Blotting Technique used for Identification of Nucleic Acid

 

3 Blotting Techniques used for the identification of Nucleic Acid

Basics techniques in the genetic engineering field

                             Image Credit to Polina Tankilevitch /Unsplash 

Nucleic acid like DNA and RNA is widely used in various research work. So, the identification of desired DNA and RNA from thousands of molecules becomes the basic requirement. The blotting technique is widely used as an analytical tool for the identification of desired DNA and RNA fragments.

The basic principle behind this blotting technique is the process based on immobilization of samples i.e. nucleic acid and solid support. 

Blot helps to transfer nucleic acid onto the carrier so they can easily be separated from the desired nucleic acid fragment. The solid support or carrier mostly used in the blotting technique are Nitrocellulose, Nylon membrane, and DBM paper that helps for the blot nucleic acid for further use in hybridization experiments for specific detection. 

Basic steps used in the Blotting techniques are : 

1. Immobilization of nucleic acid
2. Prehybridization 
3. Hybridization with specific DNA and RNA probes
4. Stringency Washes
5. Detection by autoradiograph

Different types of blotting techniques are used for the identification of target nucleic acid. The type of blotting technique is based on the identification of specific DNA, RNA, DNA/RNA both, there are three types of blotting techniques that are widely used in genetic engineering briefly discuss here are: 

1. Southern Blotting
2. Northern Blotting
3. Dot Blotting

SOUTHERN BLOTTING

The southern blotting technique is used for the detection of DNA. This technique is the first nucleic acid technique that is developed in 1975 by E.M.Southern. For performing this technique more stringent conditions are required. The southern technique is more specific and sensitive by the process.

Steps of Southern blotting for the detection of DNA

1. Isolation of DNA from specific cell /tissue.
2. To get DNA fragment restriction enzyme is used to cut the target DNA from the cell/tissue. After that treatment number of DNA fragments are formed.
3. For the separation of required DNA molecules, Gel electrophoresis is performed.
4.  DNA molecule is separated by a gel electrophoresis technique. Two different types of gel are used for electrophoresis using either polyacrylamide or agarose gel to get a required size of DNA for detection.. (*Fragments of DNA is varying according to gel, if polyacrylamide is used then we will get DNA fragments are very small on the other side if agarose gel is used then separating DNA fragments of a few hundred to 20kb in size).
5.  After gel electrophoresis, the DNA molecule is exposed to a mild alkali treatment and then transfer onto a nitrocellulose or nylon paper.
6. DNA is trapped onto the nitrocellulose membrane by placing the gel on top of a buffer saturated filter paper. Then nitrocellulose is placed on the gel.
7.  As a buffer passes through it, capillary action occurs from the bottom. During this process DNA trapped on the nitrocellulose membrane, then buffer passing through it this process is known as blotting.
8. It takes several hours to complete. 
9. Nitrocellulose membrane is then removed from the blotting stack, For permanent immobilization of DNA on membrane bake it at 80-degree celsius in vacuo. 
10. DNA embed nitrocellulose and nylon paper are exposed to labeled cDNA probes. These probe helps to hybridize cDNA molecules on the paper.
11. After the hybridization reaction membrane is washed to remove unbound probes. Then the membrane is exposed to X-ray film to develop autoradiography. This helps to reveal specific bands corresponding to the DNA fragments.

The Southern technique is an invaluable method for gene analysis. In forensic science, this technique is more useful for the detection of minute quantities of DNA. This is an important technique for the confirmation of DNA cloning results. The southern blotting is also used for the detection and identification of the transferred genes in transgenic individuals.

NORTHERN BLOTTING 

The northern technique is used for the identification and separation of the RNA molecules that are complementary to the DNA probe. The procedure is almost the same as the southern blotting. 

In short Northern blotting is an RNA preparation that is subjected to electrophoresis further RNA transferred from the gel onto a DBM paper, immobilized, and hybridized with a single-stranded radioactive probe.

*For the detection and identification of RNA, DBM ( Diazobenzyloxymethyl ) paper is used rather than nitrocellulose paper and Nylon paper because RNA can easy to covalently bind to DBM rather than nitrocellulose or nylon paper.RNA is covalently bound to DBM paper due to this reason blot- transfer is effective.

The hybridization of the RNA molecules is done with a DNA probe. which can further easy to detect with the help of autoradiography.

The northern technique is a very important technique for the detection of RNA. This is a sensitive test for the detection of transcription of a DNA sequence that is used as a probe.

DOT BLOTTING 

The dot blotting technique is the modification of southern and northern blotting techniques. The nucleic acid is directly spotted on filters without undergoing electrophoresis. The hybridization process is the same as in southern and northern blotting. 

This technique is very useful for gene expression by quantitative data. The dot blot technique is useful for the detection and the presence of a sequence that is transferred to transgenic individuals.

Generalized steps of the dot blot technique are briefly described below:

1. The target samples of DNA( or RNA) from specified tissue are transferred onto a nitrocellulose filter in the form of dots.
2. Each dot represents the sample of DNA (or RNA).
3. In the case of DNA sample first denatured, filter and then baked at 80 - degrees Celsius to fix DNA on the filter paper.
4. The filter is hybridized with a radioactive probe. wash-off unhybridized probe.
5. Detection of the dot by autoradiography. Only the dark and shaded spots appear on the X-ray film, these samples contain the sequences represented by the probe.
6. If no dark spot appears on the film, it shows that no DNA hybridization has occurred.

The blotting technique is used in molecular biology for diagnostic purposes. This technique immobilizes the molecule of interest in support. There is one more blotting technique name as Western blotting that is often used in research and for the identification of the protein.