Southern-blotting

Blotting: Steps involved & Types

What is Blotting?

Blotting techniques are the most common approaches used in biochemistry and molecular biology. In this technique, macromolecules such as DNA, RNA and proteins are set on the gel matrix, then transferred to a solid support, and then detected by molecule-specific probes. These techniques are widely used to identify proteins, DNA, and RNA and for diagnostic purposes.

Steps involved in blotting

First, run gel electrophoresis to separate the molecules in the mixture. Then bands are blotted to the membrane. Specific probes are used for specific macromolecules. They illuminate to locate the bands.

Types of Blotting

The major three types of blotting are

Western blotting

It is a technique for separating proteins based on size into straight columns. Then researchers used this to compare the amount of protein in different samples.

Western-blotting

Northern blotting

Northern blotting is used to detect RNA. This is used to study RNA-specific diseases or to show the working of disease at the RNA level.

Southern blottingĀ 

This type of blotting is used to detect DNA. It will be discussed in detail in this article.

Other blotting

Northwestern blotting is used to study protein struck in RNA. Southwestern blotting is used to determine protein struck into DNA. Far western blotting is used to detect protein molecules struck into other molecules.

Southern blotting

Southern blotting is a lab technique utilized to identify a specific DNA series in a blood or tissue sample. A restriction enzyme is utilized to cut a sample of DNA into fragments that are separated using gel electrophoresis.

The DNA pieces are transferred out of the gel to the surface of a membrane. The membrane is exposed to a DNA probe labeled with a radioactive or chemical tag. If the probe binds to the membrane, then the probe series is present in the sample.

Southern-blotting

Southern blotting was presented by Edwin Southern in 1975 as a method to find specific sequences of DNA in DNA samples.

The other blotting strategies that emerged from this approach have been called as Northern (for RNA), Western (for proteins), Eastern (for post-translational protein adjustments) and South-western (for DNA-protein interactions) blotting.

Steps involved in Southern blotting
DNA purification

To extract the DNA present inside the nucleus of a cell, we must initially lyse the whole cell to allow the expulsion of the DNA. Incubating the cell culture with detergent lyses the entire cell. Now the lysed sample includes DNA, protein, and debris. Protein is lysed by adding the proteinase enzyme and nurtured. DNA is purified and separated by alcohol precipitation and fibers are removed by using a buffer.

DNA fragmentation

Acquiring complete fragmentation of your DNA at the designated limitation enzyme sites is a vital step in Southern blot analysis. There is a wide choice of premium restriction enzymes so that you can constraint digest your DNA to assist provide clear, unambiguous Southern blot.

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Gel electrophoresis

Fragmented DNA is typically electrophoresed on an agarose gel to separate the fragments according to their molecular weights. Acrylamide gels can alternatively be utilized for good resolution of smaller DNA pieces (<800 bp).

Blotting

After electrophoresis, DNA is transferred to a positively charged nylon membrane. Conventional transfer of DNA is done overnight using an upward-transfer approach.

In the case of Southern blot but in current times nylon membranes have been executed for the blotting process due to their ability to bind more quantity of DNA efficiently which permits the Southern blot to be carried out with less amount of target DNA.

Probe labeling

A nucleic acid probe with a series homologous to the target sequence under study is labeled with radioactivity, fluorescent dye, or an enzyme that can generate a chemiluminescent signal when bred with the suitable substrate.

The choice of the label depends upon several factors such as the nature of your probe or probe template, level of sensitivity needed, metrology requirements, ease of use, and speculative time.

Hybridization and washing

Throughout hybridization, the identified probe is incubated with the DNA pieces that are immobilized on the blot under conditions that promote hybridization of complementary series. After hybridization, the unhybridized probe is gotten rid of by washing in several changes of buffer.

Detection

In the detection action, the bound, labeled probe is found using the technique required for the particular label used.

For example, radiolabeled probes may be found utilizing X-ray film or a phosphor imaging instrument, and enzymatically labeled probes are typically found by incubating with a chemiluminescent substrate and exposing the blot to X-ray film.

Uses

Southern blotting was used in numerous crucial studies like the genetic mapping of the human genome which was based upon blotting-based detection of constraint fragment length polymorphisms.

DNA fingerprinting was first established utilizing hybridization of the human DNA restriction digestion products with minisatellite probes.

Southern blotting is utilized in the measurement of copy number, and analysis of long stretches of DNA that is challenging to be magnified or sequenced using PCR.

MCQs about Blotting

  • 1. What is the primary function of blotting techniques in biochemistry and molecular biology?
    • A) Separating macromolecules into distinct bands
    • B) Amplifying DNA sequences
    • C) Identifying proteins, DNA, and RNA
    • D) Purifying cellular debris
    • Answer: C) Identifying proteins, DNA, and RNA
  • 2. What is the first step involved in blotting techniques?
    • A) Gel electrophoresis
    • B) DNA purification
    • C) Fragmentation of DNA
    • D) Labeling the probes
    • Answer: A) Gel electrophoresis
  • 3. Which of the following is not a type of blotting technique?
    • A) Western blotting
    • B) Eastern blotting
    • C) Northern blotting
    • D) Southern blotting
    • Answer: B) Eastern blotting
  • 4. What is the main purpose of Western blotting?
    • A) Detecting RNA
    • B) Separating DNA fragments
    • C) Studying post-translational protein modifications
    • D) Comparing protein amounts in different samples
    • Answer: D) Comparing protein amounts in different samples
  • 5. Who introduced the Southern blotting technique?
    • A) Edwin Northern
    • B) Edwin Western
    • C) Edwin Southern
    • D) Edwin Eastern
    • Answer: C) Edwin Southern
  • 6. What is the purpose of DNA purification in Southern blotting?
    • A) To amplify DNA fragments
    • B) To extract DNA from the membrane
    • C) To remove proteins and debris from the DNA sample
    • D) To fragment the DNA
    • Answer: C) To remove proteins and debris from the DNA sample
  • 7. Which enzyme is commonly used to fragment DNA in Southern blotting?
    • A) DNA polymerase
    • B) RNA polymerase
    • C) Restriction enzyme
    • D) Ligase
    • Answer: C) Restriction enzyme
  • 8. What is the function of gel electrophoresis in Southern blotting?
    • A) Fragmenting DNA
    • B) Labeling the probes
    • C) Separating DNA fragments based on size
    • D) Binding DNA fragments to a membrane
    • Answer: C) Separating DNA fragments based on size
  • 9. Which material is commonly used for blotting in modern techniques?
    • A) Glass
    • B) Paper
    • C) Nylon membrane
    • D) Plastic
    • Answer: C) Nylon membrane
  • 10. What is the purpose of probe labeling in Southern blotting?
    • A) To amplify DNA fragments
    • B) To separate DNA fragments
    • C) To visualize DNA fragments
    • D) To hybridize with the target sequence
    • Answer: D) To hybridize with the target sequence
  • 11. Which of the following is a common label used for probes in Southern blotting?
    • A) Radioactivity
    • B) DNA polymerase
    • C) Gelatin
    • D) Sucrose
    • Answer: A) Radioactivity
  • 12. What happens during the hybridization and washing step in Southern blotting?
    • A) DNA fragments are separated based on size
    • B) Probes are labeled with radioactivity
    • C) Unhybridized probes are removed
    • D) Gel electrophoresis is performed
    • Answer: C) Unhybridized probes are removed
  • 13. Which step in Southern blotting involves detecting the bound, labeled probe?
    • A) DNA purification
    • B) Hybridization and washing
    • C) Gel electrophoresis
    • D) Detection
    • Answer: D) Detection
  • 14. What was one of the significant applications of Southern blotting in genetic studies?
    • A) Protein sequencing
    • B) RNA synthesis
    • C) Genetic mapping of the human genome
    • D) Enzyme kinetics
    • Answer: C) Genetic mapping of the human genome
  • 15. Which blotting technique is used for detecting RNA?
    • A) Western blotting
    • B) Northern blotting
    • C) Southern blotting
    • D) Eastern blotting
    • Answer: B) Northern blotting
  • 16. What is the primary function of Southern blotting?
    • A) Detecting proteins
    • B) Identifying DNA sequences
    • C) Amplifying RNA
    • D) Studying post-translational modifications
    • Answer: B) Identifying DNA sequences
  • 17. Which step involves transferring DNA fragments from the gel to a membrane in Southern blotting?
    • A) Gel electrophoresis
    • B) DNA purification
    • C) Blotting
    • D) Detection
    • Answer: C) Blotting
  • 18. Which material is used as a solid support in Southern blotting?
    • A) Paper
    • B) Nylon membrane
    • C) Glass slide
    • D) Plastic film
    • Answer: B) Nylon membrane
  • 19. What is the purpose of DNA fragmentation in Southern blotting?
    • A) To amplify DNA sequences
    • B) To label DNA probes
    • C) To separate DNA fragments based on size
    • D) To remove proteins from DNA samples
    • Answer: C) To separate DNA fragments based on size
  • 20. Which blotting technique is used for detecting protein-DNA interactions?
    • A) Northern blotting
    • B) Southern blotting
    • C) Eastern blotting
    • D) Southwestern blotting
    • Answer: D) Southwestern blotting
  • 21. Which enzyme is commonly used for DNA purification in Southern blotting?
    • A) RNA polymerase
    • B) DNA ligase
    • C) Proteinase
    • D) Restriction enzyme
    • Answer: C) Proteinase
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Summary:

Blotting techniques are essential tools in biochemistry and molecular biology, commonly used to identify and analyze macromolecules such as DNA, RNA, and proteins. The process involves separating molecules through gel electrophoresis, transferring them onto a solid support, and detecting them using molecule-specific probes.

There are various types of blotting techniques, including Western blotting for proteins, Northern blotting for RNA, and Southern blotting for DNA detection.

Southern blotting, introduced by Edwin Southern, is particularly useful for identifying specific DNA sequences in samples. It involves several steps, including DNA purification, fragmentation, gel electrophoresis, blotting onto a membrane, probe labeling, hybridization, and detection. Southern blotting has been instrumental in genetic studies, such as mapping the human genome and DNA fingerprinting.

Overall, blotting techniques play a crucial role in molecular biology research, enabling the detection and analysis of various macromolecules for diagnostic and investigative purposes.