What is Southern Blotting?
Southern blotting is a technique used in the laboratory to detect a specific DNA sequence in a blood or tissue sample. Basically, it is a technique that was developed by Edward Southern to analyze DNA molecules. During the procedure, a restriction enzyme is used to cut a sample of DNA into fragments that are separated using electrophoresis gel. Later, the DNA fragments are transferred out of the gel to the surface of a membrane. The membrane is usually exposed to a DNA probe that is labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the probe sequence is present in the sample.
The Southern Blotting results in the transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon sheet known as the membrane. The DNA banding pattern then present in the gel is reproduced on the membrane. At this point, the DNA becomes immobilized on the membrane and can be used as a substrate for hybridization analysis with labeled DNA or RNA probes that target specific individual restriction fragments in the blotted DNA. Southern blotting detects a specific restriction fragment against a background of many other restriction fragments. The restricted DNA might be a plasmid or bacteriophage clone.
Principle of Southern Blotting
The ability of nitrocellulose powder or sheets to bind DNA has been utilized in various types of nucleic acid hybridization studies since the 1950s and the 1960s. Previously, the immobilized DNA was unfractionated and simply consisted of total DNA usually bound to nitrocellulose powder or spotted onto a nitrocellulose sheet. Gel electrophoresis methods were introduced in the early 1970s. It enables the restriction of fragments of DNA to be separated on the basis of their size. This prompted the development of techniques for the transfer of separated fragments from the gel to nitrocellulose support. The initial procedure developed in 1975 by Southern involving the capillary transfer of DNA from the gel to a nitrocellulose sheet placed on top of it was simple and effective. Although the procedure has evolved over the years the original procedure differs only slightly from the routine method applicable in many molecular biology laboratories.
Steps of Southern Blotting
- DNA digestion: The process involves obtaining complete fragmentation of the DNA at the intended restriction enzyme sites in Southern blot analysis
- Gel electrophoresis: The fragmented DNA is typically electrophoresed on an agarose gel to separate the fragments according to their molecular weights
- Blotting: After electrophoresis, the DNA is transferred to a positively charged nylon membrane
- Probe labeling: Radioactivity, Fluorescent dye, or an enzyme are used to label a nucleic acid probe with a sequence homologous to the target sequence under study to generate a chemiluminescent signal when incubated with the appropriate substrate.
- Hybridization and washing: The labeled probe is incubated with the DNA fragments that are immobilized on the blot under conditions that promote the hybridization of complementary sequences
- Detection: The bound labeled probe is detected for the particular label used
Applications of Southern Blotting
The technique is used for the analysis of gene rearrangements for instance in immunology to identify the clonal rearrangements of T cell receptor genes. The technique can also be used to identify specific fragments of DNA within a mixture of many other fragments.
Southern blotting is useful in the diagnosis of Fragile X syndrome. In addition, restriction fragment length polymorphism and variable number tandem repeat analysis.