Aim: To study following techniques through photographs

1. Southern blotting
2. Northern blotting
3. Western blotting
4. DNA Sequencing (Sanger’s Method)
5. PCR
6. DNA fingerprinting

Southern blotting: A Southern blot is a method used in molecular biology for

detection of a specific DNA sequence in DNA samples. Southern blotting combines
transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent
fragment detection by probe hybridization.

The method is named after the British biologist Edwin Southern who first published it in
1975.

Principle

Southern blot hybridization refers to the detection of specific DNA fragments that have
been separated by gel electrophoresis. After the electrophoresis the separated DNA
fragments are denaturated and transferred to a nitrocellulose (or nylon) membrane sheet
by blotting. In the blotting the gel is supported on a sponge in a bath of alkali solution, and
buffer is sucked through the gel and the sheet by paper towels stacked on top of the
nitrocellulose sheet. The buffer denaturates the DNA and transfers the single stranded
fragments from the gel to the surface of the sheet, where they adhere firmly. The
nitrocellulose sheet containing the bound single-stranded DNA fragments is peeled off the
gel and placed in a sealed plastic bag or a box together with buffer containing labelled DNA
probe specific for the target DNA sequence. The sheet is exposed to the probe under
conditions favouring hybridization. After the hybridization, the sheet is removed from the
bag, washed thoroughly to remove unhybridized probes and viewed using autoradiography
or ultraviolet light depending on the labels used (radioactive of fluorescent).

Steps for southern hybridization:

Step-1 DNA purification : isolate the DNA from the rest of the cellular material in the
nucleus. Incubate specimen with detergent to promote cell lysis. Cell lysis frees cellular
proteins and DNA.

Proteins are enzymatically degraded by incubation with proteinase.

Dna is purified from solution by alcohol precipitation .

Step-2 restriction digestion : cut the DNA into different sized fragments using restriction
endonuclease.

Step-3 gel electrophoresis : nucleic acids have a net negative charge and will move from
the left to the right. The larger molecules are held up while the smaller ones moves faster.
This results in a separation by size.

Gel can be stained Etbr. This causes DNA to fluoresce under UV light which permits
photography of the gel. This will be help us to know the exact migration of DNA standards
and the quality of the RE digestion of the test DNA.

Step-4 & 5 denaturation and blotting : DNA is then denatured with an alkaline solution
such as NaOH. This causes the double stranded to become single stranded.

The process of transferring the DNA from the gel to a membrane is called as blotting. The
blot is usually done on a sheet of nitrocellulose paper or nylon. DNA is then neutralized
with NaCl to prevent re- hybridization before adding the probe. Transferred by either
electro blotting or capillary blotting. The blot is made permanent either by

➢ Drying at 80C.
➢ Exposing to UV radiation.

Step-6 hybridization : the labelled probe is added to the membrane in buffer and
incubated for several hours to allow the probe molecules to find their targets.

Step-7& 8 wash and autoradiography : wash excess probe that bound non –specifically
to the membrane. Blot is incubated with wash buffers containing NaCl and detergent to
wash away excess probe and reduce background.

Detection : radioactive probes enable autoradiographic detection. If the probe is

radioactive the particles it emits will expose x-ray film. By pressing the filter and film, the
film will become exposed wherever probe is bound to the filter. After development , there
will be dark spots on the film wherever probe bound.

➢ Applications:
➢ To identify specific DNA in a DNA sample.
➢ To isolate desired DNA for construction of DNA.
➢ Identify mutation, deletions, and gene rearrangements.
➢ Used in prognosis of cancer and in prenatal diagnosis of genetic diseases.
➢ In RFLP
➢ Used in phylogenetic analysis
➢ In DNA finger printing: paternity and maternity testing, criminal identification and
forensic, personal identification
 Northern blotting: A northern blot is a laboratory method used to detect specific RNA
molecules among a mixture of RNA. Therefore it is also called the RNA 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. This method was named for its
similarity to the technique known as a Southern blot.

The technique was developed by Alwine and his colleagues in 1979.

Principle
In northern blot, the sample RNA is separated on the basis of size with the help of gel
electrophoresis. The separated RNA fragments are transferred to a support membrane
and then treated with a labeled DNA probe. If the sample contains the complementary
RNA sequence, the probe will bind to the membrane and it can be visualized using
various methods.

Procedure
The key steps in the northern blot technique are as follows:

➢ Process of northern blotting starts with extraction of RNA from tissue sample.

➢ mRNA is separated from this RNA by using oligo cellulose chromatography so

that only those mRNAs can be taken which have poly A tail.

➢ The RNA sample is then separated by size using agarose gel electrophoresis. RNA
molecules form streaks rather than bands on the gel as there are several small
fragments of RNA.
➢ The RNA molecules are then transferred to a special blotting paper usually made of
nitrocellulose. Membranes made up of nylon can also be used. The separation pattern of
the RNA molecules in the blotting paper remains the same as that in the gel.
➢ The blot is then exposed to a labeled, single-stranded DNA probe. The bases in this
probe will pair with their complementary RNA sequences in the blot producing a double-
stranded DNA-RNA molecule. Although the probe cannot be seen at this stage, since it
is labeled with an enzyme or radioactive tag, it can be seen after appropriate treatment
in the next step.
 ➢ Next, the probe is exposed to a colorless substrate which gets converted by the enzyme
to a colored product and is visible on an X-ray film. In case the probe is radioactive, it
can directly be seen on the X-ray film.

Applications:

• Gene expression studies – to observe overexpression of cancer-causing genes

and gene expression in case of transplant rejection
• In diagnosis of several diseases, e.g., Crohn’s disease
• For detection of viral microRNAs that play key roles in viral infection
• To screen recombinants - by detecting the mRNA formed by the transgene

Western blotting : Western blotting is a widely used technique for the detection and
analysis of proteins based on their ability to bind to specific antibodies.. It was first
described by Towbin, et.al in 1979 and has since become one of the most commonly used
methods in life science research. The specificity of the antibody- antigen interaction
enables to a target protein to be identified in the midst of a complex protein mixture.

Principle:

Western blotting is the transfer of proteins from the SDS- PAGE gel to a solid supporting
membrane. There are two types of blotting apparatus used to transfer proteins to solid
supports; these facilitate either wet transfer (tank blotting) or semidry transfer. Both of
them give good result.

Electrophoresis is used to separate complex mixtures of proteins denaturing

discontinuous one dimensional gel electrophoresis separates proteins only based on
molecular size as they move through a SDS- polyacrylamide gel(SDS PAGE) toward
the anode with the smaller protein migrating faster and bigger proteins running slower.
The SDS-PAGE is a separating gel topped by stacking gel and secured in an electrophoresis
apparatus. Sample proteins are solublized by boiling in the presence of SDS and equal
amount of the protein in solution are loaded into a gel lane, and the individual proteins
separated electrophoretically. 2-mercaptoethanol and dithiothreitol are added to reduce
disulfide bonds.

A protein sample is subjected to polyacrylamide gel electrophoresis. After this the gel is
placed over a sheet of nitrocellulose and the protein in the gel is electrophoretically
transfered to the nitrocellulose. The nitrocellulose is then soaked in blocking buffer (3%
skimmed milk solution) to "block" the non-specific binding of proteins. The nitrocellulose is
then incubated with the specific antibody for the protein of interest. The nitrocellulose is
then incubated with a second antibody, which is specific for the first antibody. For example,
if the first antibody was raised in mouse, the second antibody might be termed "goat anti-
mouse immunoglobulin". What this means is that mouse immunoglobulin were used to
elicit an antibody response in goats. The second antibody will typically have a covalently
attached enzyme which, when provided with a chromogenic substrate, will cause a color
reaction. Thus the molecular weight and amount of the desired protein can be
characterized from a complex mixture (e.g. crude cell extract) of other proteins by western
blotting.

Procedure:

Step 1: Sample Prepration

First step in the sample preparation is isolating the protein from a sample. Usually
proteins are purified from cells. Next the protein concentration is determined. Sample
buffer commonly Leammli buffer, which contains Sodium dodecyl Sulpate (SDS) and beta-
mercaptoethanol (BME) is added to the protein suspension. The sample buffer is the
centrifuge and heated to near- boiling, which denatures the protein and allows the SDS to
bind to the protein. SDS carries negative charge. Glycerol to make samples sink into wells
and the Tris base provides appropriate pH. The blue dye to visualize samples as gel is run.

Step 2: SDS-PAGE

The first step in SDS-PAGE is placing the sample, containing the protein- SDS compound, in
a well on top of the gel. A molecular weight marker is usually loaded in one of the well,
which determines the molecular weight of other proteins on the gel. And also the samples
are added in the remaining wells. The protein size is measured in kilodaltons (kDa).
Once the marker and samples are loaded, a current is run across the gel. With a negative
pole on the well of the gel, a positive pole on the opposite end of the gel. Because the
protein is bounded to negatively charged SDS, it is pulled down through the gel to the
positive pole. The larger the protein, the slower it moves.

Step 3 : Membrane Transfer (Wet Transfer)

After the gel is run, it is placed against a membrane , and current is passed across the gel to
the membrane, transferring the proteins onto the membrane. The transferring method is
also known as semi- dry method. The membrane is usually made of PVDF or nitrocellulose.

Step 4: Immunoblotting
The first step in immunoblotting is to wash the membrane and block it with non-specific
protein. The non- specific protein binds to the surface of the membrane where protein is
not already present. This will prevent antibody from binding to the membrane and giving a
non- specific signal.
Next, the primary antibody is added to the solution in which the membrane is floating.
Remember that the primary antibody recognizes a specific amino-acid sequence of a
particularProtein.
After a wash is conducted to remove unbound primary antibody, secondary antibody is
added. Secondary antibody recognizes the primary antibody, and usually is conjugated
with an enzyme, such as HRP (Horse Radish Peroxidase)
.Lastly, another wash is performed to remove unbound secondary antibody. Non-specific
binding of both the primary and secondary antibodies can occur, but thorough washing
usually minimizes this problem. The amount of time the primary and secondary antibodies
are applied, directly affects the specificity and strength of binding.

Step 5: Detection

The detection method used is dependent upon the enzyme to which the secondary
antibody is conjugated. The most common enzyme used in Western Blotting is HRP, and
the substrate used for detection is known as chemiluminescent substrate.
Once the substrate has been added, the light being emitted can be detected with film or a
photo imager. The membrane is usually stained after the detection step, so that the protein
present can be visualized, and compared to the film.

Applications:

Highly specific method to detect protein even in very low quantity.

used in clinical diagnosis(Lyme disease, Hepatitis, HIV-AIDS).

Quantifying a gene product (gene expression studies).

Polymerase chain reaction (PCR) : Polymerase chain reaction is an in vitro method

of nucleic acid synthesis, in which a specific DNA-fragment (or fragments) is amplified.
This was developed in 1983 by Kary Mullis.

Principle : PCR involves the initial denaturation of the target DNA, followed by the
annealing of target-specific oligonucleotide primers to each strand of DNA, and finally the
extension of the primers by polymerase enzyme to produce a copy of the selected region.
By repeating this cycle of denaturation, primer annealing and extension, there is an
exponential amplification of the target.
The thermal cycling is achieved by changing the reaction temperature with an automated
thermal cycler ,a machine that can be programmed to run through heating and cooling
cycles automatically. The PCR reaction mixture contains, DNA polymerase, deoxynucleotide
triphosphates (dNTP = dATP, dTTP, dCTP, dGTP), primers, PCR-buffer and template-DNA.
Primer is a complementary short nucleic acid strand that serves as a starting point for the
DNA amplification. The selection of PCR primers determines which part of the rRNA gene
will be amplified, thus determining the specificity of the detection.

Procedure:

Typically, PCR consists of a series of 20–40 repeated temperature changes, called cycles,
with each cycle commonly consisting of two or three discrete temperature steps. The
individual steps common to most PCR methods are as follows:

Denaturation : This step is the first regular cycling event and consists of heating the
reaction chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA melting,
or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds
between complementary bases, yielding two single-stranded DNA molecules.

Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F)
for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA
templates. Two different primers are typically included in the reaction mixture: one for
each of the two single-stranded complements containing the target region. The primers are
single-stranded sequences themselves, but are much shorter than the length of the target
region, complementing only very short sequences at the 3' end of each strand.

It is critical to determine a proper temperature for the annealing step because efficiency
and specificity are strongly affected by the annealing temperature. This temperature must
be low enough to allow for hybridization of the primer to the strand, but high enough for
the hybridization to be specific, i.e., the primer should bind only to a perfectly
complementary part of the strand, and nowhere else. If the temperature is too low, the
primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical
annealing temperature is about 3–5 °C below the Tm of the primers used. Stable hydrogen
bonds between complementary bases are formed only when the primer sequence very
closely matches the template sequence. During this step, the polymerase binds to the
primer-template hybrid and begins DNA formation.

Extension/elongation: The temperature at this step depends on the DNA polymerase

used; the optimum activity temperature for Taq polymerase is approximately 75–80 °C
(167–176 °F, though a temperature of 72 °C (162 °F) is commonly used with this enzyme.
In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA
template strand by adding free dNTPs from the reaction mixture that are complementary
to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs
with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand. The precise
time required for elongation depends both on the DNA polymerase used and on the length
of the DNA target region to amplify.

The processes of denaturation, annealing and elongation constitute a single cycle. Multiple
cycles are required to amplify the DNA target to millions of copies.

Applications:

• PCR allows isolation of DNA fragments from genomic DNA by selective amplification
of a specific region of DNA.
• The task of DNA sequencing can also be assisted by PCR.
• generating hybridization probes for Southern or northern hybridization and DNA
cloning.
• Bacterial colonies (such as E. coli) can be rapidly screened by PCR for correct
DNA vector constructs.
• used for genetic fingerprinting; a forensic technique used to identify a person or
organism by comparing experimental DNAs through different PCR-based methods.
• PCR permits early diagnosis of malignant diseases such
as leukemia and lymphomas.
• PCR also permits identification of non-cultivatable or slow-growing microorganisms
such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays
and animal models.

DNA Fingerprinting : DNA-fingerprinting (also called DNA typing or DNA

profiling). It is a technique of determining nucleotide sequences of certain areas of DNA
which are unique to each individual. Each person has a unique DNA fingerprint.

Alec Jeffreys (1984) invented the DNA fingerprinting technique at Leicester University,
United Kingdom.

Principle : Human genome possesses numerous small noncoding but inheritable

sequences of bases which are repeated many times. These sequences occur near telomere,
centromeres, Y chromosome and hetero-chromatic area. The area with same sequence of
bases repeated several times is called repetitive DNA.
The lengths of the repeats range from 9-40bp, and the number of repeats in the
minisatellites range from about 10-30. A minisatellite DNA sequence at a specific
chromosome location can have different lengths in different individuals. This variability is
due to either a gain or a loss of tandem repeats , probably during DNA replication. These
changes do not have any biological effect because minisatellite DNA does not encode any
proteins. Unrelated individuals generally have minisatellite that differ in length, but
children inherit one set of minisatellite DNA sequences from each parent. DNA
polymorphism is the basis of genetic mapping of human genome as well as DNA finger
printing.

Steps for DNA Fingerprinting:

1. The DNA is extracted from the nuclei of white blood cells or of spermatozoa or of the hair
follicle cells that cling to the roots of hairs that have fallen, or been pulled out.

2. The DNA molecules are first broken with the help of enzyme restriction endonuclease
(called chemical knife) that cuts them into fragments. The fragments of DNA also contain
the VNTRs.

3. Fragments are separated on an agarose gel and transferred by blotting them onto a
nylon membrane.

4. The membrane is hybridized sequentially with four or five separate labelled

minisatellite DNA probes, each of which recognizes a distinct DNA sequence. Before next
probe is used , the first probe is completely removed(stripped) from the membrane.

5. After each hybridization reaction ,the bands in which the probe has bound to digested
DNA samples are visualized by autoradiography(An X-ray film is exposed to the nylon
membrane to mark the places where the radioactive DNA probes have bound to the DNA
fragments. These places are marked as dark bands when X-ray film is developed. This is
known as autoradiography.)

6. The dark bands on X-ray film represent the DNA fingerprints (= DNA profiles).

Because of the extensive variability in human minisatellite DNA sequences, the chance of
finding two individuals in the population with the same DNA fingerfrint is about 1 in 103
to 1 in 108 Therefore , individuals’ DNA banding patterns based on minisatellite DNA
sequences are almost as unique as their fingerprints.

Applications of DNA Fingerprinting:

(i) Individuality: Like skin finger printing (der- matoglyphics), DNA finger printing can
help to distinguish one human being from another with exception of monozygotic
twins.
(ii) Paternity/Maternity Disputes: DNA finger print-ing can identify the real genetic
mother, father and the offspring.
(iii) Human Lineage: DNA from various probables is being studied to find out human
lineage.
(iv) Hereditary Diseases: The technique is being used to identify genes connected with
hereditary diseases.
(v) Forensics: DNA finger printing is very useful in the detection of crime and legal
pursuits. DNA fingerprinting has proved that Dhanu, the human bomb, was the real
murderer of Shri Rajiv Gandhi, the former Prime Minister of India
(vi) Sociology: It can identify racial groups, their origin, historical migration and
invasions. Genography is the study of migratory history of human species.

DNA Sequencing: DNA sequencing is the process of determining the sequence of

nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA.

Sanger sequencing or the chain termination method was developed by the British
biochemist Fred Sanger and his colleagues in 1977.

Principle: The Sanger method( also referred to as dideoxynucleotide sequencing) is

based on the use of dideoxynucleotide (ddNTP) in addition to the normal nucleotides
(dNTP) found in DNA. Dideoxynucleotide are essentially the same as nucleotides except
they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). These
modified nucleotides, when integrated into a DNA sequence, prevent the addition of further
nucleotides thus stop the elongation of the DNA chain. This occurs because a
phosphodiester bond cannot form between the dideoxynucleotide and the next incoming
nucleotide, and thus the DNA chain is terminated.

Procedure of the Sanger methods:

Denaturation: The DNA sample to be sequenced is combined in a tube with primer, DNA
polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled,
chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts
than the ordinary nucleotides.

Primer attachment and extension of bases: The mixture is first heated to denature the
template DNA (separate the strands), then cooled so that the primer can bind to the single-
stranded template. Once the primer has bound, the temperature is raised again, allowing
DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will
continue adding nucleotides to the chain until it happens to add a dideoxynucleotide
instead of a normal one. At that point, no further nucleotides can be added, so the strand
will end with the dideoxy nucleotide. The tube will contain fragments of different lengths,
ending at each of the nucleotide positions in the original DNA. The ends of the fragments
will be labeled with dyes that indicate their final nucleotide.

Polyacrylamide gel electrophoresis: After the reaction is done, the fragments are run
through a long, thin tube containing a gel matrix in a process called capillary gel
electrophoresis. Short fragments move quickly through the pores of the gel, while long
fragments move more slowly. As each fragment crosses the “finish line” at the end of the
tube, it’s illuminated by a laser, allowing the attached dye to be detected.

Thus, from the colors of dyes registered one after another on the detector, the sequence of
the original piece of DNA can be built up one nucleotide at a time. The data recorded by the
detector consist of a series of peaks in fluorescence intensity, as shown in
the chromatogram above. The DNA sequence is read from the peaks in the chromatogram.

Applications: Sanger DNA sequencing is widely used for research purposes like:

• Targeting smaller genomic regions in a larger number of samples

• Sequencing of variable regions
• Validating results from next-generation sequencing (NGS) studies
• Verifying plasmid sequences, inserts, mutations
• HLA typing
• Genotyping of microsatellite markers
• Identifying single disease-causing genetic variants
Diagrammatic presentation of Southern blotting procedure
Diagrammatic presentation of Northern blotting procedure

Western blotting experimental stages

Transfer of protein bands on PVDF and detection of bands by probe
Polymerase chain reaction components and steps of procedure
Picture showing variation in number and location of Variable Number of Tandem Repeats (VNTR) in
2 different individuals

DNA fingerprinting procedure using Restriction enzymes

 https://www.yourgenome.org/facts/what-is-a-dna-fingerprint

Structural difference between dideoxyribonucleotide and deoxyribonucleotide

The Sanger (chain-termination) method for DNA sequencing.