Experiment No.

Aim: To study the following techniques through photographs.

a) Southern Blotting
b) Northern Blotting
c) Western Blotting
d) DNA Sequencing (Sanger’s Method)
e) Polymerase Chain Reaction (PCR)
f) DNA Fingerprinting
a) Southern Blotting:
Southern blotting is a method used to check for the presence of a DNA sequence in a DNA
sample. The method is named after its inventor, the British biologist Edwin Southern.

Principle

Southern blotting is based on the principle of separation of DNA fragments by gel

electrophoresis followed by the identification by labelled probe hybridization. The DNA
fragments are separated based on their size and charge during electrophoresis. The technique
involves the transfer of electrophoresed DNA from the electrophoresis gel to a nitrocellulose
membrane. The nitrocellulose filter is sandwiched between the gel and the stack of paper
towels which draws the transfer buffer from the gel through capillary action. The desired
DNA is detected using a labelled probe complementary to the desired DNA.

Procedure

1. We take a DNA sample and restriction endonucleases are used to cut high-molecular-
weight DNA strands into smaller fragments, which are then electrophoresed on an
agarose gel to separate them by size.
2. If the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be
treated with an acid, such as dilute HCl, which depurinates the DNA fragments,
breaking the DNA into smaller pieces, thus allowing more efficient transfer from the
gel to membrane.
3. The DNA gel is placed into an alkaline solution (NaOH) to denature the
doublestranded DNA fragments into single strands.
4. A sheet of nitrocellulose (or nylon) membrane is placed on top of (or below,
depending on the direction of the transfer) the gel.
5. Pressure is applied evenly to the gel, to ensure good and even contact between gel and
membrane.
6. The nitrocellulose membrane is removed from the blotting stack, and the membrane
with single stranded DNA bands attached on to it is baked in a vacuum or regular
oven at 80 °C for 2-3 hours or exposed to ultraviolet radiation to permanently attach
the transferred DNA onto the membrane.
7. The membrane is then exposed to a hybridization probe.The probe DNA is labelled so
that it can be detected, usually by incorporating radioactivity or tagging the molecule
with a fluorescent or chromogenic dye.
8. After hybridization, excess probe is washed from the membrane and the pattern of
hybridization is visualized on X-ray film by autoradiography in the case of a
radioactive or fluorescent probe, or by development of colour on the membrane if a
chromogenic detection method is used.

Fig1: Southern Blotting technique.

 Fig: Southern Blotting Technique

b) Northern Blotting:

The technique that is used in molecular biology research to study gene expression by
detection of RNA or isolated mRNA in a sample is called northern blotting (RNA
blotting). It is a classical method for analysis of the size and steady state level of a
specific RNA in a complex sample.

Principle:

The fundamental principle of northern blotting is to separate RNA based on their size
using gel electrophoresis and identified on a cellular membrane by means of hybridization
probe with a base sequence corresponding to all or a part of the chain of the target
RNA.The signals are then detected and visualized using x-films and other methods.

Procedure:

1. The blotting procedure starts with extraction of total RNA from a homogenized tissue
sample.
2. The mRNA is then isolated by using oligo (dT) cellulose chromatography to maintain
only those RNAs with a poly (A) tail.
3. RNA samples are then separated by gel electrophoresis.
 4. The separated RNA sequence is transferred to the nylon membrane.
5. RNA transferred to the nylon membrane is then fixed using UV radiation.
6. The fixed nylon membrane is then mixed with probes.
7. The blot membrane is washed to remove unwanted probe
8. Labeled probe is detected by chemiluminescence or autoradiography. The result will
be dark bands in x ray film.

Fig: Northern Blotting Technique

c) Western Blotting

The western blot (alternatively, immunoblot) is used to detect specific proteins in a given
sample of tissue homogenate or extract. The method originated from the laboratory of George
Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette.
Principle:
In Western blotting (WB), target proteins are transferred to a hydrophobic membrane after
SDS-PAGE and detected using specific antibodies. After SDS-PAGE, a membrane is placed
on the gel, to which the separated proteins in the gel are electrophoretically transferred. The
membrane with transferred proteins is then probed with a primary antibody (an antibody
specific for the target protein), washed, and reacted with a secondary antibody labeled with
an enzyme, such as horseradish peroxidase (HRP). The bound enzyme activity is used to
detect the target protein and visualized by a chemiluminescent or chromogenic method.
Procedure:

1. Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also
known as tissue preparation.

2. The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide

gel electrophoresis.

3. The proteins are separated on the basis of electric charge, isoelectric point, molecular
weight, or combination of these all.

4. The nitrocellulose membrane is placed on the gel. The protein bands thus obtained are
transferred onto a nitrocellulose or nylon membrane where they are “probed” with
antibodies specific to the protein to be detected.

5. The antigen-antibody complexes that form on the band containing the protein
recognized by the antibody can be visualized in a variety of ways.

6. If the protein of interest is bound by a radioactive antibody, its position on the blot
can be determined by exposing the membrane to a sheet of X-ray film, a procedure
called autoradiography.

Fig: Western Blotting Technique

 d) DNA Sequencing (Sanger’s Method)
DNA sequencing is the process of determining the exact order of nucleotides within
a DNA molecule. This method is used to determine the order of the four bases—
adenine (A), guanine (G), cytosine (CY), and thymine (T) in a strand of DNA.
Sanger sequencing method was developed by Frederick Sanger and his colleagues
in 1977. It is also called enzymatic method or chain termination method.
Procedure
The single stranded DNA is mixed with primer and split into four reaction mixtures. Each
reaction mixture contains DNA polymerase, four deoxyribonucleotide phosphates (dNTPs)
and a replication terminator (ddNTP). Each reaction proceeds until a replication terminating
nucleotide (ddNTP) is added. The mixtures are loaded into four separate lanes of gel and
the electrophoresis is used to separate the DNA fragments.
STEPS:
1. Fragmentation and amplification: Fragment the DNA and clone the fragments into
vectors.
2. Denaturation: Denature the double stranded DNA (by heat or NaOH) into single stranded
DNA fragments.
3. Attach the primer: A primer is a synthetic oligonucleotide, containing 17 to 24
nucleotides. The primer binds to the DNA molecule and provides a 3' OH group, which is
necessary to initiate DNA synthesis. The 3'-OH group allows for DNA chain elongation.
4. Add 4 dNTPs + 1 ddNTP
5. Four different reaction vials are taken, each with the four standard dNTPs (dATP, dGTP,
dCTP and dTTP) and DNA polymerases. Difference among the vials is because of different
type of ddNTP. Each vial will have 1 ddNTP per 100 dNTPs.
6. After the occurrence of DNA synthesis, each reaction vial will have a unique set of single-
stranded DNA molecules of varying lengths. However, all DNA molecules will have the
same primer sequence at its 5' end.
7. Find the nucleotide sequence using gel electrophoresis: In the gel, we have varying
sequences, lined up according to size. In order to identify various terminated chains of DNA
(the DNA strands) the ddNTPs would have to be radioactively or fluorescently labelled
beforehand. The DNA strands are then separated using gel electrophoresis; then, the gel is
read from top to bottom (3' to 5') to obtain the sequence.
 Fig: DNA Sequencing (Sanger’s method)

e) Polymerase chain reaction

Polymerase chain reaction (PCR) is a technique in molecular biology used to amplify
(multiply) a single copy or a few copies of a piece of DNA, generating thousands to
millions of copies of that particular DNA sequence. PCR is a revolutionary method
developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA
polymerase to synthesize new strand of DNA complementary to the offered template
strand of DNA.
Principle:

The target sequence of nucleic acid is denatured to single strands, primers specific for each
target strand sequence are added, and DNA polymerase catalyzes the addition of
deoxynucleotides to extend and produce new strands complementary to each of the target
sequence strands (cycle 1). In cycle 2, both double-stranded products of cycle 1 are denatured
and subsequently serve as targets for more primer annealing and extension by DNA
 polymerase. After 25 to 30 cycles, at least 10 7 copies of target DNA may be produced by
means of this thermal cycling.
Procedure:
The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and
nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with
cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling
that allow DNA to be synthesized.

The basic steps are:

1. Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands.
This provides single-stranded template for the next step.

2. Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary
sequences on the single-stranded template DNA.

3. Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers,

synthesizing new strands of DNA.

4. This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours,

depending on the length of the DNA region being copied. If the reaction is efficient (works
well), the target region can go from just one or a few copies to billions.
 Fig: PCR

f) DNA fingerprinting

A DNA fingerprint is a DNA pattern that has a unique sequence such that it can be
distinguished from the DNA patterns of other individuals. DNA fingerprinting is also called
DNA typing or DNA profile. DNA fingerprinting is mainly used for criminal investigations
and forensic science.
Principle:
The principle of DNA fingerprinting is based on DNA polymorphism. 99.9% of the DNA of
every individual is the same. Only 0.1% of the DNA of every individual is different i.e.
unique to him/her. In DNA fingerprinting we deal with this 0.1% of DNA. When we obtain
the DNA sample from the crime scene or any other situation, we analyse it by density
centrifugation. Polymorphism (also called variation at the genetic level) arises due to
mutations. We can count the number of repeats and observe that every chromosome has a
unique number of repeats. 
Moreover, in DNA fingerprinting we consider VNTRs (variable number of tandem
repeats); they belong to minisatellite DNA. As the name indicates VNTR have a variable
number of repetitive DNA on every chromosome that shows a high degree of polymorphism.

Procedure:

1. The first step of DNA fingerprinting was to extract DNA from a sample of human
material, usually blood.
2. Molecular ‘scissors’, called restriction enzymes?, were used to cut the DNA. This
resulted in thousands of pieces of DNA with a variety of different lengths.
3. These pieces of DNA were then separated according to size by a process called gel
electrophoresis?:
4. Once the DNA had been sorted, the pieces of DNA were transferred or ‘blotted’ out
of the fragile gel on to a robust piece of nylon membrane and then ‘unzipped’ to
produce single strands of DNA.
5. Next the nylon membrane was incubated with radioactive probes.
6. The minisatellites that the probes have attached to were then visualised by exposing
the nylon membrane to X-ray film.
Fig: DNA Fingerprinting