Md.

Sabbir Hossain Khan

Pharmacy 49

Chapter 3: Tools of Genetic Engineering

Introduction

●​ Simply inserting a human gene into a bacterial cell is not sufficient for gene expression
since it will not replicate.
●​ A technique is needed to ensure replication and expression in bacterial cells.
●​ This is achieved using vectors to insert genes into bacterial DNA, creating a chimera
(composite DNA).
●​ The chimera, once inside the bacterial cell, can replicate and express the human gene,
producing proteins.

Important Tools of Genetic Engineering

1.​ Enzymes
2.​ Vectors
3.​ cDNA clone bank or cDNA library
4.​ Gene bank or genomic library

Enzymes Required for Recombinant DNA (rDNA)

Technology
Key enzymes used in genetic engineering:

1.​ Restriction Endonucleases​

○​ Enzymes that cut double-stranded DNA at specific sequences.

○​ Used for gene isolation, mapping DNA, and analyzing chromosome structures.
○​ Two types:​

■​ Type-I Endonucleases: Cut DNA randomly 1000-5000 bases away from

the recognition site; not commonly used in genetic engineering.
■​ Type-II Endonucleases: Cut DNA at specific sequences, generating
DNA fragments of different lengths.
○​ Restriction enzymes cut DNA in two ways:​

■​ Blunt Ends: Both strands cut at the same position.

■​ Sticky/Cohesive Ends: Staggered cuts with single-stranded overhangs.

Examples of Restriction Endonucleases

●​ BamHI (Bacillus amyloliquefaciens) → Cuts at GGATCC.
●​ EcoRI (E. coli RY 13) → Cuts at GAATTC.
●​ HindIII (Haemophilus influenzae Rd) → Cuts at AAGCTT.
●​ HaeII, BglI, HpaI, SalI → Various recognition sequences
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2.​ S1 Nuclease​

○​ Cleaves single-stranded DNA or the single strand of double-stranded DNA

with cohesive ends.
○​ Converts cohesive ends into blunt ends.
3.​ DNA Ligase​

○​ Seals single-strand nicks in DNA with 5′-3′ OH termini.

○​ Two main types:
■​ E. coli Ligase: Uses NADr as a coenzyme.
■​ T₄ DNA Ligase: Uses ATP and can join blunt ends.
4.​ Alkaline Phosphatase​

○​ Prevents plasmid DNA from self-ligation after being cut with restriction enzymes.
○​ Removes the 5' phosphate group, ensuring foreign DNA fragments can only
ligate with plasmid DNA.
5.​ Reverse Transcriptase​

○​ Converts RNA into complementary DNA (cDNA).

○​ Essential for expressing human genes in bacterial cells.
○​ Human genes contain both exons (coding regions) and introns (non-coding
regions).
○​ Bacteria lack mechanisms to remove introns, making direct human gene
insertion ineffective.
○​ Solution: Convert mRNA (which lacks introns) into cDNA (complementary
DNA) using Reverse Transcriptase.
○​ This enzyme synthesizes cDNA from mRNA, which is crucial for creating cDNA
libraries.
6.​ DNA Polymerase​

○​ These enzymes polymerize DNA and are essential for cDNA synthesis.
○​ Functions:
■​ 5′ → 3′ polymerase activity (DNA synthesis).
■​ 3′ → 5′ exonuclease activity (proofreading).
■​ 5′ → 3′ exonuclease activity (removal of primers or damaged DNA).
○​ Types:
■​ DNA Polymerase I (Poly I)
■​ DNA Polymerase II (Poly II)
■​ DNA Polymerase III (Poly III)

Enzymes Required for Recombinant DNA (rDNA) Technology

Enzyme Function Types & Examples
Characteristics

Restriction Cut double-stranded - Type I: Cuts - BamHI → GGATCC

Endonucleases DNA at specific randomly 1000-5000 - EcoRI → GAATTC -
sequences bases away (not HindIII → AAGCTT -
commonly used) - HaeII, BglI, HpaI,
Type II: Cuts at SalI
specific sequences
(used in rDNA
technology) - Cuts
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produce blunt ends

or sticky ends

S1 Nuclease Cleaves Converts cohesive

single-stranded ends into blunt ends
DNA or single strand
of cohesive-ended
DNA

DNA Ligase Joins DNA fragments - E. coli Ligase:

by sealing Uses NADr - T₄ DNA
single-strand nicks Ligase: Uses ATP,
joins blunt & sticky
ends

Alkaline Prevents plasmid Removes 5'

Phosphatase self-ligation phosphate group,
ensuring only foreign
DNA can ligate

Reverse Converts RNA into - Used for human

Transcriptase complementary gene expression in
DNA (cDNA) bacteria - Converts
mRNA (without
introns) into cDNA

DNA Polymerase Synthesizes and - 5′ → 3′ polymerase - DNA Polymerase I

repairs DNA activity (DNA (Poly I) - DNA
synthesis) - 3′ → 5′ Polymerase II (Poly
exonuclease II) - DNA
activity Polymerase III (Poly
(proofreading) - 5′ → III)
3′ exonuclease
activity (removes
primers or damaged
DNA)

Key Takeaways
●​ Restriction enzymes recognize specific palindromic sequences and cut DNA into
fragments.
●​ Sticky ends are more efficient for DNA ligation than blunt ends.
●​ Enzymes like alkaline phosphatase prevent unwanted self-ligation of plasmids.
●​ Reverse transcriptase is critical for expressing eukaryotic genes in bacteria.

Cloning Vectors
●​ Vectors: DNA molecules that carry foreign DNA into host cells.
●​ Functions as vehicles for gene transfer.
●​ Commonly used vectors:
1.​ Bacterial plasmids
2.​ Bacteriophages
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3.​ Artificial chromosomes

Plasmids
●​ Small, circular DNA molecules found in bacteria.
●​ Replicate independently of chromosomal DNA.
●​ Carry important genes, e.g., antibiotic resistance, toxin production.
●​ Can be transferred between bacterial cells.

Key Features of Plasmids

●​ Can be modified for cloning while retaining natural properties.

●​ Example: pBR322, an extensively used cloning plasmid.
○​ Constructed by cutting and rejoining plasmid parts using restriction enzymes.
○​ Demonstrates ideal features for gene cloning.
○​ Contains genes for ampicillin (ampr) and tetracycline (tetr) resistance.
○​ Restriction sites for over 20 restriction enzymes.
○​ If a gene is inserted into BamHI, tetracycline resistance is lost, but ampicillin
resistance remains.

Selection of Recombinant Plasmids

●​ Size of plasmid matters → Smaller plasmids are preferred as cloning vectors.
●​ Small DNA molecules are less prone to damage during isolation.
●​ Transformation efficiency is higher with smaller plasmids.

Important Cloning Vectors

Vector Source Size (kb) Marker

pACYC 177 E. coli 3.7 Ampr, Kanr

pBR322 E. coli 4.3 Ampr, Tetr

pSC 101 E. coli 5.4 Tetr

pUC 720 Derived from pBR322 6.4 Ampr

Phage M13 E. coli 6.4 SS DNA Production

SV 40 Mammalian cells 5.2 -

Key Takeaways
●​ Reverse transcriptase is essential for human gene expression in bacteria.
●​ DNA polymerases have multiple roles, including proofreading and primer removal.
●​ Cloning vectors like plasmids play a crucial role in gene transfer.
●​ pBR322 plasmid is widely used due to its selectable markers and restriction sites.
●​ Smaller plasmids improve transformation efficiency and cloning success.

Bacteriophages and Cosmids in Genetic Engineering

Bacteriophages as Vectors
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●​ Bacteriophages are viruses that infect bacteria, consisting of DNA enclosed in a
protein capsid.
●​ They can be used as cloning vectors for large DNA fragments.
●​ Types of bacteriophages infecting E. coli:
1.​ λ (Lambda) Phage – Head and tail structure.
2.​ M13 Phage – Filamentous shape.

Lambda (λ) Phage as a Cloning Vector

●​ The λ phage has a head with DNA (~49 kb) and a tail for infection.
●​ It binds to E. coli and injects its DNA into the cell.
●​ Inside the cell, the DNA circularizes using cos sites (12-base complementary ends).
●​ It replicates using the rolling-circle method, forming concatameric DNA.
●​ The DNA is cut at cos sites and packed into new phage heads.
●​ Foreign DNA can be inserted by removing non-essential genes.
●​ There are two types of λ vectors:
1.​ Insertion vectors (single cleavage site, for small DNA).
2.​ Replacement vectors (two cleavage sites, for large DNA).
●​ The modified DNA is packed into phage heads and used to infect E. coli.
●​ λ phage is useful for cloning large DNA fragments efficiently.

Cosmids – Hybrid Vectors

●​ Cosmids = Plasmid + Cos site of phage DNA.
●​ Can carry larger DNA fragments (~45 kb) than plasmids.
●​ Do not produce viral particles (no cell lysis).
●​ Key Features:
1.​ Origin of replication for bacterial growth.
2.​ Antibiotic resistance marker for selection.
3.​ Unique cleavage site for DNA insertion.
4.​ Cos site enables packaging into phage heads for efficient infection.
5.​ Replicates as a plasmid inside bacteria.
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Comparison: Plasmids, Phages, and Cosmids

Feature Plasmids Phage Vectors Cosmids

DNA Size Capacity ~15 kb ~23 kb ~45 kb

Mode of Entry Transformation Infection Infection

Replication Plasmid replication Rolling-circle Plasmid replication

Selection Marker Antibiotic resistance Plaque formation Antibiotic resistance

Used for Small DNA fragments Larger DNA Large DNA fragments
fragments

Cosmids combine the advantages of plasmids (stability, easy selection) and phages (efficient
entry into bacteria), making them ideal for cloning large DNA fragments.

Difference Between cDNA Library and Gene Bank

Both cDNA library and gene bank are essential tools in genetic engineering, but they differ in
their content, construction, and applications.

1. cDNA Library

●​ Derived from mRNA using reverse transcriptase.

●​ Contains only expressed genes (exons), meaning non-coding regions (introns) are
absent.
●​ Represents only active genes at a particular time and condition.
●​ Used for studying gene expression, identifying functional genes, and producing
recombinant proteins.
●​ Constructed by isolating mRNA, synthesizing cDNA, inserting it into a vector, and
transforming it into host bacteria.

2. Gene Bank (Genomic Library)

●​ Contains entire genomic DNA, including both coding (exons) and non-coding
(introns) regions.
●​ Represents the complete genome of an organism.
●​ Used for genome sequencing, genetic mapping, and cloning of entire genes.
●​ Constructed by digesting genomic DNA with restriction enzymes, inserting fragments
into a vector, and transforming into host cells.

Key Differences
Feature cDNA Library Gene Bank

Source mRNA Whole genomic DNA

Contains Only coding sequences Both coding (exons) and

(exons) non-coding (introns)
sequences

Method Reverse transcription Restriction enzyme digestion

Use Studying gene expression Genome mapping,

and protein production sequencing, and studying
regulatory elements
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Chapter 6: Introduction To Monoclonal Antibodies

1. Introduction

●​ Antibodies are special proteins made by the immune system to protect the body from
harmful things like germs and cancer cells.
●​ They help the body by recognizing and fighting off foreign invaders.

2. Immune System Overview

●​ The immune system fights off harmful substances called antigens.

●​ There are two main types of immune responses:
1.​ Cell-mediated immune response – T-cells attack harmful cells directly.
2.​ Humoral immune response – B-cells make antibodies to attach to harmful
substances and help remove them from the body.

3. Antigens and Their Role

●​ Antigens are substances that cause the immune system to react.

●​ Epitopes are small parts of an antigen that antibodies can recognize.
●​ Haptens are tiny molecules that can't start an immune response on their own but can
trigger one when attached to a larger protein.

4. Structure of Antibodies

●​ Antibodies (also known as Immunoglobulins) are Y-shaped proteins made of four

protein chains:
○​ Two light chains
○​ Two heavy chains
●​ The light chains are made of smaller protein parts, either kappa (κ) or lambda (λ).
●​ The heavy chains decide what type of antibody it is, such as IgG, IgA, IgM, IgE, or IgD.
●​ The antibody has a constant region that stays the same, and a variable region that
changes to bind with different antigens.
●​ When an antibody is cut with enzymes, it makes two parts:
○​ Fab (binding part) – This part attaches to antigens.
○​ Fc (constant part) – This part helps the immune system work better.
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5. Monoclonal Antibodies (mAbs) and Hybridoma Technology

●​ Polyclonal antibodies are made by many B-cells and can recognize different parts of
an antigen.
●​ Monoclonal antibodies (mAbs) come from one B-cell and target only one part of the
antigen, making them very specific.
●​ B-cells can’t live forever in labs, so they can’t make antibodies for long.
●​ Hybridoma technology helps solve this problem:
○​ B-cells are mixed with cancer cells (called myeloma cells) to create
hybridomas.
○​ Hybridomas can grow forever and keep making large amounts of monoclonal
antibodies.

6. Applications of Monoclonal Antibodies

●​ Medical tests: Monoclonal antibodies are used in tests like pregnancy tests, disease
detection, and blood tests.
●​ Treatments: They are used to treat diseases like cancer and autoimmune diseases.
●​ Research: Scientists use monoclonal antibodies to find proteins, study cells, and
understand diseases.

Chapter 7:Production of Monoclonal Antibodies

INTRODUCTION
Monoclonal antibodies (MAbs) were first produced in 1975 by Kohler and Milstein, involving
immunochemistry, in vitro cancer cell culture, and molecular biology.

PRINCIPLE INVOLVED IN HYBRIDOMA TECHNOLOGY

According to Burnet’s clonal selection theory, a B cell produces antibodies of single
specificity. Immunization of an animal stimulates plasma cells (B cell clones) to produce
antibodies against different epitopes of an antigen. However, plasma cells die in culture,
making continuous MAb production difficult.

Myeloma cells (cancerous plasma cells) grow indefinitely but lack useful antibody specificity.
By fusing normal plasma cells with myeloma cells, scientists created heterokaryons, later
known as Hybridomas. The fusion was first done using the Sendai virus, but polyethylene
glycol (PEG) proved more efficient.

SELECTION OF HYBRID CELLS USING HAT MEDIUM

Littlefield’s method ensured only hybrid cells survived. It used HAT medium (Hypoxanthine,
Aminopterin, Thymidine) to block de novo nucleotide synthesis, forcing cells to rely on the
salvage pathway, which requires HGPRT enzyme.

●​ HGPRT-negative myeloma cells died in HAT medium.

●​ HGPRT-positive plasma cells survived but died naturally.
●​ Only fused hybrid cells (Hybridomas) survived, as they complemented each other’s
deficiencies.
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FINAL RESULT

Hybridomas continuously produce monoclonal antibodies, useful in medicine, diagnostics,

and research. Hence, this process is called Hybridoma Technology.

●​ Problem: HGPRT-positive cells can survive in HAT medium alone.

●​ Solution: Littlefield used HGPRT-positive cells lacking thymidine kinase.
●​ Process:
○​ HGPRT-positive and HGPRT-negative cells are fused.
○​ Each cell supplies what the other lacks.
○​ Only fused hybridoma cells survive in HAT medium.
●​ Result: If one fused cell is a B cell, it produces monoclonal antibodies continuously.
●​ Technique: This method is called hybridoma technology.

Hybridoma Production Process

1. Immunization

●​ Definition: Immunization is the process of injecting an antigen mixed with an adjuvant

into a mouse to stimulate the production of antibodies.
●​ Key Points:
○​ Antigen + adjuvant injected multiple times.
○​ Monitor antibody levels through blood samples.
○​ Sacrifice mouse and harvest spleen for spleenocytes.

2. Cell Fusion

●​ Definition: Cell fusion involves fusing spleenocytes (immune cells) with immortal
myeloma cells to create hybridomas capable of continuous antibody production.
●​ Key Points:
○​ Spleenocytes mixed with myeloma cells.
○​ Fusion induced with polyethylene glycol (PEG).
○​ Human MAbs made through genetic engineering.

3. Selection in HAT Medium

●​ Definition: Hybridomas are selected by culturing them in HAT medium, which allows
only hybridomas to survive while other cells die.
●​ Key Points:
○​ HAT medium used for selection.
○​ Hybridomas transferred to regular culture medium.

4. Screening (ELISA Test)

●​ Definition: Screening identifies hybridomas producing the desired antibody using an

Enzyme-Linked Immunosorbent Assay (ELISA).
●​ Key Points:
○​ Antigen adsorbed on 96-well plates.
○​ Sample incubated to detect antibody binding.
○​ Enzyme-linked secondary antibody detects binding via color change.

5. Cloning
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●​ Definition: Cloning ensures monoclonal antibody production by isolating single
hybridoma cells and growing them into colonies.
●​ Key Points:
○​ Limiting dilution: Isolate single cells in separate wells.
○​ Soft agar: Grow colonies in semisolid media.
○​ Positive clones are expanded and re-screened.

6. Characterization and Storage

●​ Definition: Characterization verifies the monoclonality, specificity, stability, and suitability

of the antibody for its intended use.
●​ Key Points:
○​ Biochemical tests: Spectrometry, electrophoresis, chromatography.
○​ Stability and affinity testing.
○​ Freeze hybridoma cells in liquid nitrogen for long-term storage.

Final Step: Monoclonal Antibody Production

●​ Definition: The final product, monoclonal antibodies, is used for therapeutic, diagnostic,
and research purposes.
●​ Key Points:
○​ Used in medicine (e.g., cancer treatments).
○​ Used in diagnostics (e.g., disease detection).
○​ Used in research (e.g., protein studies).