principle and application of blotting techniques

1. Pharmaceutical biotechnology
Principle and application of blotting techniques.
By:- Drx Jayesh.M.Rajput
1. Northern blotting
Northern blotting or Northern hybridization is a widely used technique in molecular biology to
determine the molecular weight of mRNA and to measure relative amounts of mRNA present in
different samples and for identifying alternatively spliced transcripts and multigene family members.
Northern Blotting involves separation of RNA samples according to size by agarose gel
electrophoresis and detection with hybridization probe complementary to part of or the entire target
sequence. Northern Blot refers to capillary transfer of RNA from the electrophoresis gel to the
blotting membranes.
Principle:-
Northern blotting is a commonly used method to study gene expression by detection of RNA (or
isolated mRNA) in samples. Northern blot technique was developed by James Alwine and George
Starck and was named such by analogy to Southern blotting. In Northern Blotting the total RNA or
mRNA is isolated from an organism of interest, and then electrophoresed on denaturing agarose gel,
which separates the fragments on the basis of size. The next step is to transfer fragments from the
gel onto nitrocellulose filter or nylon membrane. This can be performed by the simple capillary
method. The transfer or a subsequent treatment results in immobilization of the RNA fragments, so
the membrane carries a semi permanent reproduction of the banding pattern of the gel. The RNA is
bound irreversibly to the membrane by baking at high temperature (80°C)or by UV crosslinking. For
the detection of a specific RNA sequence, a hybridization probe is used. A hybridization probe is a
short (100-500bp), single stranded nucleic acid either DNA or RNA probe that will bind to a
complementary piece of RNA. Hybridization probes are labeled with a marker (radioactive or non-
radioactive) so that they can be detected after hybridization. In non-radioactive detection the probe is
labeled with biotin or dioxigenin. The membrane is washed to remove non-specifically bound probe
and the hybridized probe is detected by treating the membrane with a conjugated enzyme, followed
by incubation with the chromogenic substrate solution. As a result a visible band can be seen on the
membrane where the probe is bound to the RNA sample.
Applications:-

2. 2. Southern blotting
The first of these techniques developed was the Southern blot, named after Dr. Edwin Southern who
developed it to identify specific DNA sequences. Southern blotting is a detection technique used to find
the target DNA sequences in the DNA sample in the field of molecular biology. The process starts from
electrophoresis of DNA molecules which are hybridized in a blotting membrane followed by a transfer
step where DNA from gel is transferred onto the blotting membrane.
Principle:-
Restriction endonucleases, which is an enzyme, is used to break the DNA into small fragments. These
fragments are then separated using electrophoresis. The fragments achieved is then classified according
to their size (kDa). Thus, DNA fragments are transferred to the blotting paper where it is incubated with
probes. Probes used in the Southern blotting can be highly selective. They can selectively bind with a
resolution of 1 in a million and the characteristics to bind to the intended target fragments
Applications:-
Southern blotting is used in a number of applications. The primary usage of Southern blotting is
to identify a specific DNA in a DNA sample. It is mostly used in the identification of viral
infection and certain bacterial infections. In rDNA technology, The Southern blotting technique
is used to isolate a particular DNA. It is also useful in the study of mutation and gene
rearrangement, this property is used to diagnose neonatal disease and genetic disease. Due to the
precision in DNA identification this technique is used in phylogenetic studies, paternity &
maternity analysis, forensic studies and personal identification.
Southern blotting can be applied in studying structure of a gene or to elucidate restriction
enzyme maps. Particularly, Southern blotting can be used in the identification of the methylated
sites present in case of some genes in particular. Applying restriction nucleases such as
MspI and HpaII, which can both identify and cleave among the identical sequence this can be
implemented. The discovery of RFLP s by southern blotting has helped in the mapping of several
genomes that were crucial to be mapped. In the arena of immunology the clonal rearrangements
of the immunoglobulins as well as the T Cell receptor genes that play an important role in
eliciting an immune response can be analysed by Southern blotting.

3. 3. Dot blotting
A dot blot (or slot blot) is a technique in molecular biology used to detect proteins. It represents
a simplification of the western blot method, with the exception that the proteins to be detected
are not first separated by electrophoresis. Instead, the sample is applied directly on a membrane
in a single spot, and the blotting procedure is performed.
The technique offers significant savings in time, as chromatography or gel electrophoresis, and
the complex blotting procedures for the gel are not required. However, it offers no information
on the size of the target protein.
Principle:-
Dot blot is a simple way to test for the presence of a protein of interest (POI) in a sample. The
technique is actually very similar to the Western blot, but dot blot, for reasons we’ll cover later,
is a faster, cheaper, and easier technique. As an aside, the dot blot can also be used for detection
of nucleic acids, but for the sake of simplicity, we’ll restrict this to a discussion of protein.
Applications:-
It is used in the detection of dna, rna, and proteins.
4. Colony hybridization
Colony hybridization is a method of selecting bacterial colonies with desired genes.[1]
This
method was discovered by Michael Grunstein and David S. Hogness. Colony hybridization
begins with culturing sparsely populated bacterial colonies on a nutrient agar plate. These
colonies are symmetrically replicated on a nitrocellulose filter by direct contact, after which the
cells on the filter membrane are lysed and their DNA is denatured, allowing it to bind to the
filter. These DNA clusters are then hybridized to a desired radioactively-labelled RNA or DNA
probe (chosen specifically beforehand) and screened by autoradiography. DNA clusters that
exhibit a desired gene are then matched up to the corresponding (living) bacterial colonies,
which can be isolated for further growth and experimentation.
Principle:-
It is a rapid method of isolating a colony containing a plasmid harboring a particular sequence or
a gene from a mixed population. The colonies to be screened are first replica-plated on to a
nitrocellulose filter disc that has been placed on the surface of an agar plate prior to inoculation.
Master plate is retained for reference set of colonies. The filter bearing the colonies is removed
and treated with alkali so that the bacterial colonies are lysed and the DNA they contain is
denatured. The filter is then treated with proteinase K to remove protein and leave denatured
DNA bound to the nitrocellulose. The DNA is fixed firmly by baking the filter at 80°C. A
labeled probe is hybridized to this DNA which is monitored by autoradiography. A colony
whose DNA print gives a positive auto radiographic result on X-ray film can then be picked from
the reference plate. Colony hybridization can be used to screen plasmid or cosmid based
libraries.

4. Applications:-
5. Plaque hybridization.
Plaque hybridization is a technique used in Molecular biology for the identification of recombinant
phages.[1]
The procedure can also be used for the detection of differentially represented repetitive DNA.
The technique (similar to colony hybridization) involves hybridizing isolated phage DNA to a label probe
for the gene of study. This is followed by autoradiography to detect the position of the label.[2]
The
plaque hybridization procedure has some advantages over colony hybridization due to the smaller and
well defined area of the filter to which the DNA binds
Principle:-
A screening method used in the identification of recombinant phages, first developed by
Benton and devis in 1977 involved in the identification of phage libraries can be lifted several
times less background signals.
Applications:-
Used to identify bacterial containing the gene of interest.
Thank you…