Northern and Southern blotting are techniques used in molecular biology, which are used to detect macromolecular changes related to the DNA. BiologyWise explains the nature and principle of these techniques, and compares the differences between them.
Etymology
Southern blotting is named after Sir E. M. Southern, a British biologist who developed this technique. Subsequent blotting techniques (Northern blotting, Western blotting, etc.), which are based on the same principle, are named eponymously.
In molecular biology and genetics, various blotting techniques are employed to detect and study changing levels of proteins, DNA, or RNA, and also to study the interactions occurring between them. Southern blotting is used in case of DNA, Northern blotting in case of RNA, Western blotting in case of proteins, and Eastern blotting in case of post-translational modifications of proteins. The purpose of each technique may be different, but all share the same principle and methodology, with a few minor deviations and modifications.
Principle
All blotting techniques are based on the same principle of specific base pairing of the probe sequences to the sequences immobilized on the membrane. Any change in the sample sequences will lead to the probes not binding or binding non-specifically. This change in binding affinity will be visualized upon secondary hybridization with a labeled probe. The intensity of the resultant dye or fluorescence will indicate the specificity of the binding along with other characteristics such as copy number, gene expression, etc.
Northern blotting is a technique used to detect and study specific RNA molecules from a mixture of different RNA, all isolated from a particular tissue or cell type. It allows the investigator to determine the molecular weight of mRNA, and also to determine the relative quantity of mRNA (gene expression) across different samples.
Southern blotting is used to detect and study specific DNA sequences. It allows one to study restricted (cut) DNA fragments, changes in the sequence, and its relative quantity across different samples.
Methodology
✦ Initially, the purified sample is loaded onto an agarose gel and electrophoresed, causing the sample to separate into bands. The bands are then transferred onto a carrier membrane by the principle of either capillary action or directed electric current. The membrane with the transferred bands is then sealed by baking in an oven or by treating it with a blocking solution. This ensures that the transfer is made permanent, and the unbound surface of the membrane is blocked so as to prevent binding of unwanted molecules and the possibility of a hampered result.
✦ Once the blocking is done, the membrane is washed gently to remove traces of the blocking solution. The membrane is then treated with specific target probes that bind to the transferred bands. The binding of the probe to the target molecule is called primary hybridization. The membrane is washed gently to eliminate the excess probes, and then it undergoes secondary hybridization, where the target probe is labeled with a radioactive element, a fluorescent dye, or even a chromogenic dye. The excess is again washed off.
✦ Depending on the type of secondary probe used, the membrane is visualized accordingly. In case of radioactive labeling or the use of a fluorescent dye, an X-ray film is exposed to the membrane for a few seconds and developed. If a chromogenic dye is used, the results can be seen on the membrane itself. Nowadays, fluorescent dyes are preferred over radioactive labeling to prevent undue exposure to radiation. However, there are some cases where one has to use radioactive labeling.
It was developed by James Alwine, David Kemp, and George Stark in 1977.
It was developed by Sir Edwin Mellor Southern in 1975.
Origin of Name
The name is a misnomer―an eponymous derivative of Southern blotting.
It is named after its inventor, E. M. Southern.
Purpose
It detects presence of specific RNA sequences.
It detects changes in specific DNA sequences.
Sample Preparation
The sample is used in its native state.
The sample has to be denatured.
Membrane
The membrane used is an amino benzyloxymethyl filter paper membrane.
The membrane used is a nitrocellulose membrane.
Type of Hybridization
RNA-DNA hybridization
DNA-DNA hybridization
Application
To study gene expression profiles.
To study genetic changes in DNA; it can be used for hom*ology-based cloning too.
Derivative
Northwestern blotting studies that detect interactions between RNA and proteins.
Southwestern blotting studies that detect DNA-binding proteins.
The two techniques stem from the same principle and differ in the type of molecules they target. The development in the blotting techniques has helped provide scientists with a tool to study molecular interactions and detect changes, if any.
While both techniques are used to identify nucleic acid sequences, Northern blotting is performed to detect RNA sequences, while Southern blotting is done to detect DNA sequences. The processes for each are similar, involving gel electrophoresis, transfer to a membrane, and hybridization
hybridization
Hybridization (biology), the process of combining different varieties of organisms to create a hybrid. Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals. Nucleic acid hybridization, the process of joining two complementary strands of nucleic acids - RNA, DNA or oligonucleotides.
A Dot blot is generally used as a quick method to see whether the target exists at all in the sample. If yes, then a Western/ Northern/ Southern blot is done for a more refined and quantifiable detection.
Different blotting is used to detect different type of macromolecules such as southern blotting is used for DNA analysis, western blotting is for protein analysis, northern blotting is for RNA analysis and eastern for carbohydrate detection.
Northern blot is used to detect a specific RNA sequence in a sample of mixed RNAs. Southern blot is used for detecting a specific DNA sequence in a sample of mixed DNA. Western blot is used to identify a specific amino acid sequence in a sample of mixed proteins. A specific RNA sequence is detected.
Southern, northern, and western blot protocols are similar, and begin with electrophoretic separation of protein and nucleic acid fragments on a gel, which are then transferred to a membrane (nitrocellulose membrane, polyvinylidene difluoride (PVDF) membrane, etc.) where they are immobilized.
The advantages of using Northern blotting include the use of probes with partial hom*ology, the quality, and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and re-probed for years after blotting [41].
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 quite labor-intensive, it's particularly useful for detecting large deletions in tumor genomes.
The primary usage of Southern blotting is to identify a specific DNA in a DNA sample. It is mostly used in the identification of viral infection and certain bacterial infections. In rDNA technology, The Southern blotting technique is used to isolate a particular DNA.
While both techniques are used to identify nucleic acid sequences, Northern blotting is performed to detect RNA sequences, while Southern blotting is done to detect DNA sequences. The processes for each are similar, involving gel electrophoresis, transfer to a membrane, and hybridization.
Northern blot is a laboratory analysis method used to study RNA. Specifically, purified RNA fragments from a biological sample (such as blood or tissue) are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments.
For dot blot hybridization, DNA or RNA is spotted directly onto a membrane, while for Southern or northern blot hybridization DNA fragments or mRNAs, respectively, are transferred to the membrane after size separation on an agarose gel by capillary-, vacuum-, pressure-or electroblotting and subsequently hybridized with ...
Advantages. Southern blotting does not rely on PCR amplification of DNA. This means that repeat expansions that are too large to amplify through PCR can be accurately sized. For some patients, it may be the only method that can provide accurate sizing of their repeat expansion.
Compared to qPCR, the major limitations of Northern blot analysis are low detection sensitivity and easy RNA degradation by contaminated exogenous ribonucleases (RNases) in the course of extensive handling of RNA prior to blotting.
The name of each blotting technique varies according to the targeted molecule. This can be easily remembered with the corresponding acronyms SNOW-DROP. S for Southern Blot corresponds to D for DNA. N for Northern Blot for R of RNA and W for Western Blot for P for Proteins.
A Southern blot is a laboratory method used to detect specific DNA molecules from among a many other DNA molecules. The technique was named after its inventor, Edward Southern.
A northern blot is a laboratory method used to detect specific RNA molecules among a mixture of RNA. Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell type in order to measure the RNA expression of particular genes.
Northern Blotting is a technique used for the study of gene expression. It is done by detection of particular RNA (or isolated mRNA). mRNA is generally represented as 5% of the overall RNA sequence. This method reveals the identity, number, activity, and size of the particular gene.
The microarray technique allows the expression rate of thousands of genes at a time (high throughput), whereas in the northern blotting technique, only a few genes can be expressed (low throughput). The northern blotting technique must be performed manually, whereas automation is possible in the microarray technique.
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