Name: Kemoy Francis 0803681 Course: Cell & Molecular Biology Lecturer: Miss D. Newby

Name: Kemoy Francis 0803681
Course: Cell & Molecular Biology

Lecturer: Miss D. Newby
Southern, Northern & Western Blotting and their uses in medicine and forensics
Blotting is the name given to the technique that is used in molecular biology for the transfer of
biological samples from a gel to a membrane and their succeeding recognition on the surface of
the membrane. The main blotting methods used in molecular biology were developed in the
1970s and are employed in the fields of evolution, gene mapping, medicine and forensics. These
techniques are Southern, Northern and Western Blotting and are used to detect DNA, RNA and
proteins respectively.
Southern blotting invented in 1975 by Edward Southern at Edinburgh University and is used to
detect specific DNA sequences from DNA sequences in a complex biological solution. This
technique involves the separation of DNA fragment, their transfer to a membrane and their
detection by probe hybridization.
This procedure begins with the breaking down of the DNA mixture into smaller fragment by the
use of restriction enzymes. The DNA fragments are then separated according to their respective
sizes by aragose gel electrophoresis with the smaller fragment migrating faster than the larger
ones from the negative to the positive end of the gel. Sodium Hydroxide is added to the gel to
separate the double-stranded DNA fragments into single strands. The single stranded DNA
molecules are then transferred from the gel onto a positively charged nylon or nitrocellulose
blotting membrane. Pressure is applied to the membrane and the DNA fragments are transferred
to the membrane. The membrane with the DNA attached is then incubated with a radioactive or
fluorescent dye coated probes which are made up of single stranded DNA molecules.
Hybridization will occur between the probes and complementary segments of DNA. When the
membrane is covered with an x-ray film, the position of the probes will be visible and will
indicate the position of the specific DNA fragment.
Southern Blotting is employed in the field of forensics where it can be used to detect an
individuals finger print among a series of other prints as wells as in paternity testing. It is also
employed in medicine where it can be used to sequence the genes in an individual. Gene
sequencing is important in diagnosing certain illness that are transmitted genetically, and will
enable individuals to know the diseases that their children will be disposed to for example; sickle
cell anaemia.
Northern Blotting is similar to southern blotting; however it is used for the detection of the
presence of mRNA molecules in a mixture of other RNA molecules (tRNA and rRNA). The
technique was developed in 1977 by Alwine et al at Stanford University.
In this procedure, mRNA is loaded onto the gel instead of DNA. The mRNA molecules are
separated into their respective sizes and are then transferred to a blotting membrane. The blotting
membrane with the mRNA molecules attached is then incubated in a buffer solution with a
probe. The probe is either is single-stranded DNA or RNA and will hybridize to form a doublestranded RNA-DNA molecule. Visualization of the molecules will be possible when an x-ray
film is applied to the membrane.
Northern blotting is used to detect for the presence of a transgenic gene in animals. A transgenic
animal is one that carries a foreign gene that has been deliberately inserted into its genome. This
enables animals incorporate foreign DNA into their cells so that they can synthesize new
proteins. For example transgenic chickens are now able to synthesize human proteins in the
"white" of their eggs. These animals can eventually prove to be valuable sources of proteins for
human therapy.
Western blotting is used to detect a specific protein from a mixture of proteins in a biological
solution while giving information on the size of that protein. This procedure was first introduced
by Towbin, et al. in 1979. The proteins are first extracted from the solution and are then
separated into their respective sizes by SDS-PAGE (sodium dodecyl sulphate polyacrilamide gel
electrophoresis) electrophoresis. The proteins are then transferred to a nylon or nitrocellulose
membrane similar to that used in Southern and Northern blotting. However enzyme labelled
antibodies are used to hybridize with the proteins instead of probes. These antibodies are specific
to the target proteins and will block any nonspecific binding of antibodies to the surface of the
membrane. A primary antibody will then be incubated with the membrane and if there are any
antibodies present which are directed against one or more of the blotted antigens,
those antibodies will bind to the protein (s) while the other antibodies will be washed away. In
order to detect the bounded proteins, an enzyme coated secondary antibody is allowed to react
with them. The location of the antibody is revealed by incubating it with a colourless substrate
that the attached enzyme converts to a coloured product that can be seen when an x-ray film is
applied. This will result in the visualization of the target proteins.
HIV testing employs Western Blotting to detect anti-HIV antibodies in a blood serum. Proteins
from HIV infected cells are separated and blotted onto a membrane. The serum to be tested is
separated by gel electrophoresis and is then transferred to a membrane with anti human
antibodies. Stained bands will be formed which will indicate the protein to which the patient
blood contains an antibody and will indicate the presence of HIV in the blood.
This technique is also used to detect Bovine spongiform encephalopathy (BSE) disease more
commonly known as mad cows disease.
These blotting techniques are very time consuming and lengthy hence, they are no longer widely
used to detect biological molecules. Other techniques are currently being developed which can
be used to detect for these molecules in a much shorter time.
References
http://www.biology.arizona.edu/human_bio/problem_sets/dna_forensics_1/01t.html
Retrieved November 5,2012
http://askabiologist.asu.edu/southern-blotting Retrieved November 5,2012
http://www.slideshare.net/amirashaalan/southern-northern-and-western-blotting Retrieved
November 5,2012
http://static.abdserotec.com/Lit-pdfs/Brochures1/westernblotbook.pdf Retrieved November
5,2012
http://en.wikipedia.org/wiki/Western_blot#Medical_diagnostic_applications Retrieved
November 5,2012
http://www.webmd.com/hiv-aids/human-immunodeficiency-virus-hiv-test Retrieved November
5,2012
http://prescottbiochem09.wikispaces.com/Southern+Blotting+-+Uses Retrieved
November 5,2012

Name: Kemoy Francis 0803681 Course: Cell & Molecular Biology Lecturer: Miss D. Newby

Uploaded by

Copyright:

Available Formats

Name: Kemoy Francis 0803681 Course: Cell & Molecular Biology Lecturer: Miss D. Newby

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Name: Kemoy Francis 0803681 Course: Cell & Molecular Biology Lecturer: Miss D. Newby

Uploaded by

Copyright:

Available Formats

Name: Kemoy Francis 0803681

Course: Cell & Molecular Biology

You might also like