DETECTION OF GENES

BLOTTING
Blotting is a common laboratory procedure in which biological
molecules in a gel matrix are transferred onto nitrocellulose or nylon
membrane for further scientific analysis. It is a technique in which nucleic
acids or proteins are immobilized onto a solid support, generally nylon or
nitrocellulose membranes. The biological molecules transferred in this
process are DNA, RNA or proteins. The blotting procedure is named
differently depending on the type of the molecules being transferred. When
DNA fragments are transferred the procedure is called a Southern blot,
named after Edward Southern that first developed it. The Northern blotting
procedure, which transfers RNA molecules, was developed shortly thereafter
and humorous named Northern blotting. Western blotting involves the
transfer of proteins.
All blotting procedures begin with a standard process called gel
electrophoresis when DNA, RNA, or proteins are loaded on to an agarose or
acrylamide gel and separated on the gel through an electric field. Two types
of gels are commonly used: agarose gels and acrylamide gels. Transfer is
initiated when nitrocellulose or nylon membrane is laid on top of the gel and
biological molecules are transfer from the gel to the membrane.
Hybridization / blotting is a technique in which biological molecular (DNA,
RNA or protein) are immobilized onto a nylon or nitrocellulose membrane. A
probe (a piece of nucleic acid with identical and specific sequence to the
organism or gene of interest) can then hybridize (join) to the biological
molecules (DNA, RNA or protein) with identical sequence on the membrane.
The hybridization between the blotted DNA and probe is visualized by
labeling the probe in some way. Short fragments of DNA that have a
nucleotide sequence complementary to the molecule being analyzed are
normally used as probes in Southern and Northern blots. Antibodies that
react with the protein being analyzed are used as probes in a Western blot.

SOUTHERN BLOTTING
Southern blot 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.

How it is done:

DNA Purification
Restriction Digestion
Gel Electrophoresis
Denaturation
Blotting
Hybridization
Wash off unbound probe
Autoradiograph

Applications:

To identify specific DNA in a DNA sample

To isolate desired DNA for construction of rDNA

Identify mutations, deletions, and gene rearrangements

Used in prognosis of cancer and in prenatal diagnosis of genetic

diseases

Used in phylogenetic analysis

Diagnosis of HIV-1 and infectious disease

In DNA fingerprinting
o Paternity and Maternity Testing
o Criminal Identification and Forensics
o Personal Identification

NORTHERN BLOTTING
The northern blot technique is used 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. This technique
was developed in 1977 by James Alwine, David Kemp and George Stark at
Stanford University. Northern blotting takes its name from its similarity to the
first blotting technique, the Southern blot. The major difference is that RNA,
rather than DNA, is analyzed in the northern blot.
The key to this method is hybridization. Hybridization is the process of
forming a double-stranded DNA-RNA hybrid molecule between a single
stranded DNA probe and a single stranded target RNA.

How it is done: (The procedure for the analysis of RNA is very similar to
that of DNA, with different reagents and purification steps owing mostly to
the fact that RNA is more prone to degradation than DNA.)

Isolate RNA from source (a lot more difficult than getting DNA)

Separate by size (gel electrophoresis)

Transfer to nitrocellulose membrane

Expose membrane with bound RNA to a radioactive or fluorescent DNA

probe the probe will hybridize to any RNA on the membrane with
complementary sequence

Wash away excess probe and visualize (e.g. autoradiography for

radioactive probe, light of a specific wavelength for fluorescent probe,
etc.)

Applications:

Detecting a specific mRNA in a sample

Used in the screening of recombinants by detecting the mRNA
produced by the transgene
In disease diagnosis
In gene expression studies

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.
How it is done:

Isolate protein from source (often use whole cell extracts).

Solubilize protein with SDS (sodium dodecyl sulfate, a detergent) to

unfold and extend individual protein molecules; otherwise they can
stick to each other, become folded in odd conformations, etc., all of
which will affect their migration through the gel in the next step.

Separate by size (gel electrophoresis).

Transfer to nitrocellulose membrane.

Expose membrane with bound protein to a "primary antibody" (1')

the 1' antibody will bind the protein (antigen) it is specific for. Wash
away excess 1' antibody.

Expose membrane to a secondary (2') antibody that is specific for the

1' antibody. The 2' antibody is conjugated to a marker molecule
(nowadays usually a flourescent dye).

Wash away excess secondary antibody and visualize (e.g., light of a

specific wavelength for fluorescent antibody, etc.).

Applications:

Quantification of specific protein levels in a sample