Download Blotting : Southern, Northern and Western techniques

Southern Blotting
Southern blotting
Southern blotting was invented by Edwin Southern
in 1975 , a biologist of Edinburgh University, UK.
Southern blotting is a technique in which the DNA molecules
are transferred from an agarose gel onto a membrane and
used to locate a specific gene within an entire genome.
Steps in blotting
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Restriction digestion
Agarose gel electrophoresis
Washing
Acid treatment
Alkali treatment
Washing
Blotting
Hybridization
Washing
Detection
1. Restriction digestion
Genomic DNA is restriction digested with
restriction endonucleases that cut high
molecular weight DNA strands into
smaller fragments.
2. Agarose gel electrophoresis
DNA fragments are electrophoresed on
an agarose gel to separate them by size.
3. Washing
Gel is first washed with sodium saline citrate buffer to remove the
broken or fragmented residues of agarose formed during banding
and accumulated contaminants.
4. Acid treatment
If DNA fragments are large in size (>15 kb), they require a longer time to
transfer from the gel to membrane.
Depurination with an acid (0.25M HCl) for 15 min takes the purines out,
breaking the DNA into smaller fragments.
5. Alkali treatment
Gel is placed in an alkali solution (0.25 M NaOH) to denature the double stranded DNA into single DNA strands, so that it improves the binding of the
negatively charged DNA to a positively charged membrane.
It is useful for later hybridization to the probe and also destroys any residual
RNA present in the DNA.
6. Washing
Gel is extensively washed with SSC buffer to remove the traces of NaOH.
7. Blotting
• DNA in the gel is placed on the filter paper
with wigs dipped in a reservoir containing
transfer buffer.
• Nitrocellulose or Nylon membrane can be
used for transfer.
• Nitrocellulose has a binding capacity of
100µg/cm, while nylon has a binding
capacity of about 500 µg/cm.
• Membrane is placed on the gel. A stack of
blotting papers soaked in transfer buffer is
placed above it and gently pressed using
glass plate or rod to remove the air that is
trapped between the gel and membrane.
• A stack of un-soaked blotting papers is then
placed above them and a glass plate with a
weight of 500 g is placed above it.
Transfer mechanism
1. Capillary method
Buffer moves from reservoir to blotting paper by capillary
action. During this process, the DNA from the gel is
transferred onto the membrane within 12-18 h and
immobilized there due to the ion exchange interactions
between negative charge of the DNA and positive charge
of the membrane.
2. Vacuum blot apparatus
A vacuum sucks the transfer buffer through the membrane
within 1 h.
3. Electro transfer system
An electrical force is used to transfer the DNA from the
gel onto the membrane.
Fixation methods
Membrane is taken out carefully and has to be fixed more strongly, as
the binding of the DNA to the membrane is weak.
1. UV cross linking method
Membrane is exposed to ultraviolet rays, so that a covalent bond is formed
between phosphate and thymine residues present in the DNA and the
positively charged amino groups on the surface of nylon membrane.
2. Vacuum oven method
Nitrocellulose or nylon membrane is baked at 80°C in a vacuum oven or
regular oven for 2 h. Use of vacuum oven is a preferrable due to the
flammable nature of nitrocellulose membrane.
8. Hybridization
A technique by which the nucleic acid immobilized on the
membrane is challenged with a known probe. It is used to
confirm the presence or absence of the specific DNA
sequence.
Probes
DNA hybridization probe is made of a single DNA
fragment with a specific DNA sequence.
DNA is labeled by incorporating radioactive 32P labeled
ATP or tagged with a fluorescent or bioluminescent or
chromogenic dye.
Labeled DNA contains sequences complementary to
DNA present on the membrane.
If the sequence of DNA probe is complementary, it
forms hydrogen bonds or converts into hybrid DNA.
This is known as base pairing or hybridization.
9. Washing
After hybridization, the membrane is washed using SSC buffer to remove the
unbounded probes, while bound probes remain attached
10. Detection
Detection limit is 0.1 pg of the DNA.
1. Autoradiography method - If the probe is radiolabeled 32P or fluorescent
labeled, the pattern of hybridization is visualized on X-ray film
2. Biotin streptavidin method - If the probe is labeled by a non-radioactive
chromogenic dye, the pattern of hybridization is visualized by development of
color on the membrane and measurement by colorimeter.
3. Bioluminescence method – If the probe is tagged with bioluminescent, the
pattern is visualized by measuring luminesence using luminometer.
Applications
• Determine the number of sequences (e.g., gene copies) in a
genome.
• Find out specific DNA sequence present in different animals. eg.
Presence of insulin gene in sea anemone.
• Detect the Restriction Fragment Length Polymorphism (RFLP).
Polymorphism refers to DNA sequence variation between
individuals of a species. If the sequence variation occurs at the
restriction sites, it could result in RFLP.
• Detect the Variable Number of Tandem Repeat Polymorphism
(VNTR). VNTR result due to unequal crossover. It is the molecular
basis of DNA fingerprinting.