Blotting is a powerful technique used in modern molecular biology. This technique is used to identify specific biomolecules present in a sample. There are several types of blotting techniques based on the target molecule that each of the technique is used to detect.
Dr. Edwin Southern developed one of the first blotting techniques called the Southern blot. This technique detects specific DNA sequences and therefore is also called a DNA blot.
The underlying principles of all blotting techniques are almost identical. In Southern blotting, DNA fragments are first separated using gel electrophoresis and then the separated molecules are transferred to a membrane surface. In the next step, hybridization analysis is carried out on the membrane using labeled probes complimentary to the target sequence to be identified, thus detecting the presence of DNA fragment of interest.
Bulky target molecules need to be broken down before electrophoretic separation so as to facilitate molecule movement through the gel. In Southern blotting, enzymes called restriction endonucleases are used to cut large DNA sequences in to smaller pieces.
There are four key steps in the Southern blotting technique:
In the first step, sample DNA is broken down or digested in to smaller pieces using a restriction enzyme. After digestion, the DNA fragments are separated using gel electrophoresis. Agarose gel is usually used for this purpose. Electrophoresis shows several bands that look like a smear due to the presence of several small restriction fragments in the gel. NaOH is then used to denature the DNA into single strands.
These bands are then transferred to the surface of a membrane with the help of an electrical gradient which is normally a sheet of paper called the blotting paper. The pattern of DNA fragments on the gel remains the same after transfer to the blotting paper.
Now, a probe made up of single stranded DNA fragments are introduced into the blot. The bases in the DNA probe will pair with complementary DNA sequences on the gel to form double-stranded DNA. The probe is usually labeled with a chemical or radioactive tag so as to enable tracking of the probe on the gel. Chemical substrates and X-ray films are used to locate the probe in case the tag is an enzyme. Radioactive tags can directly show up on x-ray films.
Southern blot finds several applications in the modern day molecular biology lab. Some of the key applications of Southern blot are listed below:
- identification of a single gene in a pool of DNA fragments
- gene mapping
- analysis of genetic patterns of DNA
- detection of specific DNA sequences in a genome
- study of gene deletions, duplications, and mutations that cause various diseases
- detection of genetic diseases and cancers such as monoclonal leukemia and sickle cell mutations
- detect the presence of a gene family in a genome
- DNA fingerprinting and forensic tests such as paternity testing and sex determination
Southern blot has been a widely used technique in molecular biology for over 30 years. It has tremendously helped researchers understand the fundamentals of molecular biology. However, in recent years, several new automated techniques with incredible sensitivity have emerged and some of them have partially replaced the laborious and complex Southern blot technique.
Techniques such as real-time PCR enable highly reliable and rapid detection of even a tiny target sequence, whereas Southern blot requires a large amount of target DNA. Also, newer techniques such as fluorescent in situ hybridization (FISH) allow highly sensitive identification of specific nucleotide sequences in a tissue sample with accurate localization. Both real time PCR and FISH provide a precise quantitation of the target as well, which cannot be fully achieved using Southern blot.