SOUTHERN BLOTTING

Southern blotting is a primary technique used in many fields of science specifically in molecular
biology. It was first developed by E M Southern in 1975 it allows the detection of a specific
DNA sequence from a bulk. Southern Blot involves the relocation of the DNA fragments
separated from electrophoresis gel to a nitrocellulose membrane. Labeled probes are then used to
hybridize with specific target or DNA fragment. Southern blotting is a process of detecting a
specific restriction fragment aligned with a background of many other restriction fragments.

Principle:
Southern blotting unites agarose gel electrophoresis used for separating DNA on the basis of size
with the technique to transferring the size separated DNA to a solid support such as
nitrocellulose or a nylon filter membrane for hybridizing with a probe. The DNA is transferred to
the sheet through capillary action. The DNA molecules move upwards by capillary action of the
buffer and on coming in contact with the membrane the get immobilized.

Methodology:

The whole procedure includes several steps such as purification of the DNA that is to be
analyzed followed by its digestion with restriction enzyme, separation by gel electrophoresis,
transfer to membrane, preparation and labeling of probe and then detection of the hybridized
label. A brief detail of the steps is as follows:

 Restriction digestion of the DNA by endonucleases to cut high-molecular-weight DNA

strands into smaller fragments.
 DNA size separation by Gel Electrophoresis
 DNA fragments larger than 15 kb are treated with an acid (such as HCl) before blotting
that depurinates the DNA fragments converting them into shorter lengths.
 In case of alkaline transfer, the DNA gel is placed in an alkaline solution for denaturing
the double-stranded DNA. The denaturation in an alkaline environment can result in
efficient binding of the negatively charged thymine residues of DNA to positively
 charged amino groups of membrane, separating it into single DNA strands
for hybridization.
 A sheet of nitrocellulose membrane is then held on top of the gel. Pressure is applied
evenly to the gel to guarantee effective contact between gel and membrane. If transferring
by suction, buffer is used to ensure a seal and avoid drying of the gel. Buffer transfer
by capillary action, from a region of high potential to a region of low water potential is
then used to move the DNA from the gel onto the membrane.
 The membrane is then baked in a vacuum or regular oven at 80 °C or exposed
to ultraviolet radiation to permanently attach the transferred DNA fragments to the
membrane.
 The membrane is then exposed to a labeled probe for hybridization. The probe is detected
radioactivity or tagging the molecule with a fluorescent or chromogenic dye.
 After hybridization, the pattern of hybridization is visualized by autoradiography
(radioactive/fluorescent probe) or by development of color (chromogenic detection).
Applications:
 Southern Blotting can be used for gene discovery and mapping, evolution and
developmental studies, for diagnostics and in forensics.
 Southern blotting aims at identifying and cloning of a specified gene. Southern blotting of
genomic DNA is used to identify one or more restriction fragments that contain the gene
being sought and after cloning and tentative identification of the desired recombinant by
colony or plaque hybridization probing, Southern blotting of restricted clone DNA is
used to confirm the clone identification and possibly to locate a shorter restriction
fragment, containing the sequence of interest from within the cloned DNA. This
application is important because the gene sequence might be just a few kb in length but
initially contained in a genomic clone of several hundred kb. SB can also identify
methylated sites in particular genes and determine the molecular weight of a restriction
fragment and measure the relative amounts in different samples.
 It can recognize a particular region of DNA and analyzing the genetic patterns in an
individual and homologues of human genes in the genomes of other primates.
 It is also involved in restriction fragment length polymorphism (RFLP) mapping and the
pattern of restriction digestion fragmentation of DNA or a biological sample can also be
studied.

NORTHERN BLOTTING

Northern blotting is used to determine the gene expression by the detection of RNA from a

sample. This technique was developed by James Alwine, David Kemp, and George Stark in
1977. Northern blotting includes the use of electrophoresis for the separation of RNA samples on
the basis of size, and then detection with a hybridization probe that is complementary to specific
or the entire target sequence. The term 'northern blot' refers to the capillary transfer of RNA from
the gel to the blotting membrane.
Principle:

Northern blot utilizes denaturing gel to separate RNA according to the size. The RNA is then
transferred to a nylon membrane while maintaining the same distribution as in the gel. After
fixing the RNA to the membrane, labeled probe complementary to the gene of interest is then
added to hybridize to the immobilized RNA. The nonspecifically bound probes are then washed
away. The solid membrane with probe specifically bound to RNA of interest is then dried,
exposed and analyzed. Hybridization is the key to this process resulting in a double stranded
DNA-RNA hybrid molecule by forming a bond between a single stranded RNA target and a
single stranded DNA probe.

Procedure:

 A typical RNA molecule is extracted by homogenizing the tissue with a commercial

reagent trizol. The RNA obtained is further subjected to more purification by passing the
RNA solution through a column of oligo(dT) probe which will interacts with only mRNA
that have ployA tail. After extensive washing with the buffer, the mRNA is now eluted
and then used for further analysis.

  RNA samples are then separated by gel electrophoresis. The RNA samples should be
treated with denaturing agents such as formaldehyde and glyoxal and heat before being
subjected to the electrophoretic separation.

 The size separated RNA samples are transferred to a nylon membrane through a
capillary or vacuum blotting system.

 A nylon membrane with a positive polarity is highly effective for use in northern blotting
since the negatively charged nucleic acids have a high affinity for them. The transfer
buffer used for the blotting usually contains formamide because it lowers the annealing
temperature of the probe-RNA interaction, thus eliminating the need for high
temperatures, which could cause RNA degradation.

 The usual step after the completion of blotting is to ensure that the RNA molecules are
properly bound to the solid matrix upon which it is adsorbed. This is done principally by
 exposing the membrane to UV. Exposure to UV causes the RNA molecule to undergo
extensive cross linking.

 The membrane containing fixed RNA molecules are then hybridized with the labeled.
The excess and un-hybridized labeled probe is washed off.

 The hybridized membrane is then studied by a suitable detector based on the type of
probe used. For radioactive probes, the membrane is detected with an autoradiography
and chemical labeling requires an appropriate reagent or substrate.

Applications:

 The major advantage of northern blot is the simplicity and flexibility involved in its
procedure. It is also give information about the size of the RNA and the characterization
of its expression. Northern blotting helps in studying the cellular control over structure
 and function by determining the expression rates of a particular gene during multiple
stages such as differentiation and morphogenesis, as well as in abnormal or diseased
conditions.
 It successfully compares the relative abundance of a particular gene expressed in cells
subjected to different experimental and physiological stress conditions.
 It can signify the roles of certain protein in the development and suppression of cancerous
growth. Results obtained from northern blot experiments could provide information in the
functioning of certain gene.
 The technique is also helpful in the studying alternative splicing of different gene
product. It is also helps in the study of abnormal gene and genetic disorders.

Comparison of Southern and Northern Blotting:

Properties Southern Blot Northern Blot

Molecule Detected DNA (double stranded) RNA (single stranded)

Gel pre-treatment Denaturation and depurination Not required

Gel Agarose gel Formaldehye treated agarose gel

Hybridization DNA-DNA DNA-RNA