Southern Blotting

What is Southern blotting and what is it used for?

Southern blotting is a technique used in molecular biology to detect specific DNA sequences in a sample. It was the first application of blotting to detect specific biomolecules. Southern blotting was developed in the early 1970s and named for its inventor, Edward Southern. Subsequent hybridization-based methodologies were then given directional names to honor their similarity to the Southern technique. “Northern” blots detect specific RNA sequences, while “Western” blots detect specific proteins. This naming convention extends to the naming of hybrid blot applications, such as “Southwestern” blotting, where a protein blot is probed with a DNA probe to identify DNA-binding proteins.

Southern blotting steps

  1. Extracting DNA from a sample
  2. Fragmenting the DNA with restriction enzymes
  3. Separating the DNA fragments by size using gel electrophoresis
  4. Transferring the separated DNA fragments from the gel to a nylon membrane
  5. Probing for the DNA sequence of interest using a nucleotide probe with a sequence complementary to the sequence of interest

When to use Southern blotting

Southern blotting is commonly used in applications that study the presence or absence of a DNA sequence in a sample, such as DNA fingerprinting or mutational analysis. It provides information about the structure of genomic DNA, and, depending on the detection method used, can be used to quantify the relative number of copies of a DNA sequence in a sample. Therefore, Southern blotting is often used for genomic analysis, including in cancer studies where rearrangement, deletion, or amplification of specific genomic regions play a role in disease diagnosis, prognosis, and/or progression.

Analysis of Southern blots

An advantage of phosphor imaging of radiolabeled probes is the potential to acquire quantitative data. However, some researchers may prefer using alternatives to radiolabels since radioactive reagents introduce additional complications and costs associated with storage, use, and disposal.

Several methods of Southern blot detection that do not require radiolabels have been popularly adopted. A common non-radioactive detection technique uses nucleotide probes labeled with digoxigenin or biotin. The probes are then detected indirectly using a labeled anti-digoxigenin antibody or labeled streptavidin. Several types of labels can be used on the antibody or streptavidin, such as visible or NIR fluorophores, or an enzyme such as horseradish peroxidase (HRP) which generates a chemiluminescent signal when a substrate is added. Most recently, researchers have developed methods to incorporate NIR fluorophores into the probes so Southern blots can be imaged directly using a NIR fluorescence imaging system.

Southern blotting remains an important technique for studying genetics and genomic structure as they relate to disease.

Azure Sapphire FL Biomolecular Imager with lid open
The new Azure Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types.

What equipment do you need for Northern blotting?

Southern blots have historically been analyzed with radiolabeled probes detected using X-ray film or phosphor imaging on an instrument such as the new Azure Sapphire FL Biomolecular Imager. To read summaries of phosphor imaging of Southern blots using the Sapphire, check out the blog posts on the topics, found here and here.

The Sapphire FL is a great choice for imaging Southern blots. It is able to perform phosphor imaging of radiolabeled probes and can detect NIR fluorescence. This makes it compatible with newer fluorescent detection methods. If you’re also interested in imaging chemiluminescent signals when using digoxigenin or biotin-labeled probes detected with HRP-conjugated secondary antibodies or streptavidin, the Sapphire FL has the ability to be upgraded to include an optional Chemiluminescence Module. The Azure 500 and Azure 600 Imagers are also excellent options for Southern blot imaging with chemiluminescence or NIR fluorescence.

Frequently Asked Questions

A hybridization technique used to detect a specific DNA sequence in a sample

To detect DNA sequences and to study genome structure

Southern blots involve extracting DNA from a sample, digesting the DNA into smaller pieces, separating the DNA pieces by size through gel electrophoresis, transferring the DNA from the gel to a membrane, and probing the membrane with a tagged nucleotide probe to determine the presence of the sequence of interest.

Related Products

Laser-customizable imaging system that carries out phosphor imaging for use with radiolabeled probes

Featured Publication
Southern blot imaging using Azure Sapphire
WHV core DNA was released from the NCs of cytoplasmic lysate by SDS-proteinase K treatment and detected by Southern blot analysis

Southern blots were used to study the effects of the WHc mutations on WHV reverse transcription in this paper from The Pennsylvania State University College of Medicine.

Looking for a full list of applications?

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