Southern blotting is the procedure for transfer of DNA from the gel to nitrocellulose filter which resembles blotting technique. It was developed by E.M. Southern in 1975 for analyzing the related genes in a DNA restriction fragment (by DNA: DNA hybridization). It is used in molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labeled probe hybridization. It is a procedure for identifying specific sequences of DNA, in which fragments separated on a gel are transferred directly to a second medium on which assay by hybridization may be carried out.
It is transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments. After immobilization, DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified.
In southern blotting process, DNA fragments are separated on the basis of size and charge during electrophoresis. The fragments are then transferred to nylon membrane and desired DNA is detected using specific DNA probe which is complementary to desired DNA. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.
It combines transfer of electrophoresis separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization, which is a key process. Hybridization is a process of forming double-stranded DNA molecule between a single-stranded DNA probe (Hybridization probe) and a single-stranded target patient DNA. Hybridization probe is short (100-500bp), single stranded DNA. These probes are labeled with a marker so that they can be detected after hybridization.
Important features of hybridization in blotting includes:
- The reactions are specific. The probes will only bind to targets with a complementary sequence.
- The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.
Restriction endonucleases, an enzyme, is used to break the DNA into small fragments. These fragments are then separated using electrophoresis and then classified according to their size. Thus, DNA fragments are transferred to the blotting paper where it is incubated with probes. Probes used in southern blotting can be highly selective. They can selectively bind with a resolution of 1 in a million and characteristically bind to the intended target fragments.
Southern Blotting is used for:
- Identification of specific DNA.
- Preparation of RFLP (Restriction Fragment Length Polymorphism) maps.
- Criminal identification and Forensic science.
- Detection and identification of trans-gene in transgenic individual.
- Diagnosis of infectious diseases, genetic diseases.
- Detection of mutation and gene rearrangement in DNA.
- DNA fingerprinting.
- Paternity/ maternity testing.
- Detection of any abnormalities in the structure of gene.
- Mapping of the restriction sites.
- Determination of molecular weight of a restriction fragment and to measure relative amounts in different samples.