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
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. This blotting technique can also be used for the growth of a tissue or organism. In different stages of differentiation and morphogenesis the abundance of an RNA changes and this can be identified using this technique. It also aids in the identification of abnormal, diseased or infected condition at the molecular level. The northern blot technique was developed in 1977 by James Alwine, David Kemp and George Stank at Stanford University. The technique got its name due to the similarity of the process with Southern blotting. The primary difference between these two techniques is that northern blotting concerns only about RNA.
As all normal blotting technique, northern blotting starts with the electrophoresis to separate RNA samples by size. Electrophoresis separates the RNA molecules based on the charge of the nucleic acids. The charge in the nucleic acids is proportional to the size of the nucleic acid sequence. Thus the electrophoresis membrane separates the Nucleic acid sequence according to the size of the RNA sequence. In cases where our target sequence is an mRNA, the sample can be isolated through oligo cellulose chromatographic techniques, as mRNA are characterized by the poly(A)-tail. Since gel molecules are fragile in nature, the separated sequences are transferred to the nylon membranes. The selection of nylon membrane is contributed to the factor that nucleic acids are negatively charged in nature. Once the RNA molecules are transferred it is immobilized by covalent linkage. The probe is then added, the probe can be complementary an ss DNA sequence. Formamide is generally used as a blotting buffer as it reduces the annealing temperature.
- The tissue or culture sample collected is first homogenized. The samples may be representative of different types of culture for comparison or it can be for the study of different stages of growth inside the culture.
- The RNA sequence is separated in the electrophoresis unit an agarose gel is used for the purpose of the nucleic acid separation.
- Now the separated RNA sequence is transferred to the nylon membrane. This is done by two mechanisms capillary action and the ionic interaction.
- The transfer operation is done by keeping the gel in the following order. First, the agarose gel is placed on the bottom of the stack, followed by the blotting membrane. On top of these paper towels a mild weight (glass plate) is placed. The entire setup is kept in a beaker containing transfer buffer.
- RNA transferred to the nylon membrane is then fixed using UV radiation.
- The fixed nylon membrane is then mixed with probes. The probes are specifically designed for the gene of interest, so that they will hybridize with RNA sequences on the blot corresponding to the sequence of interest.
- The blot membrane is washed to remove unwanted probe
- Labeled probe is detected by chemiluminescence or autoradiography. The result will be dark bands in x ray film.
GEl and Probes
The RNA samples are separated using agarose gels using formaldehyde as denaturing agents but in small RNA or micro RNA sequences, polyacrylamide sequences with urea as a denaturing agent also can be used. Ethidium bromide can be used as a staining agent. Two types of markers are for size marking. An RNA ladder and ribosomal subunit are used for the identification of the size of the RNA sequences.
Probes can be complementary to the whole or part of the RNA of interest. They can be RNA, DNA or oligonucleotides of 25 complementary basepairs to the target RNA. In case of RNA probes, invitro produced probes are used as invivo probes can denature due to the rigorous washing. In case of cDNA, the probes are labeled with radioactive isotopes, alkaline phosphatase or horseradish peroxidase in case of chemiluminescence.