Brief Introduction: Blotting Techniques
Blotting is used in molecular biology for the identification of proteins and nucleic acids and is widely used for diagnostic purposes. This technique immobilizes the molecule of interest on a support, which is a nitrocellulosic membrane or nylon. It uses hybridization techniques for the identification of the specific nucleic acids and genes.
The blotting technique is a tool used in the identification of biomolecules such ad DNA, mRNA and protein during different stages of gene expression. Protein synthesis involves expression of a DNA segment which gets converted to mRNA to produce the respective protein.
Subtypes of blotting such as northern, western & southern depend upon the target molecule that is being sought. When a DNA sequence is the foundation or code for a protein molecule, the particular DNA molecule of interest can be blotted using Southern Blotting technique. During gene expression, when the DNA is expressed as mRNA for a protein production, this process can be identified by Northern blotting. Finally, the coded mRNA produces the concerned protein, this protein identification can be done by Western Blotting.
General Procedure for blotting
- Homogenize the sample.
- Separation of the molecule of interest by an electrophoresis membrane.
- Transferring the molecules to a nitro cellulosic membrane/ nylon membrane.
- Hybridization or identification of the molecule
The first of these techniques developed was the Southern blot, named after Dr. Edwin Southern who developed it to identify specific DNA sequences. Southern blotting is a detection technique used to find the target DNA sequences in the DNA sample in the field of molecular biology. The process starts from electrophoresis of DNA molecules which are hybridized in a blotting membrane followed by a transfer step where DNA from gel is transferred onto the blotting membrane.
The electrophoresis step can be done using two different types of gels such as Polyacrylamide gel (PAGE) with urea and Sodium Dodecyl sulfate (SDS) with urea. These two gels have different application. When a PAGE gel is used, the same quantity of DNA fragments is transferred to the blotting paper. If a SDS is used the resolution of the bandwidth formed remains same. In this technique DNA molecule of size, 100 pg can be identified. The technique can be summarized as the formation of double stranded DNA which has one strand from target DNA and the other from DNA probe. DNA probe is produced in vitro for the sequence of interest.
Restriction endonucleases, which is an enzyme, is used to break the DNA into small fragments. These fragments are then separated using electrophoresis. The Fragments achieved is then classified according to their size (kDa). Thus, DNA fragments are transferred to the blotting paper where it is incubated with probes. Probes used in the Southern blotting can be highly selective. They can selectively bind with a resolution of 1 in a million and the characteristics to bind to the intended target fragments.
Step 1: DNA purification
To extract the DNA present inside the nucleus of a cell, we must first lyse the entire cell to enable the expulsion of the DNA. Incubating the cell culture with detergent lyses the entire cell. Now the lysed sample contains DNA, protein, and debris. Protein is lysed by adding the proteinase enzyme and incubated. DNA is purified and separated by alcohol precipitation and fibers are removed by using a buffer.
Step 2: Fragmentation
The long nucleotide sequences should be broken into smaller fragments for the purification or identification process. This is done by the restriction endonuclease enzyme.
Step 3: Gel Electrophoresis
Nucleic acids are negatively charged molecules. So they move towards the anode in an electrophoresis chamber. The movement of the DNA fragments differentiates the rate of the transport thus enabling the separation by size.
Step 4: Denaturation
DNA thus attained are double stranded in nature. For our purpose of probe hybridization, we need a single stranded DNA. DNA is therefore denatured in an alkaline solution. This results in the formation of denatured DNA.
Step 5: Blotting
Blotting is the transfer of the fragmented DNA sequence to the nitrocellulose membrane or nylon membrane. The process is done by either electroblotting or capillary blotting. The DNA molecule is saturated using a NaCl solution and permanently fixed using either UV radiation or drying.
Step 6: Hybridization
Labeled probe is added to the membrane buffer and incubated for as It takes several hours for the probe to find the exact target sequence.
Southern blotting is used in a number of applications. 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. It is also useful in the study of mutation and gene rearrangement, this property is used to diagnose neonatal disease and genetic disease. Due to the precision in DNA identification this technique is used in phylogenetic studies, paternity & maternity analysis, forensic studies and personal identification.