Assignment Title: Molecular Biology Techniques.
Course Title: Biochemistry and molecular Biology
Course Code: Pharm 217
Section: B
Submitted To:
Ms. Ramisa Anjum
Lecturer
Department of Pharmacy
University of Asia Pacific
Submitted By:
Reg.No:
23103090,23103092,23103093,23103094,23103095,23103096,23103097
Date of Submission: 24 November 2024
Department of Pharmacy
University of Asia Pacif
Northern Blot
1.(a) Principle of Northern Blot technique:
Definition:
The Northern blot, or RNA blot is a technique used in molecular biology research to study
gene expression by detection of RNA (or isolated mRNA) in a sample.
Principle:
• The principle of Northern Blotting is based on the ability of complementary RNA and DNA
strands to form stable complexes through base pairing .It includes transferring of
biomolecules from one membrane to another.
• The RNA samples are separated on gels according to their size by gel electrophoresis.Since
RNAs are single-stranded, these can form secondary structures by intermolecular base
pairing. The electrophoretic separation of the RNA segments is thus performed under
denaturing conditions.
• The separated RNA fragments are then transferred to a nylon membrane. Nitrocellulose
membrane is not used as RNA doesn’t bind effectively to the membrane.
• The transferred segments are immobilized into the membrane by fixing agents. The RNA
fragments on the membrane are detected by the addition of a labeled probe.
• The hybridization forms the basis of the detection of RNA as the specificity of hybridization
between the probe, and the RNA allows the accurate identification of the segments.
1.(b) Steps Involved In This Process:
1.Separate RNA by size: The RNA gel solution is prepared by adding formaldehyde to the
agarose solution The cast is assembled, and the prepared denaturing gel is poured into the
cast. As the gel begins to set, a comb with appropriate teeth is added to form wells.Once the
gel is set, the comb is removed, and the gel is equilibrated with a running buffer for 30
minutes before running.15 µg RNA sample is mixed with an equal volume of RNA loading
buffer.The samples are incubated at 65°C on a heating block for about 12-15 minutes. The
gel is then run at 125V for about 3 hours.
2. Transfer RNA to a membrane: A nylon membrane is cut that is larger than the size of
the denaturing gel, and a filter paper with the same size as the nylon membrane is also
prepared.Once the electrophoresis process is complete, the RNA gel is removed from the tank
and rinsed with water..The nylon membrane prepared is wetted with distilled water on an
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RNase-free dish for about 5 minutes.The wetted membrane is placed on the surface of the gel
while avoiding any air bubbles formation.The surface is further flooded with SSC, and a few
more filter papers are placed on top of the membrane.A glass plate is placed on top of the
structure in order to hold everything in place. The structure is left overnight to obtain an
effective transfer.
Figure :Steps of Northern blotting
3. Hybridize a probe to the RNA: The DNA or RNA probes to be used are to be labeled to a
specific activity of >108 dpm/µg, and unincorporated nucleotides are to be removed.The
membrane carrying the immobilized RNA is wetted with SSC.The membrane is placed in a
hybridization tube with the RNA-side-up, and 1 ml of formaldehyde solution is added..The
tube is placed in the hybridization oven and incubated at 42°C for 3 hours.The solution is
poured off, and the membrane is washed with a wash solution. The membrane is then observed
under autoradiography.
4. Detect the hybridized complex: The hybridized complex can be detected using various
methods, such as radioactivity, fluorescence et
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1.(c) Applications of Northern blot in Molecular biology:
Northern blotting is a key molecular biology technique used for the detection and analysis of
RNA. Here are some of its major applications:
1. Observation of a particular gene’s Expression, development stages,, pattern between
tissues, organs, environment stress levels etc.
2. Used to show overexpression of oncogenes and down regulation of tumour suppressor
gene’s in cancerous cells.
3. Detecting a specific mRNA in sample used for screening recombinant which are
successfully transformed with transfer.
4. Also used for studying mRNA Splicing
Example:
“Utilizing Northern Blot Analysis to Examine Gene Expression Alterations in Human
Tumors”
Northern blot in human tumors, changes in the expression levels of cancer-causing genes are
frequent. This analysis can be used to investigate whether a gene is overexpressed or
underexpressed in cancer cells as compared to normal cells. Overexpression means the cancer
cells produce or transcribe too much RNA from the gene. Overexpression can occur when a
gene is amplified or turned on or turned up.
Under expression means the cancer cells produce or transcribe too little RNA from the gene.
Underexpression can happen when a gene is inappropriately deleted or turned off or turned
down.
1.(d) Comparison between Northern blot and Southern blot in terms of
purpose and methodology:
Purpose:
➢ Northern blot:- Northern blotting can be used to analyze a sample of RNA from a
particular tissue or cell type in order to measure the RNA expression of particular genes.
➢ Southern blot:- Southern blots can be used to analyze an organism’s total DNA, also
known as its genome, in order to identify a specific sequence of interest.
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Fig : Southern blot and Northern blot
Methodology:
Aspects Northern Blot Southern blot
Target molecule RNA DNA
Sample Preparation Extract total RNA or mRNA Extract genomic DNA and
From cells or tissues digest it with restriction
Separation technique Agarose Gel electrophoresis Agarose or polyacrylamide gel
electrophoresis
Denaturation Not required (RNA is single DNA is denatured into single
stranded) strand using alkaline solution
RNA is transferred to a Single stranded DNA is
Transfer nitrocellulose or nylon transferred to a nitrocellulose
membrane or nylon membrane
Probe Hybridization Labeled complementary DNA Labeled complementary DNA
or RNA binds to target RNA binds to target DNA
Detection method Chemiluminescence Chemiluminescence
colorimetry and X-ray film colorimetry and X-ray film
Blotting technique Capillary transfer Capillary transfer
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Gel Electrophoresis
2(a): The working principle of gel electrophoresis :
Gel electrophoresis is a process where an electric current is applied to DNA samples
creating fragments that can be used for comparison between DNA samples. It is used to
separate mixtures of molecules, typically proteins or nucleic acids (such as DNA, RNA),
based on their size, charge, and shape.
The working principle of gel electrophoresis can be summarized in the following key points:
Electric Field Application: An electric field is applied across a gel (either polyacrylamide or
agarose) that has been immersed in a buffer solution.
Molecular Migration: Charged molecules, such as proteins, RNA, or DNA, migrate toward
the oppositely charged electrode (DNA migrates toward the positive electrode because of its
negative charge).
Separation by Size: Molecules flow through the gel at varying speeds. Larger molecules are
slowed down whereas smaller molecules move more slowly through the gel.
The Gel Matrix: The gel functions as a filter, and the velocity of migration is determined by
the size of its pores. Greater resistance is offered by a denser gel (greater concentration).
Visualization: Following electrophoresis, the molecules are dye-stained and examined,
frequently with UV light for DNA.
Band Formation: Different bands are formed by the separated molecules, and their size can
be ascertained by comparing them to a molecular weight marker.
Fig : Agarose Gel Electrophoresis Fig: Polyacrylamide gel electrophoresis
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2(b): Difference between agarose and polyacrylamide gel electrophoresis :
Parameter Agarose gel electrophoresis Polyacrylamide gel
electrophoresis
Definition Agarose gel electrophoresis is a method Polyacrylamide gel
of gel electrophoresis used in electrophoresis (PAGE) is a
biochemistry, molecular biology, technique widely used in
genetics, and clinical chemistry to biochemistry, forensic chemistry,
separate a mixed population of genetics, molecular biology and
macromolecules such as DNA or biotechnology to separate
proteins in a matrix of agarose biological macromolecules,
usually proteins or nucleic acids
Composition Agarose, a natural polymer extracted Polyacrylamide gels are
from seaweed, forms the basis of synthetic and formed by the
agarose gels polymerization of acrylamide
and bis-acrylamide monomers
Properties suitable for the separation of large Suitable protein electrophoresis
molecules such as DNA fragments, and the separation of small DNA
ranging from 100 base pairs to several or RNA fragments.
megabases in size.
Mechanism of Size-based Separation Molecular Sieving
Separation
Applications Preferred method for analyzing DNA Widely used for the analysis of
fragments generated in PCR, restriction proteins and small nucleic acids.
digestion, or RNA samples from It is essential in techniques such
transcription experiments as SDS-PAGE for determining
protein molecular weight
Resolution Lower resolution PAGE has higher resolution
Gel preparation Agarose gels are poured horizontally PAGE gels are poured vertically
Casting Set as it cools Set by chemical reaction
methodology
Concentration 0.5-2% 6-15%
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2.(c) The roles of the buffer and loading dye in electrophoresis:
Gel electrophoresis is a method to sort DNA, RNA, or proteins by size.
• A gel electrophoresis buffer is an electrolyte solution used in gel electrophoresis to
provide a stable environment for the separation of nucleic acids. In electrophoresis,
buffers like TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are used most
commonly.
• A loading dye is a colored dye that is mixed with a DNA sample prior to running
electrophoresis.
The roles of buffer in electrophoresis:
Conducts electricity: Buffers provide the ions (e.g., Tris, acetate, and EDTA) required for
conducting the electric current across the gel.
Maintains pH: The buffer, by providing a reservoir of weak acid and base, also keeps the pH
within a narrow range. This is important because the structure and charge of a protein or nucleic
acid will change if subjected to significant pH changes, thus preventing proper separation.
Prevents gel damage: Protects the gel from overheating by evenly dissipating heat generated
during the run.
The roles of loading dye in electrophoresis:
Tracks migration: Includes visible dyes (e.g., bromophenol blue, xylene cyanol) that migrate
with the sample, allowing you to monitor the progress of electrophoresis in real-time.
Increases the density of the sample: Contains substances that assist the sample sink into the
wells by making it dense, such as sucrose or glycerol.
Makes the sample visible: The dye adds color to the sample, making it easier to see if it's
loaded into the well.
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2.(d) Gel electrophoresis can be used to analyze DNA:
A solution of DNA is colorless, and except for being viscous at high concentrations, is visually
indistinguishable from water. Therefore, techniques such as gel electrophoresis have been
developed to detect and analyze DNA.
Process:
Gel Preparation: Agarose is dissolved in a buffer solution, poured into a mold, and allowed
to solidify to create a gel for loading DNA samples.
DNA Loading: DNA samples are put onto agarose gel wells, frequently accompanied by a
loading buffer to aid in the DNA's sinking and movement tracking.
Electric Current Application: By applying an electric field, the DNA fragments move
through the gel matrix according to their size, with smaller pieces traveling more quickly than
bigger ones.
Visualization: A DNA-specific dye, like ethidium bromide or SYBR Green, which fluoresces
under UV light and makes the DNA bands visible, is used to stain the gel after electrophoresis.
Size Determination: Based on their migratory distance, the fragment sizes are estimated by
comparing the DNA bands to a known-size DNA ladder or marker.
Application: Gel electrophoresis is used in genetic research for PCR analysis and mutation
detection, forensics for DNA fingerprinting, RT-PCR gene expression investigations,
diagnostics for genetic disorders, and education for molecular biology instruction.
In conclusion, gel electrophoresis is an essential molecular biology technique used in forensic
investigations, diagnostics, and research to separate, examine, and study DNA fragments
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3.(a) Role of SDS in SDS-PAGE :
Sodium dodecyl sulfate (SDS) is an organic compound that is used as a detergent and protein
denaturant in biochemical research. It is a crucial component in SDS-PAGE ( Sodium Dodecyl
Sulphate - Polyacrylamide Gel Electrophoresis). It’s primary role is to denature & linearize
proteins, ensuring that they are separated solely based on their molecular weight.
Here are it’s key functions -
1. Denaturing Proteins : SDS disrupts non covalent bonds within proteins, such as hydrogen
bonds, hydrophobic interactions and ionic interactions. This linearizes the proteins into a rod
like shape that might affect migration through the gel.
2. Uniform Charges : SDS molecules bind to the hydrophobic regions of the denatured
proteins, coating them with a negative charge. This negative charge overwhelms the proteins
inherent charge, making the uniformly negatively charged.
3. Facilitating Electrophoresis Separation : It enables the proteins to migrate through the gel
matrix under the influence of an electric field.
4. Size based Separation : Once proteins are negatively charged, they will migrate through
the gel matrix towards the positive electrode. Their migration will be determined by their size,
with smaller proteins moving faster than larger ones.
By performing these functions, SDS-PAGE becomes a powerful tool in molecular biology and
biochemistry.
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3.(b) Steps Involved In Performing SDS-PAGE :
1. Sample Preparation -
i) Protein Extraction : Proteins are extracted from cells and tissues using appropriate buffers.
ii) Denaturation : Proteins are denatured using heat and a reducing agent to break the disulfide
bonds.
iii) SDS- Treatment : SDS added to the denatured proteins, coating them with a negative charge
and ensuring uniform migration based on size.
2. Gel Preparation -
i) Casting: A gel casting apparatus is used to assemble glass plates and spacers to create a mold.
ii) Gel Solution Preparation: A solution containing acrylamide, bis-acrylamide, SDS, and a
buffer is prepared. The percentage of acrylamide determines the gel's pore size and,
consequently, its resolving power.
iii) Gel Pouring: The gel solution is poured into the mold and overlaid with isopropanol or
water to prevent oxygen inhibition of polymerization.
iv) Comb Insertion: A comb is inserted into the gel solution to create wells for sample loading.
v) Polymerization: The gel polymerizes, forming a solid matrix.
3. Gel Electrophoresis :
i) Sample Loading: The protein samples are loaded into wells at the top of the gel.
ii) Electric Field Application: An electric field is applied across the gel, causing the negatively
charged proteins to migrate towards the anode.
iii) Protein Separation: Smaller proteins migrate faster through the gel pores than larger
proteins, resulting in separation based on molecular weight.
4. Staining & Visualization :
i) Gel Staining: After electrophoresis, the gel is stained with a dye like Coomassie Blue or
silver stain to visualize the protein bands.
ii) Destaining: Excess stain is removed by washing the gel with a destaining solution.
iii) Visualization: The stained gel is visualized, and the protein bands can be analyzed to
determine their molecular weight and relative abundance
It is used in various branches of biology such as molecular biology, genetics, forensics, and
biotechnology for protein separation
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3.(c) Visualization of Proteins after SDS-PAGE:
Proteins separated by SDS-PAGE are visualized using staining techniques that highlight them
in the gel. Two commonly used methods are:
1. Coomassie Brilliant Blue Staining: This is an easy-to-use technique where the gel is
stained with a Coomassie dye solution, which binds to proteins. Excess dye is removed
using a destaining solution of water or methanol-acetic acid, leaving blue protein bands
visible.
2. Silver Staining: A highly sensitive technique that can detect even small amounts of
protein. The gel is treated with a series of reagents including silver nitrate, making
proteins visible as dark bands. Silver staining provides greater sensitivity but is more
time-intensive than Coomassie staining.
3.(d) Advantages of SDS-PAGE:
▫ High Resolution: SDS-PAGE can separate proteins with high molecular weight
resolution, enabling detailed analysis.
▫ Versatility: It is compatible with downstream applications like Western blotting and
mass spectrometry.
▫ Quantitative and Qualitative Analysis: SDS-PAGE allows for both the identification
and quantification of protein samples.
▫ Broad Range of Applications: It is used in research, clinical diagnostics, and
biotechnology for studying protein purity, expression, and molecular weight
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Pulse-Field Gel Electrophoresis (PFGE)
4.(a) Differences Between PFGE and Traditional Gel Electrophoresis
− Ability to Separate Large DNA Molecules: PFGE is designed for separating very
large DNA fragments (up to several megabases), which is not possible with traditional
gel electrophoresis.
− Alternating Electric Field: Unlike conventional methods, PFGE applies an alternating
electric field to reorient the DNA, enabling its separation based on size.
− Enhanced Resolution: PFGE achieves higher resolution for large DNA fragments,
making it valuable in genomic studies, such as bacterial typing and chromosome
mapping.
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4(b): The Significance of Altering Electric Field in PFGE
The alternating electric field is important in pulsed field gel electrophoresis (PFGE) because it
causes DNA fragments to travel and reorient at various rates, which makes it possible to
separate them:
Reorientation: DNA pieces reorient to the new electric field direction due to the alternating
electric field. The size of the DNA fragment determines how long it takes for the DNA to
reorient.
Migration :The reorientation causes DNA pieces to travel at varying rates. Larger DNA
segments travel more slowly because they require longer to reorient.
Separation: DNA fragments can be separated because they travel at varying rates depending
on their size. Only until the pulse duration is sufficiently lengthy for all of the DNA
fragments to reorient can the separation be accomplished.
Resolution :DNA fragments up to roughly 10 megabases (Mb) in size can be resolved with
PFGE. The process of PFGE breaks down bacterial DNA into big pieces using restriction
enzymes, which are subsequently separated by an alternating electric field. Food-borne
infections have been tracked using PFGE, which is regarded as the gold standard in molecular
subtyping.
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4(c): Two Specific Applications of PFGE in Genomic Studies
1. DNA Fingerprinting in Epidemiology: -
The genetic profiles of bacterial, fungal, and other microbial diseases are frequently analysed
by PFGE. It is essential for identifying and monitoring particular organism strains, such
Salmonella, Escherichia coli, or Listeria monocytogenes, in epidemiological research and
outbreak investigations. Researchers are able to compare and distinguish strains thanks to the
distinct DNA banding patterns that are produced.
2. Chromosomal Mapping and Genome Analysis:
PFGE plays a critical role in constructing physical maps of chromosomes by determining the
sizes of large DNA fragments. This technique is essential for studying the organization of
genomes, particularly in organisms with large or complex genomes such as yeast, plants, or
mammals. It also aids in identifying structural variations like insertions, deletions, or
translocations.
Fig: Geonome analysis in PFGE Method
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4(d): Why PFGE Commonly Used for Separating Large DNA?
Large DNA molecules are separated using Pulsed-Field Gel Electrophoresis (PFGE) because
it can resolve fragments larger than 20–50 kilobases (kb), something that regular gel
electrophoresis is unable to do.
1.Alternating Electric Fields: PFGE forces big DNA molecules to reorient and flow through
the gel according to size by using an electric field that alternates in direction on a regular basis.
2.Size-Dependent Reorientation: Larger DNA fragments take longer to reorient compared to
smaller ones, enabling size-based separation of fragments up to several megabases.
3.High Resolution: The reorientation mechanism ensures clear separation of large DNA
molecules, avoiding the overlapping bands common in standard electrophoresis.
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