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
1970’s 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

Northern blotting is used to detect for the presence of a transgenic gene in animals. The probe is either is single-stranded DNA or RNA and will hybridize to form a doublestranded RNA-DNA molecule. and will enable individuals to know the diseases that their children will be disposed to for example. Gene sequencing is important in diagnosing certain illness that are transmitted genetically. Hybridization will occur between the probes and complementary segments of DNA. The technique was developed in 1977 by Alwine et al at Stanford University. mRNA is loaded onto the gel instead of DNA. Southern Blotting is employed in the field of forensics where it can be used to detect an individual’s finger print among a series of other prints as wells as in paternity testing. A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. Visualization of the molecules will be possible when an x-ray film is applied to the membrane. sickle cell the membrane. The blotting membrane with the mRNA molecules attached is then incubated in a buffer solution with a probe. The mRNA molecules are separated into their respective sizes and are then transferred to a blotting membrane. In this procedure. Northern Blotting is similar to southern blotting. When the membrane is covered with an x-ray film. It is also employed in medicine where it can be used to sequence the genes in an individual. This . 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. the position of the probes will be visible and will indicate the position of the specific DNA fragment. however it is used for the detection of the presence of mRNA molecules in a mixture of other RNA molecules (tRNA and rRNA).

However enzyme labelled antibodies are used to hybridize with the proteins instead of probes. in 1979. et al. 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. an enzyme coated secondary antibody is allowed to react with them. The proteins are then transferred to a nylon or nitrocellulose membrane similar to that used in Southern and Northern blotting. 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.enables animals incorporate foreign DNA into their cells so that they can synthesize new proteins. This procedure was first introduced by Towbin. In order to detect the bounded proteins. 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. 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. For example transgenic chickens are now able to synthesize human proteins in the "white" of their eggs. those antibodies will bind to the protein (s) while the other antibodies will be washed away. These animals can eventually prove to be valuable sources of proteins for human therapy. These antibodies are specific to the target proteins and will block any nonspecific binding of antibodies to the surface of the membrane. HIV testing employs Western Blotting to detect anti-HIV antibodies in a blood serum. The serum to be tested is separated by gel electrophoresis and is then transferred to a membrane with anti human . This will result in the visualization of the target proteins. Proteins from HIV infected cells are separated and blotted onto a membrane.

html Retrieved November 5.2012 .pdf Retrieved November 5. 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. References http://www.wikispaces.biology.wikipedia.2012 Retrieved November 5.2012 http://static. This technique is also used to detect Bovine spongiform encephalopathy (BSE) disease more commonly known as mad cow’s disease. they are no longer widely used to detect biological Retrieved November Retrieved November 5.2012 Other techniques are currently being developed which can be used to detect for these molecules in a much shorter Retrieved November 5.2012 http://www.2012 Retrieved November 5.slideshare.abdserotec. These blotting techniques are very time consuming and lengthy hence.arizona.