polymorphism occurring at a frequency of
about 1 per 350 bp and accounting for 90-95 percent of DNA sequence
variation.
(ii) Variable number of tandem repeats (VNTR). A tandem repeat is a
short sequence of DNA that is repeated in a head-to-tail fashion at a
specific chromosomal location. For many tandem repeats, the number
of repeated units vary between individuals. Such loci are termed
VNTRs. A single VNTR locus from unrelated individuals is normally
different
(iii) Short tandem repeats (STRs) are also called microsatellites and simple
sequence repeats (SSR). They are 1-13nucleotides in length that is
repeated several times (usually 5-20 times) in a tandem array. There
are at least 6,50,000 STRs in human genome and the most common
type is the dinucleotide [CA]n where n is the number of repeats.
(5) To prepare DNA fingerprint,
DNA must be recovered from the cells or tissues of the body. Only a small
amount of tissue - like blood, hair, or skin - is needed. For example, the
amount of DNA found at the root of one hair is usually sufficient.
(6) Isolated DNA is digested with restriction enzymes and analysed by agarose
gel electrophoresis.
(7) Restriction enzyme digested DNA is transferred from gel to nylon membrane
by Southern blotting.
(8) Radioactive or fluorescent probes added to the nylon membrane produces a
pattern called the DNA fingerprint. Each probe typically sticks in only one or
two specific places on the nylon membrane, based on sequence
complementarity.
(9) The final DNA fingerprint is built by using several probes (5-10 or more)
simultaneously. It resembles the bar codes used by grocery store scanners.
Probes for Fingerprint: A stretch of labeled DNA used to detect and locate a
target complementary sequence in the genome is called a probe. There are
mainly two types of probes.
(a) Multilocus Probe: A subset of human satellites share a common core
sequence embedded in each repeat unit, which is involved in the
 generation of hypervariable tandem repeated loci by serving as polymerase
slippage signal. These core sequence probes detect variable number of
tandem repeats (VNTR) in the genomes of other vertebrates as well as
plants. In India Bkm probe is used in paternity and crime investigations.
(b) Bkm Probe: Singh et.al. isolated a sex chromosome associated repetitive
DNA from female Indian banded krait Bungarus fasciatus as minor
satellite DNA component and designated it as Bkm. This sequence is
present in many eukaryotes but absent in prokaryotes. These are
preferentially concentrated in the sex determining region of the sex
chromosomes of Drosophila, snakes, birds, mouse and man. The
conserved components of Bkm are long arrays of the repeats of the
tetranuleotide GATA.
(c) Single Locus Probe: Locus specific probe. A probe that detects a single
hypervariable locus is called a locus specific or single locus probe.

Applications of DNA Fingerprints

DNA fingerprints are useful in several applications of human health care

research, as well as in the justice system.
(1) Diagnosis of Inherited Disorders
DNA fingerprinting is used to diagnose inherited disorders in both prenatal
and newborn babies in hospitals around the world. These disorders may
include cystic fibrosis, hemophilia, Huntington's disease, familial
Alzheimer's, sickle cell anemia, thalassemia, and many others. Early
detection of such disorders enables the medical staff to prepare themselves
and the parents for proper treatment of the child. In some programs,
genetic counselors use DNA fingerprint information to help prospective
parents understand the risk of having an affected child. In other programs,
prospective parents use DNA fingerprint information in their decisions
concerning affected pregnancies.
(2) Developing Cures for Inherited Disorders Research programs to locate
inherited disorders on the chromosomes depend on the information
contained in DNA fingerprints. By studying the DNA fingerprints of
relatives who have a history of some particular disorder, or by comparing
large groups of people with and without the disorder, it is possible to
identify DNA patterns associated with the disease in question. This work is
a necessary first step in designing an eventual genetic cure for these
disorders.
(3) Biological Evidence: FBI and police labs around the U.S. use DNA
fingerprints to link suspects to biological evidence - blood or semen stains,
hair, or items of clothing - found at the scene of crime. Since 1987,
hundreds of cases have been decided with the assistance of DNA
fingerprint evidence.

DNA microarray

A DNA microarray is a high-throughput technology used to simultaneously

measure the expression levels of thousands of genes or to detect variations in
genomic DNA. Here's a brief explanation:
1. *Microarray Design:* A DNA microarray consists of a solid support, often
a glass slide or silicon chip, onto which thousands of DNA sequences, known
as probes, are attached in a grid-like pattern.

2. *Sample Preparation:* RNA or DNA from the biological sample of interest

is extracted and labeled with fluorescent dyes or other detectable markers.

3. *Hybridization:* The labeled sample is then allowed to hybridize (bind) to

the complementary DNA probes on the microarray. The degree of
hybridization reflects the abundance of specific RNA or DNA sequences in
the sample.

4. *Detection:* The microarray is scanned to detect the fluorescent signals,

indicating the level of hybridization at each spot on the array.

5. *Data Analysis:* The intensity of signals is quantified and analyzed to

determine the relative abundance of specific nucleic acid sequences. This
information can be used to understand gene expression patterns or identify
genetic variations.

DNA microarrays have applications in gene expression profiling, comparative

genomic hybridization (CGH) for detecting chromosomal abnormalities, and
genotyping. They provide a powerful tool for studying the molecular basis of
diseases, identifying biomarkers, and exploring the functional aspects of genes
on a large scale.

Northern blotting

Northern blotting is a laboratory technique used to study gene expression by

detecting and analyzing RNA molecules. Here's a brief overview:

1. *RNA Separation:* First, RNA molecules are separated based on size using
gel electrophoresis. The gel contains small pores that allow RNA molecules to
migrate through when an electric field is applied.

2. *Transfer to Membrane:* After electrophoresis, the separated RNA

molecules are transferred from the gel to a membrane, typically made of nylon
or nitrocellulose. This transfer process preserves the spatial arrangement of the
RNA bands.

3. *Hybridization:* The membrane is then exposed to a labeled probe, which

is a single-stranded DNA or RNA molecule complementary to the RNA of
interest. This allows the probe to bind specifically to the target RNA sequence.

4. *Detection:* The presence of the labeled RNA-probe complex is detected.

This can be done using autoradiography, chemiluminescence, or other
methods depending on the labeling technique.
 5. *Analysis:* The intensity of the signals on the membrane is analyzed to
quantify the amount of specific RNA molecules present. This provides
information about the expression levels of the targeted genes.

In summary, Northern blotting is a technique used to study RNA expression

patterns, allowing researchers to identify and quantify specific RNA molecules
in a sample.

Production of cloned and transgenic animals

The production of cloned animals involves a process called somatic cell nuclear
transfer (SCNT). Here are the key steps:

1. *Donor Cell Collection:*

- Somatic cells (cells other than reproductive cells) are collected from the individual
that is to be cloned. This donor cell contains the complete set of genetic information.

2. *Egg Cell Preparation:*

- Egg cells (ova) are obtained from a female donor. The nucleus of the egg cell is
removed, leaving an enucleated egg.

3. *Nuclear Transfer:*
- The nucleus of the somatic cell is extracted and transferred into the enucleated egg
cell. This creates a reconstructed cell with the complete genetic material of the donor.

4. *Stimulation of Division:*
- The reconstructed cell is then stimulated to divide and develop into an early-stage
embryo. This can be done through chemical or electrical means to trigger cell division.

5. *Implantation:*
- The developing embryo is implanted into the uterus of a surrogate mother, where it
continues to grow and develop.

6. *Birth of the Cloned Animal:*

- If successful, the surrogate mother gives birth to an animal that is genetically
identical to the donor of the somatic cell.

This process has been used to clone various animals, including sheep (e.g., Dolly the
sheep was the first mammal cloned from an adult somatic cell), cows, and other
mammals. Cloning has applications in agriculture, preservation of endangered species,
and biomedical research. However, it comes with ethical and technical challenges.

The production of transgenic animals involves introducing foreign genes into

their genome. Here's an overview of the key steps:

1. *Gene Selection:*
- A specific gene of interest is selected for introduction into the animal. This gene
could come from the same species or a different one, depending on the desired traits
or purposes.
2. *Isolation of the Gene:*
- The selected gene is isolated from the source organism's DNA. This can be done
using various molecular biology techniques, such as polymerase chain reaction (PCR).

3. *Gene Insertion:*
- The isolated gene is then inserted into the DNA of the target animal. Techniques
like microinjection are often used, where the gene is directly injected into a fertilized
egg.

4. *Integration of the Gene:*

- The introduced gene integrates into the DNA of the host organism. This step is
crucial for the transgenic organism to express the desired traits encoded by the foreign
gene.

5. *Embryo Transfer:*
- The genetically modified embryo is then transferred into the uterus of a surrogate
mother for gestation.

6. *Birth of Transgenic Offspring:*

- If successful, the surrogate mother gives birth to transgenic offspring that carry the
inserted gene in their DNA.

Transgenic animals have been created for various purposes, including the production
of pharmaceuticals (using animals as "bioreactors"), studying gene function, and
enhancing specific traits for agricultural or medical applications. Ethical
considerations and potential environmental impacts are important aspects to address
in the development and use of transgenic organisms.

Nuclear transplantation, also known as somatic cell nuclear transfer (SCNT), is a

technique used in cloning. It involves transferring the nucleus of a somatic cell (a
non-reproductive cell) into an enucleated egg cell (an egg cell with its nucleus
removed). The process was famously demonstrated in the cloning of Dolly the sheep.

Here are the key steps in nuclear transplantation, using the cloning of Dolly as an
example:

1. *Donor Cell Collection:*

- A somatic cell is collected from the animal that is to be cloned. In the case of
Dolly, this was a mammary gland cell taken from a Finn Dorset sheep.

2. *Egg Cell Enucleation:*

- An egg cell is obtained from another sheep, and its nucleus is removed, creating an
enucleated egg.

3. *Nuclear Transfer:*
- The nucleus from the somatic cell is then transferred into the enucleated egg cell.
This creates a reconstructed cell with the complete genetic material of the donor.
4. *Stimulation of Division:*
- The reconstructed cell is stimulated to divide and develop into an early-stage
embryo. This can be achieved through chemical or electrical means.

5. *Implantation:*
- The developing embryo is implanted into the uterus of a surrogate mother, where it
continues to grow and develop.

6. *Birth of Dolly:*
- If successful, the surrogate mother gives birth to an individual (in this case, Dolly)
that is genetically identical to the animal from which the somatic cell was taken.

Dolly, born in 1996, was the first mammal cloned from an adult somatic cell,
demonstrating the possibility of creating genetically identical animals through nuclear
transplantation. This breakthrough had significant implications for biotechnology,
genetics, and the understanding of cellular reprogramming.

Retroviral method

The retroviral method is a technique used for the introduction of foreign genes into
the genome of an organism, including the creation of transgenic animals. Here's an
overview of the process:

1. *Selection of Gene and Vector:*

- A gene of interest is chosen for insertion into the host organism's genome.
Additionally, a retroviral vector is selected. Retroviruses are a type of virus whose
genetic material is RNA, and they can integrate their genetic material into the host
cell's DNA.

2. *Modification of Retroviral Vector:*

- The selected gene is incorporated into the retroviral vector, creating a genetically
engineered retrovirus. This engineered virus is typically modified to eliminate its
ability to cause disease.

3. *Introduction into Target Cells:*

- The genetically modified retrovirus is introduced into the target cells of the
organism. This is often done through methods like microinjection or by infecting cells
in culture.

4. *Integration of Genetic Material:*

- The retrovirus infects the host cells, and its genetic material, including the gene of
interest, is integrated into the host cell's DNA. This step is crucial for the expression
of the introduced gene.

5. *Propagation of Modified Cells:*

- The genetically modified cells are cultured and allowed to multiply. This ensures
that the introduced gene is present in a sufficient number of cells.

6. *Implantation or Development:*
 - The modified cells or embryos are then implanted into the host organism or
allowed to develop further. If the process is successful, the organism will carry the
integrated gene in its DNA.

The retroviral method is valuable for creating transgenic animals and has been used in
various research applications. However, there are challenges and considerations, such
as the potential for random integration of the gene into the host genome, which may
lead to unpredictable effects. Ethical and safety aspects also need careful
consideration in the use of retroviral methods for genetic modification.

DNA microinjection

DNA microinjection is a biotechnological technique used to introduce foreign genetic

material (DNA) into a host cell or organism. It is commonly employed in the creation
of transgenic organisms, including animals. Here's an overview of the DNA
microinjection process:

1. *Isolation of DNA:*
- The gene of interest is isolated from the source organism's DNA. This gene could
code for a specific trait, protein, or function.

2. *Preparation of DNA Solution:*

- The isolated DNA is then prepared in a solution suitable for microinjection. This
solution typically contains the genetic material, buffer solution, and other necessary
components.

3. *Selection of Host Cells or Embryos:*

- The host cells or embryos to be genetically modified are carefully chosen. This
could be a fertilized egg in the case of creating transgenic animals.

4. *Microinjection:*
- Using a fine glass needle, the prepared DNA solution is injected directly into the
nucleus of the host cell or embryo. This is done under a microscope to ensure
precision.

5. *Integration of DNA:*
- The introduced DNA integrates into the host cell's genome. The cell then carries
the foreign genetic material and will express it as part of its normal functions.

6. *Culturing and Development:*

- The genetically modified cells or embryos are cultured in a suitable environment
to allow for growth and development. This can involve cell culture or implantation
into a surrogate organism.

7. *Birth or Development of Transgenic Organism:*

- If the process is successful, the organism that develops from the genetically
modified cells or embryos will be transgenic, expressing the traits encoded by the
introduced DNA.
DNA microinjection is a precise method for creating transgenic organisms, and it has
been widely used in scientific research and biotechnology. However, it may have
limitations such as low efficiency and the possibility of random integration of the
introduced DNA. Advanced techniques like CRISPR/Cas9 have become increasingly
popular for genetic modifications due to their higher precision and efficiency.

Applications of transgenic animals

Transgenic animals, which carry foreign genes in their genome, have various
applications across different fields. Some key applications include:

1. *Biomedical Research:*
- Transgenic animals serve as valuable models to study human diseases and test
potential therapies. They help researchers understand gene functions and the
mechanisms underlying various health conditions.

2. *Pharmaceutical Production:*
- Animals can be genetically modified to produce human proteins or antibodies in
their milk or blood, serving as "bioreactors." This facilitates the large-scale
production of pharmaceuticals for medical treatments.

3. *Agricultural Enhancement:*
- Transgenic animals can be engineered for improved traits, such as increased
resistance to diseases, enhanced growth rates, or better meat quality. This has
implications for more efficient and sustainable agriculture.

4. *Organ Transplants (Xenotransplantation):*

- Genetic modification can be used to reduce the risk of organ rejection in
xenotransplantation. Transgenic pigs, for example, have been developed with organs
more compatible with human recipients.

5. *Biotechnology and Industry:*

- Transgenic animals may be used in the production of industrial products. For
instance, genetically modified goats have been created to produce spider silk proteins
in their milk, which can be harvested for various applications.

6. *Environmental Monitoring:*
- Some transgenic animals are designed to act as environmental sensors. They can
be engineered to exhibit specific responses to environmental pollutants, helping
researchers monitor and assess environmental conditions.

7. *Behavioral Studies:*
- Transgenic animals with modified genes related to behavior provide insights into
the genetic basis of certain behaviors. These studies contribute to our understanding
of neurological and psychological processes.

While transgenic technology offers numerous possibilities, ethical considerations,

ecological impact assessments, and careful regulation are essential to ensure
responsible and safe applications in these diverse fields.
The production of pharmaceuticals using transgenic animals involves genetically
modifying these animals to produce specific proteins or substances that have
therapeutic value. Here's a brief overview of the process:

1. *Gene Selection:*
- A gene encoding a therapeutic protein or substance is selected for insertion into
the genome of the transgenic animal. This gene is often chosen for its ability to
produce a substance with medical applications.

2. *Genetic Modification:*
- The selected gene is incorporated into the DNA of the animal. This can be
achieved through methods like DNA microinjection or using advanced gene-editing
techniques such as CRISPR/Cas9.

3. *Generation of Transgenic Animals:*

- The genetically modified embryos are implanted into surrogate animals, where
they develop into transgenic animals. These animals carry the foreign gene in their
DNA.

4. *Expression of the Gene:*

- The introduced gene is activated, and the transgenic animals start producing the
therapeutic protein or substance. This production often occurs in specific tissues, such
as milk glands, where the substance can be easily harvested.

5. *Harvesting the Pharmaceuticals:*

- The pharmaceutical substance is collected from the milk or other appropriate
tissues of the transgenic animals. This substance is then purified and processed to
obtain the final pharmaceutical product.

6. *Quality Control:*
- Rigorous quality control measures are implemented to ensure that the
pharmaceutical product meets safety and efficacy standards. This includes testing for
purity, potency, and absence of contaminants.

7. *Scale-Up Production:*
- Once the production process is optimized and validated, it can be scaled up for
large-scale manufacturing to meet commercial demand. This often involves using a
herd of transgenic animals to produce pharmaceuticals efficiently.

8. *Regulatory Approval:*
- The pharmaceutical product undergoes regulatory approval processes to ensure its
safety and efficacy. Regulatory agencies assess the production process, quality control
measures, and clinical trial data before granting approval for market distribution.

Examples of pharmaceuticals produced using transgenic animals include therapeutic

proteins like insulin, anticoagulants, and antibodies used in the treatment of various
medical conditions. The use of transgenic animals in pharmaceutical production offers
advantages in terms of scalability and cost-effectiveness.
Production of transgenic plants

The production of transgenic plants involves the introduction of foreign genes into
the genome of a plant species to impart specific traits or characteristics. Here's a
general overview of the process:

1. *Gene of Interest Selection:*

- A gene that codes for a desired trait, such as resistance to pests, tolerance to
herbicides, or enhanced nutritional content, is identified and selected.

2. *Vector Construction:*
- The selected gene is incorporated into a vector, often a plasmid or a viral vector.
The vector serves as a carrier to deliver the gene into the plant cells.

3. *Transformation:*
- The vector containing the gene of interest is introduced into the target plant cells.
Various methods can be used for transformation, including Agrobacterium-mediated
transformation, particle bombardment (gene gun), or electroporation.

4. *Integration of the Gene:*

- The introduced gene integrates into the plant's genome. This step is crucial for the
stable expression of the foreign gene in subsequent generations.

5. *Selection of Transgenic Plants:*

- Selective pressure, such as exposure to a specific antibiotic or herbicide, is applied
to identify and isolate only those plant cells that have successfully integrated the
foreign gene.

6. *Regeneration of Transgenic Plants:*

- The selected transgenic cells are cultured to regenerate whole plants. This involves
the development of shoots and roots from the transformed cells.

7. *Propagation:*
- Transgenic plants are propagated through normal plant breeding methods or tissue
culture to produce a population of plants with the desired trait.

8. *Field Testing:*
- Transgenic plants undergo field trials to assess their performance under natural
conditions. This includes evaluating factors like yield, resistance to pests or diseases,
and environmental impact.

9. *Regulatory Approval:*
- Before commercialization, transgenic plants typically undergo regulatory scrutiny
to ensure they meet safety and environmental standards. Regulatory agencies assess
the potential risks and benefits of the transgenic plants.

10. *Commercialization:*
- Once regulatory approval is obtained, transgenic plants can be commercially
cultivated and used in agriculture for various purposes, such as improving crop yields,
reducing pesticide use, or enhancing nutritional content.
Examples of transgenic plants include genetically modified crops like Bt cotton
(expressing a bacterial toxin for insect resistance) and Golden Rice (engineered to
produce beta-carotene, a precursor of vitamin A).

Agrobacterium-mediated transformation is a widely used method for introducing

foreign genes into the genome of plants. This technique is particularly valuable for
creating transgenic plants with desired traits. Here's an overview of the
Agrobacterium-mediated transformation process:

1. *Agrobacterium tumefaciens as a Vector:*

- Agrobacterium tumefaciens, a soil bacterium, is used as a vector in this method.
The bacterium naturally transfers a part of its DNA (T-DNA) into the plant host,
causing the formation of crown gall tumors.

2. *Gene of Interest and Vector Construction:*

- The gene of interest is inserted into a plasmid vector, along with other genetic
elements necessary for the transformation process. This engineered plasmid is
introduced into Agrobacterium tumefaciens.

3. *Cocultivation:*
- The Agrobacterium carrying the gene of interest is cocultivated with plant tissues.
This can be done by immersing plant explants (small sections of plant tissues) in a
culture medium containing the Agrobacterium.

4. *Transfer of T-DNA:*
- Agrobacterium transfers the engineered T-DNA, including the gene of interest,
into the plant cells. This transfer is facilitated by specific signals and proteins that
allow the integration of the T-DNA into the plant genome.

5. *Integration of Foreign Gene:*

- The T-DNA integrates into the plant genome, and the gene of interest becomes
part of the plant's DNA. The integration is often stable and can be passed on to
subsequent generations.

6. *Selection of Transgenic Cells:*

- Selective pressure, such as the presence of antibiotics or herbicides, is applied to
identify and isolate plant cells that have successfully integrated the foreign gene.

7. *Regeneration of Transgenic Plants:*

- The selected transgenic cells are cultured to regenerate whole plants. This involves
inducing the development of shoots and roots from the transformed cells.

8. *Verification and Characterization:*

- The transgenic plants are verified to confirm the presence and expression of the
introduced gene. Further characterization may involve assessing the phenotype and
functionality of the transgenic plants.
9. *Propagation and Field Testing:*
- Transgenic plants are propagated through normal plant breeding methods or tissue
culture. They undergo field trials to assess their performance under natural conditions.

10. *Regulatory Approval and Commercialization:*

- Before commercialization, transgenic plants typically undergo regulatory scrutiny
to ensure they meet safety and environmental standards. Once approved, they can be
commercially cultivated for various agricultural purposes.

Agrobacterium-mediated transformation is a versatile and widely applicable method,

enabling the creation of genetically modified crops with traits such as pest resistance,
herbicide tolerance, and improved nutritional content.

Applications of transgenic plants

Creating insect-resistant plants involves incorporating specific genes into the plant's
genome, often derived from the bacterium Bacillus thuringiensis (Bt), which produces
insecticidal proteins. Here's a brief overview of the process:

1. *Gene Selection:*
- The gene(s) encoding insecticidal proteins from Bacillus thuringiensis (Bt) are
selected. These proteins are toxic to certain pests.

2. *Vector Construction:*
- The selected Bt gene is inserted into a vector, typically a plasmid. This engineered
plasmid will serve as a carrier to introduce the gene into the plant cells.

3. *Transformation:*
- The plasmid containing the Bt gene is introduced into the plant cells. Various
methods can be used for transformation, such as Agrobacterium-mediated
transformation or particle bombardment.

4. *Integration of the Bt Gene:*

- The introduced Bt gene integrates into the plant's genome. This integration is
essential for stable expression in subsequent generations.

5. *Selection of Transgenic Plants:*

- Selective pressure is applied to identify and isolate plant cells that have
successfully integrated the Bt gene. This is often achieved by exposing the
transformed cells to a specific antibiotic or herbicide.

6. *Regeneration of Transgenic Plants:*

- The selected transgenic cells are cultured to regenerate whole plants. This involves
inducing the development of shoots and roots from the transformed cells.

7. *Verification of Transgenic Plants:*

- The transgenic plants are verified to confirm the presence and expression of the Bt
gene. This may involve molecular techniques to analyze the plant's DNA and confirm
the production of the insecticidal protein.
8. *Field Testing:*
- Transgenic plants undergo field trials to assess their performance under natural
conditions. This includes evaluating their resistance to target pests and the overall
impact on plant health and yield.

9. *Regulatory Approval:*
- Before commercialization, transgenic plants typically undergo regulatory scrutiny
to ensure they meet safety and environmental standards. Regulatory agencies assess
the potential risks and benefits of the insect-resistant plants.

10. *Commercialization:*
- Once regulatory approval is obtained, insect-resistant plants can be commercially
cultivated for agricultural purposes. These plants help reduce the need for chemical
insecticides and provide a more sustainable approach to pest control.

Insect-resistant plants, often expressing Bt proteins, have been successfully developed

for crops such as cotton, corn, and soybeans, offering a valuable tool for pest
management in agriculture.

The production of edible vaccines from transgenic plants involves introducing genes
encoding vaccine antigens into the plant's genome. When the transgenic plants are
consumed, they express the vaccine antigens, eliciting an immune response in the
recipient. Here's a brief overview of the process:

1. *Selection of Vaccine Antigen:*

- A gene encoding a specific vaccine antigen is selected. This antigen is typically a
protein or part of a pathogen that can stimulate an immune response.

2. *Vector Construction:*
- The selected gene is inserted into a plant expression vector, often derived from a
plasmid. This engineered vector serves as a carrier for the vaccine gene and contains
regulatory elements for proper expression in plants.

3. *Transformation:*
- The plant expression vector is introduced into plant cells through methods like
Agrobacterium-mediated transformation or particle bombardment. This introduces the
vaccine gene into the plant's genome.

4. *Integration of the Vaccine Gene:*

- The introduced vaccine gene integrates into the plant's genome. This integration
ensures the stable expression of the vaccine antigen in subsequent generations of the
transgenic plant.

5. *Selection of Transgenic Plants:*

- Selective pressure, such as exposure to a specific antibiotic or herbicide, is applied
to identify and isolate plant cells that have successfully integrated the vaccine gene.
6. *Regeneration of Transgenic Plants:*
- The selected transgenic cells are cultured to regenerate whole plants. This involves
inducing the development of shoots and roots from the transformed cells.

7. *Verification of Vaccine Expression:*

- The transgenic plants are verified to confirm the presence and expression of the
vaccine gene. Molecular techniques may be used to analyze the plant's DNA and
confirm the production of the vaccine antigen.

8. *Harvesting and Processing:*

- The transgenic plants are grown to maturity, and the edible parts (such as leaves,
fruits, or seeds) containing the vaccine antigen are harvested. These plant tissues are
then processed to extract and purify the vaccine antigen.

9. *Vaccine Delivery through Consumption:*

- The processed plant material, containing the vaccine antigen, can be consumed by
individuals. Once ingested, the vaccine antigen is presented to the immune system,
initiating an immune response.

10. *Immune Response and Protection:*

- The immune response stimulated by the consumed edible vaccine provides
protection against the targeted disease. This approach offers an alternative and
potentially more accessible method of vaccination, particularly in regions with limited
access to traditional vaccines.

Edible vaccines from transgenic plants offer advantages in terms of ease of

administration, reduced dependence on cold storage, and potentially lower production
costs. However, research and development, regulatory approval, and public
acceptance are important considerations in advancing this innovative vaccination
approach.

The production of Golden Rice involves the genetic modification of rice plants to
produce beta-carotene, a precursor of vitamin A. This approach aims to address
vitamin A deficiency, particularly in regions where rice is a staple food. Here's a brief
overview of the process:

1. *Selection of Genes:*
- Genes responsible for the synthesis of beta-carotene, typically derived from
bacteria or other plants, are selected. The key genes include those encoding enzymes
such as phytoene synthase and phytoene desaturase, which are involved in the
carotenoid biosynthetic pathway.

2. *Vector Construction:*
- The selected genes are inserted into a plant expression vector, often derived from a
plasmid. This engineered vector serves as a carrier for the beta-carotene biosynthetic
genes and includes regulatory elements for proper expression in rice plants.

3. *Transformation:* - The plant expression vector is introduced into rice plant cells
using methods such as Agrobacterium-mediated transformation or particle
bombardment. This delivers the beta-carotene biosynthetic genes into the rice plant's
genome.

4. *Integration of Genes:*
- The introduced genes integrate into the rice plant's genome. This integration
ensures the stable expression of the beta-carotene biosynthetic genes in subsequent
generations.

5. *Selection of Transgenic Plants:*

- Selective pressure, often involving exposure to a specific antibiotic or herbicide, is
applied to identify and isolate rice plant cells that have successfully integrated the
beta-carotene biosynthetic genes.

6. *Regeneration of Transgenic Plants:*

- The selected transgenic cells are cultured to regenerate whole rice plants. This
involves inducing the development of shoots and roots from the transformed cells.

7. *Verification of Beta-Carotene Production:*

- The transgenic rice plants are verified to confirm the presence and expression of
the beta-carotene biosynthetic genes. Analytical techniques may be used to measure
the production of beta-carotene in the plant tissues.

8. *Field Testing:*
- Transgenic Golden Rice plants undergo field trials to assess their performance
under natural conditions. This includes evaluating the levels of beta-carotene
production, agronomic characteristics, and overall plant health.

9. *Regulatory Approval:*
- Before commercialization, Golden Rice typically undergoes regulatory scrutiny to
ensure it meets safety and environmental standards. Regulatory agencies assess the
potential risks and benefits of Golden Rice.

10. *Commercialization:*
- Once regulatory approval is obtained, Golden Rice can be cultivated and
distributed for consumption. The goal is to provide a nutritional intervention that
addresses vitamin A deficiency, especially in regions where rice is a dietary staple.

Golden Rice is an example of a genetically modified crop designed to address a

specific nutritional deficiency, and its development involves rigorous research, testing,
and regulatory processes to ensure safety and efficacy.

Meta-genomics: an introduction

In metagenomics, scientists extract genetic material directly from environmental

samples, such as soil, water, or human microbiome. This genetic material, often
composed of DNA or RNA, is then sequenced to obtain a vast amount of genetic
information. Unlike traditional genomics, which focuses on the DNA of a single
organism, metagenomics looks at the genetic material of an entire community of
microorganisms.
Key steps in metagenomic analysis include:

1. *Sample Collection:* Collecting samples from diverse environments, ranging from

oceans to human intestines, to capture the microbial diversity.

2. *DNA Extraction:* Isolating and extracting the total genomic DNA from the
collected samples.

3. *Library Preparation:* Fragmenting the DNA into smaller, manageable pieces and
creating libraries that can be sequenced.

4. *Sequencing:* Employing high-throughput sequencing technologies to read the

genetic information in the libraries, generating massive amounts of data.

5. *Bioinformatics Analysis:* Processing and analyzing the sequencing data using

computational tools to identify genes, predict functions, and assess microbial diversity.

Metagenomics has various applications, such as:

- *Microbial Diversity:* Revealing the diversity of microorganisms in different

environments.

- *Functional Analysis:* Understanding the functional capabilities of microbial

communities, including metabolic pathways.

- *Bioprospecting:* Identifying novel genes and enzymes with potential applications

in industry, medicine, or environmental remediation.

- *Disease Association:* Investigating the role of microbial communities in health

and disease, particularly in the human microbiome.

Metagenomics has revolutionized our ability to study microbial ecosystems, providing

valuable insights into the unseen world of microorganisms and their roles in various
ecological and biological processes.

Molecular diagnosis of genetic diseases (Cystic fibrosis, Sickle cell anaemia)

Cystic fibrosis is a genetic disorder that affects the respiratory, digestive, and
reproductive systems. It leads to the production of thick and sticky mucus, causing
respiratory and digestive problems. Symptoms include persistent cough, difficulty
breathing, and digestive issues. It is a lifelong condition with treatments focused on
managing symptoms and improving quality of life.

Cystic fibrosis is caused by mutations in the CFTR (cystic fibrosis transmembrane

conductance regulator) gene. These mutations disrupt the normal function of the
CFTR protein, which plays a crucial role in controlling the flow of salt and fluids in
and out of cells. As a result, thick and sticky mucus accumulates in various organs,
particularly the lungs and digestive system. The condition is inherited in an autosomal
recessive manner, meaning an individual needs to inherit two faulty copies of the
CFTR gene (one from each parent) to develop cystic fibrosis.

In cystic fibrosis, the malfunctioning CFTR protein disrupts the balance of salt and
fluids in cells, leading to several complications:

1. *Respiratory Issues:* Thickened mucus clogs the airways, making it difficult to

breathe. This can result in persistent coughing, lung infections, and progressive lung
damage.

2. *Digestive Problems:* The thick mucus affects the pancreas, impairing the
production and release of digestive enzymes. This leads to difficulty absorbing
nutrients from food, causing malnutrition and poor growth.

3. *Other Organs:* Cystic fibrosis can also affect the liver, reproductive organs, and
sweat glands, leading to a range of complications such as liver disease and infertility.

The severity of symptoms varies among individuals, and advancements in medical

care have improved life expectancy and quality of life for those with cystic fibrosis.
Treatment typically involves managing symptoms, preventing infections, and
addressing nutritional needs.

Molecular diagnostics for cystic fibrosis involve various techniques to identify

mutations in the CFTR gene. Here's an overview of some methods:

1. *Polymerase Chain Reaction (PCR):* PCR is used to amplify specific regions of

the CFTR gene, making it easier to analyze for mutations.

2. *Restriction Fragment Length Polymorphism (RFLP):* RFLP is a technique that

detects variations in DNA sequence. It involves cutting the amplified DNA with
restriction enzymes, and the resulting fragments are separated to identify mutations in
the CFTR gene.

3. *Fluorescent In Situ Hybridization (FISH):* FISH can be employed to visualize

and map the location of the CFTR gene on chromosomes using fluorescent probes.
This technique helps identify large-scale chromosomal abnormalities.

4. *Multiplex Ligation-dependent Probe Amplification (MLPA):* MLPA detects

deletions or duplications in the CFTR gene. It involves ligating specific probes to the
target DNA, followed by PCR amplification and analysis of fragment lengths.

5. *Sanger Sequencing:* This method directly reads the DNA sequence of the CFTR
gene, allowing identification of specific mutations.

6. *Next-Generation Sequencing (NGS):* NGS technologies enable high-throughput

sequencing of the entire CFTR gene, providing comprehensive information on genetic
variations.

7. *Real-Time PCR (qPCR):* qPCR monitors the amplification of DNA in real-time

and can be used for quantitative analysis of CFTR mutations.
These molecular diagnostic techniques play a crucial role in identifying the specific
mutations responsible for cystic fibrosis. Understanding the genetic basis helps in
providing personalized and targeted therapies for individuals with this condition.
Keep in mind that the choice of method depends on factors like cost, speed, and the
level of detail required for a particular diagnosis.

Sure, let's delve into the details of Restriction Fragment Length Polymorphism
(RFLP) and Fluorescent In Situ Hybridization (FISH):

### Restriction Fragment Length Polymorphism (RFLP):

1. *Principle:*
- RFLP is based on variations in DNA sequences that result in different patterns of
restriction enzyme cleavage.
- The technique exploits polymorphisms in the lengths of DNA fragments generated
by cutting DNA with restriction enzymes.

2. *Procedure:*
- DNA is extracted from the sample and then digested with specific restriction
enzymes.
- The resulting fragments are separated by gel electrophoresis, creating a pattern of
bands on the gel.
- The gel is then treated to visualize the DNA fragments, often using a DNA stain.

3. *Analysis:*
- Variations in the lengths of DNA fragments can be observed as distinct banding
patterns.
- Comparing these patterns among individuals helps identify the presence or
absence of specific genetic variations, including mutations in the CFTR gene in the
case of cystic fibrosis.

4. *Application:*
- RFLP has been widely used for genetic linkage mapping, paternity testing, and
identifying genetic mutations associated with diseases.

### Fluorescent In Situ Hybridization (FISH):

1. *Principle:*
- FISH is a cytogenetic technique that uses fluorescent probes to bind to specific
DNA sequences.
- It allows visualization of the location and number of specific DNA sequences
within chromosomes.

2. *Procedure:*
- Cells, often from a tissue sample, are fixed on a slide.
- Fluorescent DNA probes, complementary to the target DNA sequence, are applied
to the fixed cells.
- The probe binds to its complementary sequence, and the fluorescence is visualized
under a fluorescent microscope.
3. *Analysis:*
- FISH can reveal the spatial organization of genes, detect chromosomal
abnormalities, and identify specific genetic markers.
- In the context of cystic fibrosis, FISH can be used to locate the CFTR gene on
chromosomes and detect any chromosomal rearrangements.

4. *Application:*
- FISH is widely employed in cytogenetics for research and clinical purposes,
including the detection of genetic disorders, cancer diagnosis, and studying
chromosomal abnormalities.

Both RFLP and FISH have been instrumental in genetic research and diagnostics,
offering valuable insights into genetic variations associated with diseases such as
cystic fibrosis.

Sickle cell anemia is a genetic blood disorder characterized by the presence of

abnormal hemoglobin, known as hemoglobin S. This causes red blood cells to become
rigid and take on a crescent or "sickle" shape. These misshapen cells can lead to
various complications, such as pain, anemia, and organ damage. It is an inherited
condition, with symptoms ranging from mild to severe.

Sickle cell anemia is caused by a genetic mutation that affects hemoglobin, the protein
responsible for carrying oxygen in red blood cells. The mutation leads to the
production of abnormal hemoglobin called hemoglobin S. Individuals inherit the
gene for sickle cell anemia from both parents, meaning they must have two copies of
the mutated gene (one from each parent) to develop the disorder.

When oxygen levels are low, the abnormal hemoglobin S causes red blood cells to
become rigid and take on a sickle shape. These sickle cells can then block blood flow,
leading to various complications such as pain, organ damage, and anemia. The genetic
mutation is more prevalent in certain populations, particularly those with ancestry
from regions where malaria is or was prevalent, as carrying one copy of the sickle cell
gene can provide some resistance to malaria.

In sickle cell anemia, the abnormal hemoglobin S causes red blood cells to become
rigid and assume a crescent or sickle shape. These misshapen cells can lead to several
complications:

1. *Vaso-occlusive Crises:* The sickle-shaped cells can block blood vessels, causing
episodes of severe pain known as vaso-occlusive crises. These painful episodes can
affect various organs and tissues.

2. *Anemia:* The sickle cells are fragile and can rupture, leading to a shortage of red
blood cells, a condition known as anemia. Anemia can result in fatigue, weakness,
and shortness of breath.

3. *Organ Damage:* The blocked blood flow can damage organs, especially the
spleen, liver, lungs, and kidneys. Over time, this can contribute to chronic organ
damage.
4. *Stroke:* Sickle cells can also block blood vessels in the brain, increasing the risk
of stroke, especially in children with sickle cell anemia.

5. *Infections:* People with sickle cell anemia are more susceptible to infections due
to a compromised immune system, particularly affecting the spleen, which is often
damaged in this condition.

6. *Delayed Growth:* Children with sickle cell anemia may experience delayed
growth and puberty due to the effects on bone development.

It's important to note that the severity of symptoms can vary, and individuals with
sickle cell anemia may experience different complications to different extents.
Management and treatment focus on alleviating symptoms and preventing
complications.

Molecular diagnostics for sickle cell anemia involves various techniques to identify
the specific genetic mutations associated with the disorder. Here's a detailed overview
of some key methods:

1. *Polymerase Chain Reaction (PCR):*

- PCR is used to amplify a specific region of the DNA that includes the HBB gene,
which encodes the beta-globin chain of hemoglobin.
- PCR allows for the detection of the specific point mutation responsible for sickle
cell anemia (A to T substitution in the HBB gene).

2. *Restriction Fragment Length Polymorphism (RFLP):*

- After PCR amplification, RFLP is often employed to analyze the DNA fragments.
- The mutation that causes sickle cell anemia creates a recognition site for certain
restriction enzymes. RFLP can identify the presence or absence of these sites.

3. *Allele-Specific Oligonucleotide (ASO) Hybridization:*

- ASO probes are short, synthetic DNA sequences designed to hybridize specifically
with the normal or mutated HBB gene.
- This technique helps identify the specific mutation by detecting the hybridization
pattern.

4. *DNA Sequencing:*
- Sanger sequencing or newer sequencing technologies can be used for direct
sequencing of the HBB gene. This provides a comprehensive analysis of the DNA
sequence, confirming the presence of the sickle cell mutation.

5. *Fluorescence In Situ Hybridization (FISH):*

- FISH is a cytogenetic technique that uses fluorescent probes to bind specific DNA
sequences.
- While less commonly used for sickle cell anemia, FISH can be applied to detect
chromosomal abnormalities, such as large deletions or rearrangements involving the
HBB gene.

6. *Multiplex Ligation-Dependent Probe Amplification (MLPA):*

 - MLPA is a technique that can identify deletions or duplications in the HBB gene.
- It involves the use of probes targeting specific regions, and the resulting PCR
products are quantified to assess copy number variations.

7. *Next-Generation Sequencing (NGS):*

- NGS technologies allow for high-throughput sequencing and can be applied to
analyze multiple genes simultaneously, providing a more comprehensive genetic
profile.

These molecular diagnostic techniques play a crucial role in confirming the diagnosis
of sickle cell anemia, determining carrier status, and facilitating genetic counseling.
The choice of method depends on factors such as cost, availability, and the specific
information required.

Recombinant DNA in medicines

Recombinant DNA technology has revolutionized medicine by enabling the

production of various therapeutic proteins, vaccines, and drugs. Here are a few
examples:

1. *Human Growth Hormone (HGH):*

- Recombinant DNA technology is used to produce synthetic human growth
hormone, which is crucial for normal growth and development. This has been
particularly beneficial for individuals with growth disorders.

2. *Erythropoietin (EPO):*
- Recombinant EPO is employed to stimulate the production of red blood cells. It is
used in the treatment of anemia, especially in patients with kidney failure or
undergoing certain medical treatments like chemotherapy.

3. *Clotting Factors:*
- Hemophiliacs, who lack certain clotting factors, benefit from recombinant DNA-
derived clotting factors. This helps in managing and preventing bleeding episodes.

4. *Vaccines:*
- Recombinant DNA technology is instrumental in the development of vaccines. For
example, the hepatitis B vaccine is produced using yeast cells that have been
genetically engineered to produce viral antigens.

5. *Monoclonal Antibodies:*
- Monoclonal antibodies used in cancer therapy and autoimmune diseases are often
produced through recombinant DNA technology. These antibodies are designed to
target specific cells or molecules in the body.

6. *Enzyme Replacement Therapy:*

- Enzyme deficiencies, such as in lysosomal storage disorders, can be treated with
recombinant enzymes. Patients receive regular infusions of the missing enzyme to
alleviate symptoms.
7. *Antibody Therapeutics:*
- Antibodies produced through genetic engineering are used in treating conditions
like rheumatoid arthritis and certain cancers. These antibodies can be designed to
target specific markers on diseased cells.

8. *Insulin (as previously mentioned):*

- Recombinant insulin is a classic example of using genetic engineering to produce a
crucial medicine for diabetes treatment.

Recombinant DNA technology allows for the precise manipulation of genes to

produce therapeutic agents, often with improved safety and efficacy compared to
traditional methods. This technology continues to play a vital role in advancing
medical treatments and improving patient outcomes.

Recombinant insulin

Certainly! Recombinant DNA technology involves the manipulation of genetic

material to create new combinations of genes. In the case of recombinant insulin,
here's a more detailed explanation:

1. *Isolation of the Insulin Gene:*

- The human insulin gene is isolated from human DNA. This gene contains the
instructions for producing insulin, a hormone crucial for regulating blood sugar.

2. *Creation of Recombinant DNA:*

- The isolated insulin gene is combined with a small, circular piece of DNA
called a plasmid. This plasmid acts as a vector, a carrier for the foreign gene. The
result is recombinant DNA, a hybrid molecule containing genetic material from
both the insulin gene and the plasmid.

3. *Introduction into Host Organism:*

- The recombinant DNA is introduced into a host organism, often a bacterium or
yeast, which will serve as a living factory to produce insulin. This is usually done
using a process called transformation.

4. *Replication and Expression:*

- The host organism replicates the recombinant DNA as it multiplies. As a result,
the insulin gene is also replicated. The host cell then reads the insulin gene and
uses it as instructions to produce insulin.

5. *Harvesting Insulin:*
- Once the host cells have produced a sufficient amount of insulin, the insulin is
harvested. The host cells are typically separated, and the insulin is purified from
the cell culture.

6. *Quality Control:*
- Rigorous quality control measures are taken to ensure that the produced insulin
is pure, safe, and biologically active. This includes testing for any contaminants
and confirming that the final product is structurally identical to human insulin.
7. *Medical Application:*
- The purified recombinant insulin is then used as a medication for people with
diabetes. It serves as a synthetic replacement for the insulin that the body would
normally produce to regulate blood sugar levels.

This process allows for the mass production of insulin in a controlled and efficient
manner, reducing dependence on animal-based insulin and ensuring a safer and
more sustainable supply for patients with diabetes.

From NCERT

Genetically Engineered Insulin

Management of adult-onset diabetes is possible by taking insulin at regular time
intervals. What would a diabetic patient do if enough human-insulin was not available?
If you discuss this, you would soon realise that one would have to isolate and use
insulin from other animals. Would the insulin isolated from other animals be just as
effective as that secreted by the human body itself and would it not elicit an immune
response in the human body? Now, imagine if bacterium were available that could
make human insulin. Suddenly the whole process becomes so simple. You can easily
grow a large quantity of the bacteria and make as much insulin as you need.
Think about whether insulin can be orally administered to diabetic people or not. Why?
Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and
pigs. Insulin from an animal source, though caused some patients to develop allergy
or other types of reactions to the foreign protein. Insulin consists of two short
polypeptide chains: chain A and chain B, that are linked together by disulphide
bridges. In mammals, including humans, insulin is synthesised as a pro-hormone (like
a pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully
mature and functional hormone) which contains an extra stretch called the C peptide.
This C peptide is not present in the mature insulin and is removed during maturation
into insulin.The main challenge for production of insulin using rDNA techniques was
getting insulin assembled into a mature form. In 1983, Eli Lilly an American
company prepared two DNA sequences corresponding to A and B, chains of human
insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A
and B were produced separately, extracted and combined by creating disulfide bonds
to form human insulin.

Human growth hormone (HGH)

Certainly! Here's a more detailed explanation of how recombinant DNA technology is

used to produce human growth hormone (HGH) for medicinal purposes:

1. *Isolation of the HGH Gene:*

- The process begins by isolating the gene that codes for human growth hormone
from human DNA. This gene contains the instructions for synthesizing the HGH
protein.
2. *Construction of Recombinant DNA:*
- The isolated HGH gene is combined with a vector, often a plasmid, which acts as a
carrier for the gene. This creates recombinant DNA, a hybrid molecule containing
genetic material from both the HGH gene and the vector.

3. *Introduction into Host Organism:*

- The recombinant DNA is introduced into a host organism, frequently a bacterium
or yeast. The host organism will serve as a living factory to produce HGH using the
instructions encoded in the inserted gene.

4. *Expression of HGH:*
- The host organism replicates the recombinant DNA during its normal growth and
division. As a result, the HGH gene is also replicated. The host cells then read the
HGH gene and translate it into the HGH protein, which is subsequently expressed
within the host organism.

5. *Harvesting and Purification:*

- Once the host cells have produced a sufficient amount of HGH, the hormone is
harvested. The host cells are separated, and the HGH is purified from the cell culture.
This purification process is crucial to obtaining a highly concentrated and pure form
of HGH.

6. *Quality Control:*
- Rigorous quality control measures are implemented to ensure the safety, purity,
and efficacy of the produced HGH. This involves testing for any contaminants and
verifying that the final product is structurally identical to natural human growth
hormone.

7. *Medical Application:*
- The purified recombinant HGH is used as a therapeutic agent. It is administered to
individuals with growth disorders, such as children with growth hormone deficiency,
to stimulate normal growth and development.

The use of recombinant DNA technology in producing human growth hormone has
significantly improved the availability and safety of HGH for medical applications,
offering a more reliable source compared to previous methods that involved
extraction from human pituitary glands.

Gene therapy

Certainly! Gene therapy is a medical approach that involves the introduction,

alteration, or replacement of genetic material within an individual's cells to treat or
prevent disease. Here's a detailed explanation of gene therapy:

1. *Identification of Target Gene:*

- The process begins with identifying the specific gene associated with a particular
disease. This could be a faulty or missing gene causing a genetic disorder, or it might
involve modifying a gene to enhance a therapeutic effect.
2. *Development of Therapeutic Gene:*
- Scientists design a therapeutic gene that will either replace, supplement, or modify
the function of the target gene. This therapeutic gene is often introduced into a vector,
which is a delivery system used to transport the gene into the target cells.

3. *Selection of Delivery Vehicle (Vector):*

- Vectors are typically viruses that have been modified to serve as delivery vehicles
for the therapeutic gene. Commonly used viruses include adenoviruses, lentiviruses,
and adeno-associated viruses (AAVs). These viruses are engineered to be safe and
effective in delivering the therapeutic gene to the target cells.

4. *Administration to Patient:*
- The modified vector containing the therapeutic gene is then introduced into the
patient's body. This can be done through various methods, such as direct injection into
the target tissue, intravenous injection, or even inhalation depending on the type of
disease being treated.

5. *Cellular Uptake:*
- The vector carries the therapeutic gene into the targeted cells. Once inside the cells,
the therapeutic gene is expressed, meaning it is used as instructions to produce the
desired protein or to regulate gene activity.

6. *Expression of Therapeutic Gene:*

- The therapeutic gene functions within the cells to produce a functional protein or
to modify cellular processes. This is aimed at correcting the underlying cause of the
disease or alleviating its symptoms.

7. *Monitoring and Adjustments:*

- The patient's response to gene therapy is closely monitored, and adjustments may
be made as needed. This monitoring involves assessing the levels of the therapeutic
protein, the impact on symptoms, and potential side effects.

8. *Long-Term Effects and Follow-Up:*

- Gene therapy aims for a long-term therapeutic effect. Follow-up assessments are
conducted to evaluate the sustained impact of the treatment, potential side effects, and
whether additional interventions or adjustments are necessary.

Gene therapy holds promise for treating a variety of genetic and acquired disorders,
ranging from rare genetic diseases to certain types of cancer. Ongoing research and
technological advancements continue to refine and expand the applications of gene
therapy in the field of medicine.
From NCERT

Gene Therapy
If a person is born with a hereditary disease, can a corrective therapy be taken for
such a disease? Gene therapy is an attempt to do this. Gene therapy is a collection of
methods that allows correction of a gene defect that has been diagnosed in a
child/embryo. Here genes are inserted into a person’s cells and tissues to treat a
disease. Correction of a genetic defect involves delivery of a normal gene into the
individual or embryo to take over the function of and compensate for the non-
functional gene. The first clinical gene therapy was given in 1990 to a 4-year old
girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the
immune system to function. The disorder is caused due to the deletion of the gene for
adenosine deaminase. In some children ADA deficiency can be cured by bone
marrow transplantation; in others it can be treated by enzyme replacement therapy, in
which functional ADA is given to the patient by injection. But the problem with both
of these approaches that they are not completely curative. As a first step towards gene
therapy, lymphocytes from the blood of the patient are grown in a culture outside the
body. A functional ADA cDNA (using a retroviral vector) is then introduced into
these lymphocytes, which are subsequently returned to the patient. However, as these
cells are not immortal, the patient requires periodic infusion of such genetically
engineered lymphocytes. However, if the gene isolate from marrow cells producing
ADA is introduced into cells at early embryonic stages, it could be a permanent cure.