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Southern Blot: Principles, Example Workflow, & Applications

by Tom S. Lang

Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye
Direct labeling of nucleic acid using Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye. DNA ladder was labeled with Helixyte™ iFluor® 350 Nucleic Acid Labeling Dye (Lane 4) and analyzed alongside unlabeled DNA (Lane 1) on 1% agarose gel using gel electrophoresis.
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.


TechniqueWestern BlotNorthern BlotSouthern Blot
Target MoleculeProteinRNADNA
Sample PrepProtein extractionRNA isolationDNA extraction
SeparationGel Electrophoresis/SDS-PAGEGel ElectrophoresisGel Electrophoresis
Membrane MaterialNitrocellulose/PVDFNylonNylon
ProbeUses a single primary antibody or a combination of primary and secondary antibodies conjugated to either a fluorophore or enzymeRNA, DNA or oligodeoxynucleotideNucleic acid probe
DetectionFilm, CCD camera, LED or infrared imaging systemX-Ray film, chemiluminescenceX-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:

S-adenosyl-methionine (SAM) removes methyl groups
S-adenosyl-methionine (SAM) removes methyl groups from gene promoter. Loss of DNA methylation results in aberrant transcription of target gene that lacks these groups.
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


Southern blot
Simplified steps of Southern blot, from initial DNA extraction through processing and final visualization of labeled DNA sequences. Figure made in BioRender.
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.



Resources:

 

Examples of Southern Blot Analysis Applications


BCR/ABL chromosomal translocation
Illustration of BCR/ABL chromosomal translocation, or the 'Philadelphia Chromosome', which results in unique alleles. Figure made in BioRender.
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


Telomere quantitative fluorescence in situ hybridization
Telomere quantitative fluorescence in situ hybridization in metaphase HeLa cells using iFluor® 647-dUTP labeled telomere probes. Probes were created using the ReadiLink™ iFluor® 647 FISH Fluorescence Imaging kit.
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 ApplicationsLimitationsAdvantages
Identification of a single gene in a pool of DNA fragmentsIt is labor-intensiveMultiple similar sequences in the genome can be analyzed with a single probe
Gene mapping and analysis of genetic patterns of DNAIt 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 genomeRarely, variants in restriction enzyme cutting sites can lead to atypical resultsTargets 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 mutationsRequires large amounts of extracted DNAStill provides results if a novel partner is involved
DNA fingerprinting and forensic tests such as paternity testing and sex determinationProcess is time-consuming and cannot be acceleratedVery useful for highly complex samples

 

Products



Table 1. Nucleic Acid Staining Reagents and Assay Kits

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
17590Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* 498522100 uL
17590Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution* 4965391 mL
17507DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *10 mM solution in water*3584612 mL
17589Gelite™ Green Nucleic Acid Gel Staining Kit4975201 Kit
17594Gelite™ Orange Nucleic Acid Gel Staining Kit4975201 Kit
17520Hoechst 33258 *CAS 23491-45-4*352461100 mg
17530Hoechst 33342 *Ultrapure Grade*350461100 mg
17537 Hoechst 34580 *CAS 911004-45-0*3684375 mg
17548 Nuclear Blue™ DCS1 *5 mM DMSO Solution*3504610.5 mL
17550 Nuclear Green™ DCS1 *5 mM DMSO Solution*5035260.5 mL
17540 Nuclear Green™ LCS1 *5 mM DMSO Solution*5035260.5 mL
17551 Nuclear Orange™ DCS1 *5 mM DMSO Solution*5285760.5 mL
17541 Nuclear Orange™ LCS1 *5 mM DMSO Solution*5145550.5 mL
17552 Nuclear Red™ DCS1 *5 mM DMSO Solution*6426600.5 mL
17542 Nuclear Red™ LCS1 *5 mM DMSO Solution*6226450.5 mL
17543 Nuclear Violet™ LCS1 *5 mM DMSO Solution*4014590.5 mL
17539 Nuclear Yellow [Hoechst S769121] *CAS 74681-68-8* 35549525 mg

Table 2. Gelite™ DNA Ladders

Cat#
Product Name
Unit Size
17750Gelite™ 50 bp DNA Ladder0.5 mL
17753Gelite™ 100 bp DNA Ladder0.5 mL
17756Gelite™ 1 kb DNA Ladder0.5 mL
17760Gelite™ 100 bp-1 kb DNA Ladder0.5 mL

Table 3. ReadiUse™ DNA ladders

 

References



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


Original created on March 8, 2024, last updated on April 29, 2024
Tagged under: blot, DNA, nucleic acids, gel electrophoresis