Concept, methodology and applications

SOUTHERN BLOTTING
🔍 Concept
Southern blot is a method used to detect specific DNA sequences by transferring
electrophoresis-separated DNA fragments to a filter membrane and detecting them through
probe hybridization.

Southern blotting is a molecular biology technique used to detect specific DNA sequences
within a complex mixture of DNA.

 Developed by Edwin Southern in 1975

 Involves transfer of DNA fragments from an agarose gel onto a membrane (usually
nylon or nitrocellulose)
 A labeled DNA probe is then hybridized to the membrane to identify the fragment of
interest
🔍 Methodology (Step-by-Step)

1. DNA Extraction & Digestion

 Extract genomic DNA

 Cut with restriction enzymes to generate fragments

2. Gel Electrophoresis

 Separate DNA fragments by size using agarose gel electrophoresis

3. Denaturation

 Soak the gel in an alkaline solution (e.g., NaOH) to:

o Denature double-stranded DNA into single strands
o Necessary for probe hybridization
4. Blotting (Transfer)

 Transfer DNA from gel to nylon or nitrocellulose membrane

 Capillary action, vacuum, or electroblotting is used
 DNA fragments are immobilized on the membrane

5. Hybridization with Probe

 Incubate the membrane with a labeled probe (radioactive, fluorescent, or

chemiluminescent)
 The probe is complementary to the DNA sequence of interest

6. Washing

 Remove excess probe with high-stringency washes to ensure specificity

7. Detection

 Use a detection system (e.g., chemiluminescence) to visualize the bands where

hybridization occurred

🔍 Applications of Southern Blotting

✅1. Gene Detection

 Identify presence or absence of specific DNA sequences

 Confirm gene insertions or deletions

✅2. DNA Fingerprinting

 Compare DNA samples for forensic analysis or paternity testing

 Based on variation in restriction fragment length polymorphisms (RFLPs)

✅3. Diagnosis of Genetic Disorders

 Detect mutations, deletions, or rearrangements in disease-related genes (e.g., sickle cell

anemia, thalassemia)

✅4. Confirmation of Cloning

 Verify the presence of a cloned gene in a vector

✅5. Mapping Genomes

 Identify location of genes in large genomes (used before whole-genome sequencing

became common)

✅6. Transgene Analysis

 Check whether a transgene has integrated into an organism’s genome and how many
copies are present

🧪 NORTHERN BLOTTING:

🔍 Concept

Northern blotting is a molecular biology technique used to detect and study specific RNA molecules
(especially mRNA) in a sample.

 Developed by analogy to Southern blotting (hence the name)

 Helps in studying gene expression by analyzing RNA levels

🧠 Key Idea:
If a gene is actively being expressed, its mRNA will be present and detectable.

🔍 Methodology (Step-by-Step)

1. RNA Extraction

 Isolate total RNA or mRNA from cells or tissues

 Ensure RNase-free conditions (RNA is fragile and degrades easily)

2. Gel Electrophoresis

 Separate RNA molecules using formaldehyde-agarose gel electrophoresis

o Formaldehyde is used to denature RNA and prevent secondary structure formation

3. Blotting (Transfer)

 Transfer separated RNA onto a nylon or nitrocellulose membrane

 Usually done via capillary action or vacuum transfer
4. UV Cross-Linking or Baking

 RNA is immobilized on the membrane by:

o UV light cross-linking, or
o Baking at high temperature

5. Hybridization with a Probe

 Membrane is incubated with a labeled single-stranded DNA or RNA probe

 Probe is complementary to the RNA sequence of interest

6. Washing

 Excess, non-specifically bound probe is washed off

7. Detection

 Use autoradiography (radioactive probes) or chemiluminescence/fluorescence to detect bound

probe
 The signal corresponds to the presence and quantity of specific RNA

🧪 Applications of Northern Blotting

✅1. Gene Expression Analysis

 Determine if a gene is being transcribed in a specific cell or tissue type

 Compare mRNA levels across different developmental stages, treatments, or disease states
✅2. mRNA Size Determination

 Determine the length of mRNA transcripts

 Detect alternative splicing variants

✅3. Tissue-Specific Expression

 Identify which tissues express a particular gene

✅4. Effect of Drugs or Mutations

 Analyze how external treatments, mutations, or gene knockouts affect gene expression

🧪 WESTERN BLOTTING:

🔍 Concept

Western blotting (also called immunoblotting) is a technique used to detect specific proteins in a
complex mixture.

 Uses antibodies to bind specifically to the target protein

 Combines protein separation by size (via SDS-PAGE) and detection by antibody-based probing

🧠 Key Idea:
Detects protein expression — tells you whether a protein is present, and gives info about its size and
abundance

🔍 Methodology (Step-by-Step)

1. Protein Extraction

 Extract total protein from cells or tissues

 Use protease inhibitors to prevent degradation

2. Protein Quantification (optional but common)

 Measure concentration using BCA or Bradford assay to load equal amounts

3. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

 Separates proteins based on molecular weight

 SDS denatures proteins and gives them uniform negative charge

4. Blotting (Transfer)

 Transfer proteins from gel to a membrane (nitrocellulose or PVDF)

 Usually done using electroblotting

5. Blocking

 Incubate membrane with blocking solution (like BSA or non-fat milk)

 Prevents non-specific binding of antibodies

6. Primary Antibody Incubation

 Incubate membrane with a primary antibody specific to the target protein

7. Secondary Antibody Incubation

 Incubate with a secondary antibody that binds to the primary antibody

 This antibody is labeled with:
o Enzymes (e.g., HRP (horse radish peroxidase) or alkaline phosphatase)
o Or fluorescent tags

8. Detection

 Add substrate to visualize bands:

o Chemiluminescence (e.g., ECL system)
o Colorimetric or fluorescence methods
 The intensity of the band reflects protein abundance
🧪 Applications of Western Blotting

✅1. Protein Expression Analysis

 Detect whether a specific protein is expressed in a sample

✅2. Protein Size Determination

 Estimate the molecular weight of the protein

✅3. Post-Translational Modifications

 Identify phosphorylated, acetylated, or glycosylated forms of proteins using specific antibodies

✅4. Disease Diagnosis

 Detect disease-specific proteins (e.g., HIV confirmatory test)

 Used in diagnostics for Lyme disease, prion diseases, etc.

✅5. Validation of Other Experiments

 Confirm results from proteomics, ELISA, or mass spectrometry

✅6. Comparative Expression

 Compare expression levels across different tissues, time points, treatments, or conditions

Sure! Here's a clear and concise breakdown of Mendel’s Laws of Inheritance — super
important for any genetics section in molecular biology.

🧪 Mendel’s Laws of Inheritance

Formulated by Gregor Mendel in the 1860s based on his experiments with pea plants (Pisum sativum).

🔍 1. Law of Dominance

"In a heterozygous condition, one allele (dominant) masks the expression of the other (recessive)."

 When two different alleles for a trait are present, the dominant allele is expressed.
 The recessive allele is only expressed if both alleles are recessive.

🧪 Example:

 Cross between RR (round seeds) and rr (wrinkled seeds) gives Rr (round), not intermediate.

🔍 2. Law of Segregation

"Each individual has two alleles for a trait, which segregate (separate) during gamete formation, so
that each gamete gets only one allele." (Alleles seggregate)
  Occurs during meiosis.
 Each parent contributes only one allele to the offspring.
 Explains the 3:1 ratio seen in monohybrid crosses.

🧪 Example:

 F1 genotype: Rr
 F2 gametes: R and r segregate, so Rr × Rr gives:
o Genotypes: RR, Rr, Rr, rr
o Phenotypes: 3 round : 1 wrinkled

🔍 3. Law of Independent Assortment

"Genes for different traits are inherited independently of one another, if they are on different
chromosomes."

 Applies to dihybrid crosses or more

 Each trait’s allele pair segregates independently during gamete formation
 Explains the 9:3:3:1 ratio in F2 generation of a dihybrid cross

🧪 Example:

 Cross: YyRr × YyRr

o Y = yellow, y = green
o R = round, r = wrinkled
 F2 phenotypes:
o 9 Yellow Round
o 3 Yellow Wrinkled
o 3 Green Round
o 1 Green Wrinkled

🔍 Difference Between Meiosis and Mitosis

Feature Mitosis Meiosis

Growth, repair, asexual Sexual reproduction (formation of

Purpose
reproduction gametes)
 Feature Mitosis Meiosis

Location Somatic (body) cells Germ cells (testes & ovaries)

Number of Divisions 1 (single division) 2 (Meiosis I & Meiosis II)

Daughter Cells Formed 2 4

Genetically identical to parent Genetically different from parent & each

Genetic Identity
cell other

Chromosome Number Diploid → Diploid (2n → 2n) Diploid → Haploid (2n → n)

Crossing Over ❌ Absent ✅ Occurs in Prophase I

Homologous
Do not pair Pair up & segregate in Meiosis I
Chromosomes

Cell renewal, tissue repair,

Function in Organism Genetic variation, gamete formation
growth

Examples Skin cell division, wound healing Sperm and egg formation

Type Equational division Reductional division

🔍 What is a Vector?

A vector is a DNA molecule that acts as a carrier or vehicle to deliver a gene of interest (GOI)
into a host cell. Eg. natural plasmids used as vectors- colE1 and RSF 2124. Natural plasmids did
not fulfill all suitable criteria hence artificially constructed plasmid were used. The best example
is pBR 322
✂️ What Are Restriction Enzymes?

🔍 Definition:

Restriction enzymes (also called restriction endonucleases) are molecular scissors — enzymes that cut
DNA at specific nucleotide sequences, called recognition sites.

 They are naturally found in bacteria, where they serve as a defense mechanism against viral
DNA (bacteriophages).
 In biotechnology, they are used to cut and manipulate DNA for cloning, mapping, and analysis.
Refer notebook further.

Expression vector: li
🔍 Joining of DNA Molecules

In recombinant DNA technology, after DNA fragments are cut (e.g., with restriction enzymes), they
must be joined together to form a stable recombinant molecule. This joining is achieved using:

1. DNA Ligases
2. Linkers
3. Adaptors
4. Homopolymer tailing

1️⃣ DNA Ligases

🔍 Function:

DNA ligases are enzymes that catalyze the formation of phosphodiester bonds between adjacent
nucleotides, effectively joining DNA strands.
🔍 Types Used in rDNA Technology:
Type Source Best For

E. coli DNA Ligase Escherichia coli Joins DNA with sticky ends

T4 DNA Ligase T4 bacteriophage Joins both sticky and blunt ends

🧪 T4 DNA Ligase:

 Most commonly used in genetic engineering.

 Requires ATP as a cofactor.
 Can ligate blunt ends, which is more challenging.

🧪 E. coli DNA Ligase:

 Requires NAD⁺ as a cofactor.

 Less efficient, especially for blunt end ligation.

2⃣ Linkers

🔍 What are Linkers?

 Short, synthetic double-stranded oligonucleotides with recognition sites for restriction

enzymes.
 Added to blunt-ended DNA to create sticky ends.

🔍 How They Work:

1. Linkers are ligated to blunt-ended DNA.

2. Then treated with a restriction enzyme to generate sticky ends.
3. Allows the DNA to be inserted into a plasmid/vector with compatible sticky ends.

3️⃣ Adaptors

🔍 What are Adaptors?

 Pre-made synthetic oligonucleotides with one sticky end and one blunt end.
 Help attach DNA fragments to plasmids without cutting the fragment with restriction enzymes.
🔍 Key Point:

 Adaptors must be phosphorylated before ligation to allow bond formation.

4️⃣ Homopolymer Tailing

🔍 What is it?

 A technique where homopolymers (repeats of the same nucleotide) are added to DNA ends to
create complementary sticky tails.

🔍 Enzyme Used:

 Terminal deoxynucleotidyl transferase (TdT) adds nucleotides to 3′ ends without a template.

🔍 Example:

 One DNA strand gets a poly-A tail

 Another gets a poly-T tail
 They can then base pair and be ligated together

✅Summary Table:
Method Purpose Best For

DNA Ligase (T4) Forms phosphodiester bond Sticky or blunt end joining

DNA Ligase (E. coli) Sticky-end joining (needs NAD⁺) Less commonly used

Linkers Add restriction sites to blunt ends When site-specific cloning needed

Provide sticky ends without restriction

Adaptors Prevents need for cutting DNA
sites

Homopolymer When restriction enzymes are

Adds complementary tails to blunt ends
Tailing unavailable
🔍 Chemical Methods to Transfer Recombinant DNA into
Bacterial Host Cells
Chemical transformation methods are commonly used because they are simple, cost-effective, and
widely applicable, especially for E. coli.

🧪 1. CALCIUM CHLORIDE (CACL₂) MEDIATED TRANSFORMATION

🧪 Concept:

This method uses CaCl₂ to make bacterial cell walls permeable to DNA by neutralizing the negative
charges on both the DNA and the bacterial cell membrane.

🧪 Procedure:

1. Bacterial cells are made competent by incubation in ice-cold 0.1️ M CaCl₂.

2. Recombinant DNA is added to the competent cells.
3. The mixture is heat shocked (usually at 42°C for ~30–90 seconds) to allow DNA uptake.
4. Cells are then incubated in nutrient media to allow recovery and expression of the recombinant
gene.

✅Advantages:

 Simple and inexpensive

 Effective for plasmid transformation in E. coli
 No special equipment required

🧪 Limitations:

 Only works efficiently for competent cells

 Not suitable for all bacterial species
 Less effective for large plasmids

🧪 2. L IPOSOME -M EDIATED DNA TRANSFER

🧪 Concept:

Liposome-mediated transformation uses artificial lipid vesicles (liposomes) to encapsulate DNA and
facilitate its delivery into cells by fusing with the cell membrane.
🧪 Procedure:

1. DNA is encapsulated within liposomes (spherical lipid bilayers).

2. Liposomes are incubated with bacterial cells.
3. Fusion between liposomes and bacterial membranes allows DNA to enter the cells.

✅Advantages:

 Can protect DNA from degradation

 Useful for transfection of animal and plant cells
 May be adapted for bacteria with special conditions

🧪 Limitations:

 Not commonly used in bacteria — more popular in eukaryotic systems

 Preparation and delivery systems can be complex
 Low efficiency in standard bacterial transformation

🔍 Summary Table:
Method Principle Used In Pros Cons

CaCl₂- Calcium ions + heat shock Easy, low-cost, Lower efficiency

E. coli, basic bacteria
Mediated → DNA uptake widely used for some bacteria

Eukaryotes,
Liposome- Lipid vesicles fuse with Protects DNA, Less efficient in
experimental in
Mediated membrane to deliver DNA useful in research bacteria, costly
bacteria

🔍Physical Methods to Transfer Recombinant DNA into Bacterial Host

Cells

Once recombinant DNA is constructed, it must be introduced into a host cell (usually E. coli) for cloning
or expression. These methods are called transformation techniques.

There are physical, chemical, and biological methods — but here we’ll focus on physical methods:
⚡ 1. ELECTROPORATION

🧪 Concept:

Electroporation uses a brief, high-voltage electric pulse to create temporary pores or destabilize the
membrane in the bacterial cell membrane, allowing DNA to enter the cell.

🧪 Procedure:

1. Mix recombinant DNA with competent bacterial cells in a chilled electroporation cuvette.
2. Apply a short electric pulse (typically 1.8–2.5 kV).
3. DNA enters the cell through the membrane pores.
4. Cells are incubated for 1 hour before plating on nutrient media to allow recovery and
expression.

✅Advantages:

 High transformation efficiency

 Works for many types of cells (bacteria, yeast, plant, animal)
 Can be used for plasmid DNA, BACs, or even linear DNA

🧪 Limitations:

 Requires specialized equipment

 High voltage may kill cells if not optimized

🧪 2. GENE GUN (BIOLISTICS OR PARTICLE BOMBARDMENT)

🧪 Concept:

DNA is coated onto microscopic particles (usually gold or tungsten) and physically shot into cells at high
velocity using a "gene gun".

🧪 Procedure:

1. DNA is precipitated onto microscopic particles of gold or tungsten.

2. These particles are loaded into a gene gun device.
3. A burst of gas (helium or nitrogen) fires the particles into the target cells.
4. Some DNA enters the host cell and integrates into the genome.

✅Advantages:

 Useful for cells with tough walls (e.g., plant cells, some bacteria)
 Doesn’t require chemical competence or electroporation
  Useful in transgenic plant and animal research

🧪 Limitations:

 Low transformation efficiency in bacteria

 Can damage target cells
 Equipment is expensive and delicate

🔍 Summary Table:
Method Principle Used In Pros Cons

Electric pulse opens Bacteria, yeast, High efficiency, Requires expensive

Electroporation
membrane pores mammalian cells fast equipment

DNA-coated particles Plants, bacteria (rare), Works on tough Low efficiency, cell
Gene Gun
are shot into cells animal tissues cells/walls damage possible

🧪 Methods of Screening Recombinants

Once recombinant DNA is successfully introduced into a host cell, it’s essential to identify which cells
actually took up the recombinant plasmid. This process is known as screening.

🧪 1. SCREENING USING SELECTIVE MARKERS

🧪 Concept:

Selective markers are genes included in the vector that allow only those host cells with the vector to
grow under specific conditions.

✅Common Selective Markers:

 Antibiotic resistance genes (e.g., amp^r, tet^r): allow growth on antibiotic-containing media
 Nutritional markers: used in auxotrophic mutant strains (e.g., lacZ, his, ura)
🧪 How It Works:

1. After transformation, cells are spread on media containing a selective agent (e.g., ampicillin).
2. Only cells that contain the plasmid (with the antibiotic resistance gene) survive.
3. This tells us which cells took up the plasmid — but not whether the plasmid has the insert
(GOI).

➡️ That's where blue-white screening comes in.

🧪 2. BLUE-WHITE SCREENING

🧪 Principle:

Blue-white screening is a visual method to distinguish between:

 Recombinant plasmids (with insert)

 Non-recombinant plasmids (without insert)

It uses the lacZ gene, which encodes β-galactosidase, and a chromogenic substrate, X-gal.

🧪 How It Works:

1. lacZ gene is part of the plasmid's multiple cloning site.

 2. When an insert is ligated into this site, it interrupts the lacZ, inactivating β-galactosidase.
3. Bacteria are plated on media containing:
o X-gal (substrate)
o IPTG (induces lac operon)
o Antibiotic (e.g., ampicillin)
4. After incubation:
o Blue colonies = have plasmid without insert (goi) → functional lacZ → cleaves X-gal.
o White colonies = have plasmid with insert → disrupted lacZ → no cleavage → X-gal
stays colorless.

✅Summary Table:
Colony Color lacZ Status Insert Present? Interpretation

Blue Active ❌No Non-recombinant plasmid

White Disrupted (inactive) ✅Yes Recombinant (with insert)

🧪 Bacteriophage Life Cycles

Bacteriophages (phages) are viruses that infect bacteria. They follow two main types of replication
cycles:

 Lytic cycle → leads to host cell destruction

 Lysogenic cycle → viral DNA integrates into host genome

⚡ 1. LYTIC CYCLE (VIRULENT PHAGES)

🔍 Definition:

The lytic cycle is a replication process where the phage hijacks the host’s machinery to produce new
virus particles, ultimately lysing (bursting) the bacterial cell to release progeny.

🔍 Stages of the Lytic Cycle:

1. Attachment (Adsorption) – Phage attaches to specific receptors on bacterial surface.

2. Penetration – Phage injects its DNA into the host cell.
3. Biosynthesis – Host cell's machinery makes viral components (DNA and proteins).
4. Assembly (Maturation) – New phage particles are assembled inside the host.
5. Lysis – Host cell bursts, releasing new phages to infect other bacteria.
Lytic cycle of a T even bacteriophage

🔍 Virulent Phages & T-Series Phages

 Virulent phages only follow the lytic cycle (e.g., T4, T2 phages).
 T-series phages (T1–T7) are well-known E. coli phages used extensively in research.
o T4 phage is the classical example of a virulent phage.

Structure of T4 phage:
🌀 Concept of Plaque Formation

🧪 Plaques are clear zones formed on a bacterial lawn (solid culture) due to lysis of bacterial cells by
phages.

 One phage infects a cell → multiple lytic cycles → clears a zone around it.
 Each plaque theoretically arises from a single phage.

🧠 Used to quantify phages via plaque-forming units (PFUs).

🔍 2. Lysogenic Cycle (Temperate Phages)

🔍 Definition:

In the lysogenic cycle, phage DNA is integrated into the host bacterial genome and is replicated along
with the host DNA, without killing the host.

🔍 Temperate Phage (λ phage)

 λ (lambda) phage is a classic temperate phage that infects E. coli.

 Has the ability to switch between lytic and lysogenic cycles.
🔍 Steps in the Lysogenic Cycle:

1. Attachment & Penetration – Similar to lytic.

2. Integration – Phage DNA integrates into host chromosome as a prophage.
3. Replication – Host replicates, copying both bacterial and prophage DNA.
4. Induction – Under stress (e.g., UV light), prophage excises and enters the lytic cycle.

🔍 Comparison: Lytic vs. Lysogenic Cycle

Feature Lytic Cycle Lysogenic Cycle

Phage Type Virulent (e.g., T4) Temperate (e.g., λ phage)

Host Cell Fate Destroyed (lysed) Survives and divides normally

DNA Integration No Yes (forms a prophage)

Viral Multiplication Immediate Delayed (until induction)

Plaque Appearance Clear plaques May appear turbid (if partial lysis occurs)

🔍 Bacteriophage Mutants

Bacteriophages can undergo mutations just like any other organisms. Studying these phage mutants
helps in understanding viral genetics, protein function, and host-virus interactions.

🔍 1. Plaque Morphology Mutants (r-type mutants)

🧪 Concept:

Mutations can cause changes in plaque appearance due to differences in infection rate and lysis
efficiency.

 "r" stands for rapid lysis.

 r-type mutants produce larger, clearer plaques than wild-type phages.
🧪 Characteristics:

 r⁺ (wild type): Small, turbid plaques (normal lysis rate)

 r (mutant): Large, clear plaques (rapid lysis)

🧠 These mutations typically occur in genes controlling lysis timing or adsorption rate.

🔍 2. Host Range Mutants

🧪 Concept:

These mutants can infect a different set of bacterial strains than the wild-type phage.

 Due to mutations in genes encoding tail fibers or other binding proteins.

 Helps study phage-bacteria specificity.

🧪 Example:

A phage that usually infects E. coli strain A might, after mutation, also infect strain B. This is a host range
extension.

🧠 Used in mapping bacterial surface receptors and phage adsorption mechanisms.

🔍 3. Conditional Lethal Mutants

These mutants can survive and replicate only under specific conditions, making them "conditionally
viable".

🧪 Types:

a. Ts mutants (Temperature-sensitive)

 Grow at permissive temperatures (e.g., 30°C)

 Fail to grow at non-permissive temperatures (e.g., 42°C)

🧠 Useful for studying essential phage genes:

 Allow mutant to grow and be studied at lower temperatures.

 Gene function is shut down at higher temperatures, revealing its role.
b. Am mutants (Amber mutants)

 Contain nonsense mutations introducing a premature stop codon (UAG).

 Cannot grow unless the host has a suppressor tRNA that reads through the stop codon.

🧠 Used in:

 Studying gene function

 Mapping genetic code
 Understanding translation regulation

🔍 Summary Table:
Mutant Type Characteristic Use in Research

Study lysis mechanism, mutation

r-type (rapid lysis) Large, clear plaques due to fast cell lysis
effects

Understand host specificity,

Host range mutants Infect new/different bacterial strains
receptor binding

Ts (Temperature- Functional studies of essential

Replication depends on temperature
sensitive) phage genes

Stop codon introduced; growth needs Gene function, genetic code,

Am (Amber mutants)
suppressor tRNA translation study

🔍 Holliday Model for Homologous Recombination

🔍 Definition:

The Holliday model, proposed by Robin Holliday in 1964, explains how homologous
recombination occurs between two similar or identical DNA molecules — especially during
meiosis or DNA repair.
🔍 Steps of the Holliday Model:

1. Alignment of Homologous DNA:

 Two homologous DNA duplexes (usually from homologous chromosomes or sister

chromatids) align precisely.

2. Single-Strand Nicking:

 Identical positions on one strand of each duplex are nicked (cut).

 Creates free 3' ends for strand invasion.

3. Strand Invasion and Exchange:

 The free 3' end of each strand invades the opposite duplex and base-pairs with the
complementary strand, displacing the original strand.
 Forms a cross-shaped structure called the Holliday junction.

4. Branch Migration:

 The junction moves along the DNA in a process called branch migration, enlarging the
region of heteroduplex (mixed base pairs from both DNA molecules).

5. Resolution of Holliday Junction:

 The crossed strands are cut in one of two ways:

o Horizontal cut (same strands as nicked originally): Produces non-recombinant
(patch) DNA.
o Vertical cut (opposite strands): Produces recombinant (splice) DNA.

🧠 Result: Genetic material is exchanged between the homologous molecules.

🔍 Role of Proteins in Recombination (E. coli system)

1. 🧪 REC PROTEINS (INITIATION AND PROCESSING)

Protein Function

Binds single-stranded DNA; promotes strand invasion and pairing of homologous

RecA
sequences. Central to recombination!
 Protein Function

Complex with helicase and nuclease activity; prepares DNA ends for recombination
RecBCD
by producing 3' overhangs (end resection)

2. 🧪 RUV PROTEINS (BRANCH MIGRATION AND RESOLUTION)

Protein Function

RuvA Binds the Holliday junction and forms a complex with RuvB

RuvB Helicase that drives branch migration using ATP

Resolvase that cuts the Holliday junction to resolve the recombination intermediate
RuvC
into two separate DNA molecules

🔍 Summary Table:
Stage Involved Proteins

Strand invasion RecA

End resection RecBCD

Branch migration RuvA + RuvB

Junction resolution RuvC

🔍 What is End Resection?

🔍 Definition:

End resection is the process by which the ends of a DNA double-strand break (DSB) are processed to
generate single-stranded 3' overhangs that are essential for homologous recombination.

This exposes single-stranded DNA (ssDNA) that can invade a homologous DNA molecule — a critical
step in DNA repair and recombination.
🧪 Joining of DNA Molecules in RDT (Recombinant DNA
Technology)

Once you cut your gene of interest and your vector with restriction enzymes, you need to join them
together to make a recombinant DNA molecule. This is where DNA ligases and other techniques come
into play.

🧪 1. DNA LIGASES: M OLECULAR GLUE

DNA ligase is an enzyme that joins two DNA strands by forming a phosphodiester bond between the 3′-
OH and 5′-phosphate ends.

🧪 Two commonly used ligases:

Ligase Source Function

T4 DNA ligase T4 bacteriophage Can join both sticky ends and blunt ends

E. coli DNA ligase E. coli Mainly joins sticky ends; requires NAD⁺ instead of ATP

🧪 Use in RDT:

 Joins insert DNA and vector DNA to form recombinant plasmid

🧪 2. USE OF ADAPTORS
🔍 What are Adaptors?

Short synthetic double-stranded DNA fragments with a restriction site built in.

🧪 Why use them?

 When your DNA fragment doesn’t have compatible cohesive ends, adaptors provide the
needed site for ligation.
🧪 3. HOMOPOLYMER TAILING

🔍 What is it?

Adding long stretches of the same nucleotide (a homopolymer) to the 3' ends of DNA using an enzyme
called terminal deoxynucleotidyl transferase (TdT).

🧪 Example:

 Add poly-dG tail to the insert

 Add poly-dC tail to the vector
 G and C will pair via base pairing → DNA can now anneal and be joined

Homopolymer tailing is like giving DNA pieces matching zippers so they can connect.

🔍 Summary Table:
Method Used For How it Works Special Tools

Forms phosphodiester
DNA Ligase Join compatible DNA ends T4 DNA ligase
bonds

Synthetic DNA +
Adaptors Add restriction sites Ligate synthetic DNA pieces
ligase

Homopolymer Join blunt ends without Add G/C/A/T tails for

TdT enzyme
tailing restriction sites annealing

🔍 What Are Linkers?

🔍 Definition:

Linkers are short, synthetic double-stranded DNA sequences that contain a specific restriction enzyme
recognition site.

They are blunt-ended, so they can be ligated to any blunt-ended DNA fragment — and then cut with
restriction enzymes to create sticky ends.

🔍 Why Use Linkers?

Imagine this situation:

  You have a blunt-ended DNA fragment (from PCR or certain restriction enzymes)
 Your vector requires sticky ends (for better ligation)

🧠 You can ligate a linker to the blunt-ended DNA, and then digest it with a restriction enzyme to create
a sticky end that matches the vector.