|MoBio||Blotting Methods and Applications||Chapter 9|
Following gel electrophoresis, probes are often used to detect specific molecules from the mixture. However, probes cannot be applied directly to the gel. The problem can be solved by three types of blotting methods: Southern blotting, Northern blotting and Western blotting.
Southern blotting is a technique for detecting specific DNA fragments in a complex mixture. The technique was invented in mid-1970s by Edward Southern. It has been applied to detect Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandem Repeat (VNTR) Polymorphism. The latter is the basis of DNA fingerprinting.
Restriction Fragment Length Polymorphism (RFLP)
Polymorphism refers to the DNA sequence variation between individuals of a species. If the sequence variation occurs at the restriction sites, it could result in RFLP. The most well known example is the RFLP due to β globin gene mutation.
Variable Number of Tandem Repeat (VNTR) Polymorphism
Northern blotting is used for detecting RNA fragments, instead of DNA fragments. The technique is called "Northern" simply because it is similar to "Southern", not because it was invented by a person named "Northern".
In the Southern blotting, DNA fragments are denatured with alkaline solution. In the Northern blotting, RNA fragments are treated with formaldehyde to ensure linear conformation.
Western blotting is used to detect a particular protein in a mixture. The probe used is therefore not DNA or RNA, but antibodies. The technique is also called "immunoblotting".