Southern Blot: Principles, Example Workflow, & Applications

One of the 'directional blots', in simple terms, Southern blot analysis is a laboratory method used to study DNA to determine the identity, size, and abundance of specific DNA sequences. Southern blotting was designed to locate a particular DNA sequence within a complex mixture. For example, it could be used to locate a specific gene within an entire genome. It is eponymously named after its inventor Edwin Southern, and the other similar methodologies were named after compass directions as a kind of scientific humor playing on his name. Blotting techniques are commonly used to identify and separate a target molecule from a complex mixture of molecules. While they all follow a similar type of workflow, each blotting technique differs in the type of molecule it can identify.

Technique	Western Blot	Northern Blot	Southern Blot
Target Molecule	Protein	RNA	DNA
Sample Prep	Protein extraction	RNA isolation	DNA extraction
Separation	Gel Electrophoresis/SDS-PAGE	Gel Electrophoresis	Gel Electrophoresis
Membrane Material	Nitrocellulose/PVDF	Nylon	Nylon
Probe	Uses a single primary antibody or a combination of primary and secondary antibodies conjugated to either a fluorophore or enzyme	RNA, DNA or oligodeoxynucleotide	Nucleic acid probe
Detection	Film, CCD camera, LED or infrared imaging system	X-Ray film, chemiluminescence	X-Ray film, chemiluminescence

Northern and Southern blotting techniques are both used to identify nucleic acid sequences and so are the most similar. However, there are several crucial differences between them: Northern blotting is performed to detect RNA sequences, while Southern blotting is done to detect DNA sequences. The processes for each are comparable, involving gel electrophoresis, transfer to a membrane, and hybridization. However, while agarose gel is typically used for electrophoresis in Southern blotting, formaldehyde or polyacrylamide gels are commonly used in Northern blotting. Southern blotting also requires DNA to be digested with restriction enzymes before running the gel; this is not necessary for RNA in Northern blotting.

Southern blot hybridization was the original go-to technique of the molecular pathology laboratory for the detection of DNA alterations however one of the disadvantages of this technique is that it requires the extraction of a fairly large amount of DNA (5 to 10 μg) from the fresh sample in a time-consuming process.

Assaywise Letter:

Overview of DNA Extraction Methods

Due to these disadvantages, Southern blot analysis has been replaced with PCR techniques for most applications, but Southern blot is still the gold standard for assessing immunoglobulin or T-cell receptor gene rearrangements. The amount of DNA needed for the Southern blot depends on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, the researcher can expect to detect approximately 0.1 pg of the target DNA.

Southern Blot is also very useful for detecting gene rearrangements if breakpoints are highly variable, or scattered over a large genomic region, or if a novel partner is involved. Another advantage of Southern blot is that it is very useful for the analysis of repetitive DNA sequences because multiple similar sequences in the genome can be analyzed with a single probe. On the other hand, methylation-sensitive restriction enzyme-based techniques analyze a limited number of CpG sites located within restriction recognition sites, and Southern blot analysis requires a large amount of high-quality DNA. Although these techniques were frequently used before bisulfite conversion-based techniques became popular, they are used only occasionally now.

Basic Southern Blot Workflow

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This is an example, and should be adjusted and optimized depending on experimental requirements.

1. Prepare purified DNA extraction from a biological sample (such as blood or tissue)
2. DNA extraction is then enzymatically digested with one or more restriction enzymes to produce DNA fragments
3. DNA fragments will vary in size, so they are separated accordingly using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move further than larger fragments.
4. The DNA is then denatured, usually while it is still on the gel, often by soaking it in about 0.5M NaOH, to separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.
   
   
   Note: Fragments larger than 15kb can be challenging to transfer to the membrane. If necessary, adding HCl can remove the purines, fragmenting it further. This step is aptly called depurination. The acid must be neutralized afterwards.
   
   
   
5. After electrophoresis is completed, the DNA fragments on the gel are then transferred out of the gel, or matrix, onto a solid membrane, typically nylon. Nitrocellulose can be used as well, but has a binding capacity of approximately 100 ug/cm, in comparison to the 500 ug/cm of nylon. Transfer can be done via either capillary action or a vacuum apparatus.
6. Once the DNA has been transferred, it is treated with UV light to covalently bind it to the membrane.
7. The membrane is then incubated with a nucleic acid probe that has a sequence homologous to the target sequence and is labeled with a fluorescent dye, radioactive tag, or a chemical tag, such as an enzyme capable of generating a chemiluminescent signal. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence, to be visualized on the Southern blot.
8. The unhybridized probe is removed by washing with a buffer. The fully hybridized labeled probe molecules stay bound to the blot and can then be detected and analyzed.

Detection methods differ based on the probe label. For example, radiolabeled probes are visualized with X-ray film or phosphorimaging, while enzymatically labeled probes are require a chemiluminescent substrate.

Examples of Southern Blot Analysis Applications

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Southern blotting is still routinely used in molecular biology for the detection of a specific DNA sequence in DNA samples. It has been used in the discovery and validation of several hematological gene fusions. DNA isolated from tumor samples are subjected to restriction endonuclease digestion and separated onto agarose gels by electrophoresis. The digested DNA fragments transferred to the nylon membrane are then probed by hybridization with known DNA sequences adjacent to the breakpoint.

A novel rearranged band, in addition to the germ line band derived from the normal allele, indicates the presence of rearrangement. Probes in the vicinity of the suspected breakpoints are always used. The detection of rearrangements depends on clustering of the breakpoints in one of the rearranging chromosomes within a small region.

For example, the immunoglobulin heavy chain (IGH)-associated gene fusions in lymphomas were first identified with the confirmation of rearrangement in the IGH gene using Southern blotting, followed by genomic screening of phage libraries with selected IGH probe, resulting in the identification of a clone containing the rearranged segment from the tumor genome. Translocations such as BCR/ABL with multiple breakpoints require multiple probes and multiple enzyme digestions of DNA, resulting in a costly and time consuming process.

In human tumors, changes in the number of copies, or the structure, of cancer-causing genes are frequent. Southern blot analysis can be used to investigate whether a gene is amplified, deleted, or structurally rearranged in cancer cells as compared to normal cells. Although this technique is labor-intensive, it's especially useful for detecting large deletions in tumor genomes. Southern blotting continues to be useful for the analysis of the immunoglobulin and T-cell receptor loci in leukemias and lymphomas. It's valuable because it can scan thousands of base pairs of DNA. It is likely to remain a preferred method in clinical applications in which the complexity, or primary sequence, of the locus makes DNA amplification difficult.

 

Southern Blot Summary

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For more than 30 years, Southern blot has been a widely used technique in molecular biology and has helped researchers to understand the fundamentals of molecular biology. Recently, several new automated techniques with incredible sensitivity have partially replaced the labor intensive and complex Southern blot technique. For example, the real-time PCR technique enables highly reliable and rapid detection of even a tiny target sequence, whereas Southern blot requires a large amount of target DNA.

Newer techniques such as FISH (fluorescent in situ hybridization) allow for highly sensitive identification of specific nucleotide sequences in a tissue sample with accurate localization. Both real time PCR and FISH provide a precise quantification of the target as well, which simply cannot be fully achieved using Southern blot.

Common Applications	Limitations	Advantages
Identification of a single gene in a pool of DNA fragments	It is labor-intensive	Multiple similar sequences in the genome can be analyzed with a single probe
Gene mapping and analysis of genetic patterns of DNA	It is not scalable (only a limited number of samples can be processed simultaneously)	Ideal for detecting gene rearrangements if breakpoints are highly variable
Detection of specific DNA sequences or gene family within a genome	Rarely, variants in restriction enzyme cutting sites can lead to atypical results	Targets can be scattered over a large genomic region
Study of gene deletions, duplications, and mutations that cause various diseases, including cancers such as monoclonal leukemia and sickle cell mutations	Requires large amounts of extracted DNA	Still provides results if a novel partner is involved
DNA fingerprinting and forensic tests such as paternity testing and sex determination	Process is time-consuming and cannot be accelerated	Very useful for highly complex samples

Products

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References

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Southern Blot
Southern vs Northern vs Western Blotting Techniques
Southern Blotting
Southern Blotting
Southern Blot - Lab Technique Used to Detect Specific Biomolecules
Chapter 8 - Analysis of Gene-Specific DNA Methylation