Southern blotting is the first detection technique which is used to identify the specific DNA sequences in the provided tissue or blood sample and is named after the scientist who developed it, ‘Ed Southern in the year 1975.
Northern blotting is the technique used to know the gene expression of the provided sample by identifying the specific RNA or mRNA molecules; it was developed by David Kemp, James Alwine and George Stark in 1977 at Stanford University. Western blotting is a sensitive assay used in the detection of proteins in the given sample, and Stark’s group developed it.
Blotting techniques are a widely used method for the specific identification of desired nucleic acid (DNA and RNA fragments) and proteins from the numerous molecules.
This technique involves immobilization of the sample of molecules of nucleic acid on the support of nylon membranes or nitrocellulose. It is a hybridization technique for the identification of specified genes, proteins or nucleic acid (DNA, mRNA) during multiple stages of gene expression.
Therefore, these types of blotting like Southern, Northern and Western are the subtypes of blotting and rely on the target molecule that is being separated.
The general process that is followed in blotting is in simple four steps, and firstly the sample is homogenized; secondly, separation of target molecules is done by an electrophoresis membrane; thirdly transferring of molecules to nylon or nitrocellulosic membrane and lastly, identification or hybridization of molecules is made.
In this article, we will observe the critical differences that lie between these three techniques, along with their description.
Content: Southern Vs Northern Vs Western Blotting Techniques
|Basis for Comparison||Southern Blotting||Northern Blotting||Western Blotting|
|Meaning||The technique used in the laboratory to detect the specific sequence of DNA in the provided tissue or blood sample.||The technique that identifies a specific sequence of RNA or mRNA molecules for gene expression in the given blood sample.||The most widely and accepted technique that identifies the sequence of amino acid in the protein mixture of the given sample.|
|Developed by||Edward M. Southern in 1975.||Alwine and colleagues in 1979.||George Stark's group in 1979 at Stanford University.
|Sample preparation||DNA extraction.||RNA isolation.||Protein extraction.|
|Separation||Agarose gel electrophoresis.||Denaturating formaldehyde agarose gel.||SDS PAGE.|
|The material used as a membrane||Nylon.||Nylon.||Nitrocellulose or PVDF.|
|Probe||The nucleic acid probe or DNA probe.||DNA, RNA or oligodeoxynucleotide.||Primary and secondary antibodies.|
|Probe label||The enzyme, Radiolabel.||The enzyme, Radiolabel.||The enzyme.|
|It detects||Specific DNA sequences.||Specific RNA sequences.||Specific proteins.|
|Detection methods||Chemiluminescence, X-ray film.||Chemiluminescence, X-ray film.||Camera, LED, cooled CCD, film, or infrared imaging system.|
|Applications||Used in DNA fingerprinting and to identify specific gene sequences.||Gene analysis expression.||In diagnosing disease.|
Definition of Southern blotting technique
This is the first nucleic acid blotting technique. Ed Southern developed it in 1975. The process is briefly described in the following points:
- The genomic DNA is digested with a restriction enzyme (one or more); this DNA was isolated from cells/tissues.
- The digest or mixture of DNA is loaded into a well of polyacrylamide or agarose gel. The mixture is subjected to electrophoresis.
- As DNA is negatively charged, it migrates towards the positively charged electrode (anode).
- The DNA molecules which are separated get denatured by exposure to mild alkali, and then further transferred to nylon paper or nitrocellulose.
- This result in a replica of the pattern of DNA fragment on the gel.
- Later the DNA is annealed to the paper on exposure to heat (80 degree Celsius).
- The nylon or nitrocellulose paper is exposed to labelled cDNA probes. These probes are hybridized with complementary DNA molecules on the paper.
- Lastly, the paper is roughly washed and is exposed to X-ray film for the developed autoradiography.
- Finally, this shows or reveals the specific bands corresponding to the DNA fragments recognized by cDNA probes.
There is numerous application of the Southern Blotting like it is used in gene analysis, genetic disease, infectious agent, mutation, RFLP, identification of any specific gene, and also in victim identification, DNA fingerprinting, criminal and rapist identification, paternity testing.
Definition of Northern blotting technique
Northern Blotting technique is almost similar to the Southern blotting technique; the essential and only difference is that in northern blotting, there is the specific identification of the RNA or mRNA molecules for the gene expression.
Likewise the southern blotting the process is same here, as RNA is subjected to the electrophoresis, and then by blot transfer and hybridization and autoradiography.
The only issue which arises here is that the RNA molecules do not easily bind with the nylon membrane or nitrocellulose papers. So, blot transfer is performed by advanced or modified nylon membranes or nitrocellulose papers and are used widely. In autoradiography, the blot-transferred RNA molecules are easy to detect, after they hybridize with a labelled oligonucleotide or DNA probe.
Although, this technique theoretically seems to be good, for determining the number of genes in the given DNA sequence. Still, there are few drawbacks like the presence of introns and exons in the sequence; secondly, genes may give origin to one or more transcripts.
This technique is widely used in disease diagnosis and gene expression.
Definition of Western blotting technique
Western Blotting technique is used for the identification of the amino acid sequence in the mixture of proteins. It is also known as immunoblotting or protein blotting. Here proteins are separated into SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis), as per their molecular weight and it follows the principle of immunochromatography.
Then proteins are transferred to nitrocellulose paper and are detected by using primary and secondary antibodies and substrate. George Stark’s group developed this technique in 1979 at Stanford University.
It is widely used in HIV test to check anti-HIV antibody in the sample of human serum; also used in Hepatitis B, HSV 2 infections, Lyme disease, Creutzfeldt-Jakob disease test, and to test the amount of protein present in any sample.
Stark’s group developed this technique. Still, Towbin’s group developed the other useful techniques in this method like the use of secondary antibodies, buffers, etc. while Burnette gave the name to this technique and other modification of this method; so still it is arguable that who should be contributed for this technique.
Key Differences Between Southern, Northern and Western Blotting Technique
Upcoming points will exhibit the essential differences between the three types of techniques used in the laboratory for detection of DNA, RNA and protein and gene expression.
- The technique used in the laboratory to detect the specific sequence of DNA in the provided tissue or blood sample is known as Southern blotting; On the other hand, a technique that identifies the specific sequence of RNA or mRNA molecules for gene expression is known as Northern blotting; and The most widely accepted technique that determines the sequence of amino acid in protein mixture of the given sample is Western blotting.
- Southern blotting was developed by Edward M. Southern in 1975, and credit for the northern blotting goes to Alwine and colleagues in 1979, while George Stark’s group in 1979 in Stanford University developed western blotting.
- Southern blotting targets DNA, northern blotting target is RNA and western blot targets are protein.
- Separation of a sample in southern blotting is done by agarose gel electrophoresis, and in northern blotting, it is done by denaturating formaldehyde agarose gel. In contrast, in western blotting it is done is the SDS PAGE.
- The nucleic acid probe or DNA probe is used in southern blotting, whereas DNA, RNA or oligodeoxynucleotide in case of northern blotting and western blotting primary and secondary antibodies.
- The detection method used in southern and northern blotting are chemiluminescence, X-ray film and in western blotting camera, LED, cooled CCD, film, or infrared imaging system are used.
- Southern blotting is used in DNA fingerprinting and to identify specific gene sequences, while northern blotting is used in gene analysis expression and western blotting is used in diagnosing disease.
From this article, we can conclude that these techniques were developed to identify different molecules present in blood like DNA, RNA and protein, they can also be used for identifying particular gene expression. Hence, they all are equally important and have vital aspects from a biology point of view.