The complementary base-pairing of DNA double helix or DNA-RNA heteroduplex has been

extensively utilised by the scientists in various methods of recombinant DNA technology. The ability
of DNA & RNA strands to denature & re-anneal depending upon the surrounding conditions in vivo is
well established, & this ability when used in vitro has come to be known as hybridisation technique.

Southern blotting

THEORETICAL ASPECTS

Southern blotting is a method routinely used in molecular biology for detection of a specific DNA
sequence in DNA samples. Southern blotting combines transfer ofelectrophoresis-separated DNA
fragments to a filter membrane and subsequent fragment detection by probe hybridization. The
method is named after its inventor, the Britishbiologist E. M. Southern. Other blotting methods
(i.e., Western blot,Northern blot, Eastern blot, South-western blot) that employ similar principles,
but using RNA or protein, have later been named in reference to E. M. Southern's name.

Method

1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller
fragments.

2. The DNA fragments are then electrophoresed on an agarose gel to separate them by size.

3. If some of 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.

4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution
(typically containing sodiumhydroxide) to denature the double-stranded DNA. The
denaturation in an alkaline environment may improve binding of the negatively charged
DNA to a positively charged membrane, separating it into single DNA strands for
later hybridization to the probe, and destroys any residual RNA that may still be present in
the DNA.

5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below,
depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel
(either using suction, or by placing a stack of paper towels and a weight on top of the
membrane and gel), to ensure good and even contact between gel and membrane. If
transferring by suction 20X SSC buffer is used to ensure a seal and prevent drying of the gel.
Buffer transfer by capillary action from a region of highwater potential to a region of low
water potential (usually filter paper and paper tissues) is then used to move the DNA from
the gel on to the membrane; ion exchangeinteractions bind the DNA to the membrane due
to the negative charge of the DNA and positive charge of the membrane.

6. The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard
conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon
membrane) to permanently attach the transferred DNA to the membrane.
 7. The membrane is then exposed to a hybridization probe—a single DNA fragment with a
specific sequence whose presence in the target DNA is to be determined. 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. In some cases, the hybridization probe may
be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to
the sample DNA, most common hybridization methods use salmon or herring sperm DNA for
blocking of the membrane surface and target DNA, deionized formamide, and detergents
such as SDSto reduce non-specific binding of the probe.

8. After hybridization, excess probe is washed from the membrane (typically using SSC buffer),
and the pattern of hybridization is visualized on X-ray film byautoradiography in the case of a
radioactive or fluorescent probe, or by development of colour on the membrane if a
chromogenic detection method is used.

Result & Applications

Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this
fragment contains DNA sequence that is complementary to the probe.

The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of
the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the
target-probe hybrids, required for analysis by autoradiography or other detection methods.

Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine
the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single
DNA segment that has not been cut by the restriction enzyme will produce a single band on a
Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several
highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of
the hybridization conditions (for example, increasing the hybridization temperature or decreasing
salt concentration) may be used to increase specificity and decrease hybridization of the probe to
sequences that are less than 100% similar.

Southern blots are used in gene discovery and mapping, evolution and development studies
(comparing gene sequences across species), diagnostics (e.g. prenatal screening and diagnosis) and
forensics (DNA fingerprinting). The technique can locate particular fragments of DNA in a complex
mixture and can quickly identify a gene or gene segment within the genome. In regards to
genetically modified organisms, Southern blotting is used as a definitive test to ensure that a
particular section of DNA of known genetic sequence has been successfully incorporated into the
genome of the host organism.Southern blotting can be used in either direct or indirect detection
strategies. Due to its versatility and accuracy, this technique has become an indispensable molecular
genetics tool for medicine (testing for emerging infectious diseases, hereditary conditions, etc.),
research, and public health.
Northern blotting

THEORETICAL ASPECTS

The Northern blot is a technique used in molecular biology research to study gene expression by
detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe
cellular control over structure and function by determining the particular gene expression levels
during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting
involves the use of electrophoresis to separate RNA samples by size, and detection with a
hybridization probe complementary to part of or the entire target sequence.

The term 'northern blot' actually refers specifically to the capillary transfer of RNA from the
electrophoresis gel to the blotting membrane, however the entire process is commonly referred to
as northern blotting. The northern blot technique was developed in 1977 by James Alwine, David
Kemp, and George Stark . Northern blotting takes its name from its similarity to the first blotting
technique, the Southern blot, named for biologist Edwin Southern. The major difference is that RNA,
rather than DNA, is analyzed in the Northern blot.

Method

1. A general blotting procedurestarts with extraction of total RNA from a homogenized tissue
sample. The mRNA can then be isolated through the use of oligo (dT) cellulose
chromatography to maintain only those RNAs with a poly(A) tail.

2. RNA samples are then separated by gel electrophoresis.The RNA samples are most
commonly separated on agarose gels containing formaldehyde as a denaturing agent for the
RNA to limit secondary structure.

3. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples,
now separated by size, are transferred to a nylon membrane through a capillary or vacuum
blotting system.A nylon membrane with a positive charge is the most 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 preventing RNA degradation by high
temperatures.

4. Once the RNA has been transferred to the membrane it is immobilized through covalent
linkage to the membrane by UV light or heat.

5. Probes for northern blotting are composed of nucleic acids with a complementary sequence
to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a
minimum of 25 complementary bases to the target sequence. The probes need to be
labelled either with radioactive isotopes or with chemiluminescence in which alkaline
phosphatase or horseradish peroxidase breakdown chemiluminescent substrates producing
a detectable emission of light. The chemiluminescent labelling can occur in two ways: either
the probe is attached to the enzyme, or the probe is labelled with a ligand (e.g. biotin) for
which the antibody (e.g. avidin or streptavidin) is attached to the enzyme.
 After a probe has been labeled, it is hybridized to the RNA on the membrane. The
membrane is washed to ensure that the probe has bound specifically and to prevent
background signals.

X-ray film can detect both the radioactive and chemiluminescent signals.

Applications

Northern blotting allows one to observe a particular gene's expression pattern between tissues,
organs, developmental stages, environmental stress levels, pathogen infection, and over the course
of treatment. The technique has been used to show over-expression of oncogenes and down-
regulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissueas well as
the gene expression in the rejection of transplanted organs. If an up-regulated gene is observed by
an abundance of mRNA on the northern blot the sample can then be sequenced to determine if the
gene is known to researchers or if it is a novel finding. The expression patterns obtained under given
conditions can provide insight into the function of that gene. Since the RNA is first separated by size,
if only one probe type is used variance in the level of each band on the membrane can provide
insight into the size of the product, suggesting alternative splice products of the same gene or
repetitive sequence motifs. The variance in size of a gene product can also indicate deletions or
errors in transcript processing, by altering the probe target used along the known sequence it is
possible to determine which region of the RNA is missing.
Western blotting

THEORETICAL ASPECTS

The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their
subsequent detection on the surface of the membrane. The Western blot (alternatively, protein
immunoblot) is a widely used analytical technique used to detect specific proteins in the given
sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured
proteins usually by the length of the polypeptide. The proteins are then transferred to a membrane
(typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the
target protein.

The name Western blot was given to the technique because it is similar to Southern blotting which
was invented by and named after the inventor E. M. Southern. Western blotting is also called
immunoblotting because an antibody is used to specifically detect its antigen. The specificity of the
antibody-antigen interaction enables a target protein to be identified in the midst of a complex
protein mixture. Western blotting can produce qualitative and semiquantitative data about that
protein.

Method

1. Tissue/Protein sample preparation

Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first
broken down mechanically using a blender (for larger sample volumes), using a homogenizer
(smaller volumes), or by sonication. Assorted detergents, salts, and buffers may be employed to
encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often
added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done
at cold temperatures to avoid protein denaturing and degradation.

2. Electrophoretic separation of proteins

Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to characterize
individual proteins in a complex sample or to examine multiple proteins within a single sample.
Several forms of PAGE exist and can provide different types of information about the protein(s).For
example, denaturing PAGE is used to separate proteins by their length while native/non-denaturing
PAGE separates proteins by their 3-D structure.

3. Transfer of proteins to the membrane (blotting)

In order to make the proteins accessible to antibody detection, they are moved from within the gel
onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is
placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed
in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.
Another method for transferring the proteins is called electroblotting and uses an electric current to
pull proteins from the gel into the PVDF or nitrocellulose membrane. The protein move from within
the gel onto the membrane while maintaining the organization they had within the gel. As a result of
this "blotting" process, the proteins are exposed on a thin surface layer for detection Protein binding
is based upon hydrophobic interactions, as well as charged interactions between the membrane and
protein.

4. Blocking non-specific sites on membrane

The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after
the transfer of the proteins from the gel, it is important to block the remaining surface of the
membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A
variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been
used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the
assay by reducing background interference and improving the signal to noise ratio.

5. Probing with antibodies & detection

The final step is probing the protein (antigen) of interest with an antibody that specifically recognizes
it. With the direct detection method, the primary antibody that is used to detect an antigen on the
blot is labeled with an enzyme or fluorescent dye. This detection method is not widely used as most
researchers prefer the indirect detection method for a variety of reasons. In the indirect detection
method, a primary antibody is added first to bind to the antigen. This is followed by a labeled
secondary antibody that is directed against the primary antibody. Labels include radio-isotopic
labeling, biotin, fluorescent probes such as fluorescein or rhodamine, and enzyme conjugates such
as horseradish peroxidase or alkaline phosphatase. The indirect method offers many advantages
over the direct method: Secondary antibody can amplify signal; a variety of labeled secondary
antibodies are available; one secondary may be used with many primary antibodies; labeling does
not affect primary antibody immunoreactivity; changing secondary antibody allows change of
detection method. The detection of binding/interaction of the labeled antibody to the protein of
interest would vary according to the specific label that is attached to the antibody. In case of
enzymatic labels, chromogenic or chemi-luminescent substrates can be used.

Like other blotting procedures, Western blotting consists of a series of incubations with different
immunochemical reagents separated by wash steps. Washing steps are necessary to remove
unbound reagents and reduce background, thereby increasing the signal:noise ratio. Wash buffer
usually consist of a physiological buffer such as Tris buffered saline (TBS) or phosphate buffered
saline (PBS) with a etergent such as Tween 20 is to help remove nonspecifically bound material.

DEMONSTRATION OF WESTERN BLOTTING……………………….