BLOTTING TECHNIQUE

Blotting is the molecular biology technique that allows molecular biologists to transfer the products of a PCR reaction (particularly nucleic acids and protein molecules) from a gel strip onto a matrix or specialized chemically reactive paper for further separation and/or studies. It is the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane.

Blotting techniques are important molecular biology procedures, and they have been used in recombinant DNA technology since the early 1970s. In blotting technique, separated nucleic acid molecules and proteins are immediately transferred from the agarose gel matrix onto a different matrix (usually made up of nitrocellulose paper) where they are made readily accessible and identified.

Blotting technique generally combine the electrophoresis technique and some immunological technique for the separation of proteins and nucleic acid molecules. In blotting technique, separated unstained nucleic acid fragments (DNA and RNA) and proteins are transferred onto a nitrocellulose membrane after been stacked in a blotting sandwich pattern- which allows the deposition of the separated nucleic acid molecules or protein fragments onto the membrane by capillary action as the buffer is drawn through the gel (Figure 1).

There are three types of blotting techniques employed in molecular biology, and they include southern blotting, northern blotting and western blotting.

• Southern blotting technique is used to identify the DNA sequence (gene) of interest in a biological sample; it allow investigators to determine the molecular weight of a restriction fragment and to measure its relative amounts in different samples.

• Northern blotting technique is used to identify the RNA sequence (i.e. mRNA) of interest in a biological sample; and it allow investigators to determine the molecular weight of an mRNA and to measure the relative amounts of the mRNA present in different samples.

• Western blotting technique is used to identify specific antibody proteins that have been separated from one another in a sample; and it allow investigators to determine the molecular weight of a protein molecule and to measure the relative amounts of the protein present in different samples.

The blotting sandwich (shown in Figure 1) provides a more stable and permanent platform for the transfer of proteins and nucleic acid fragments to a nitrocellulose membrane. Gel slab (after electrophoresis) is moved into a buffer solution; and a nitrocellulose membrane or paper is placed on top of the gel; after which, layers of specialized papers (which draws the buffer solution) are stacked onto it like a sandwich. Denatured fragments of nucleic acids (DNA and RNA) and proteins are carried along into the membrane filter.

References

Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson J.D (2002). The molecular Biology of the Cell. Fourth edition. New York, Garland, USA.

Chen I and Dubnau D (2004). DNA uptake during bacterial transformation. Nat. Rev. Microbiol. 2 (3): 241–249.

Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.

Dale J (2003). Molecular genetics of bacteria. Jeremy W. Dale and Simon Park (4^th eds.). John Wiley & Sons Ltd, West Sussex, UK. Pp. 312-313.

Das H.K (2010). Textbook of Biotechnology. Fourth edition. Wiley edition. Wiley India Pvt, Ltd, New Delhi, India.

Lewis R (2004). Human Genetics: Concepts and Applications. Sixth edition. McGraw Hill Publishers, USA.

Lodish H, Berk A, Matsudaira P, Kaiser C.A, Kreiger M, Scott M.P, Zipursky S.L and Darnell J (2004). Molecular Cell Biology. Fifth edition. Scientific American Books, Freeman, New York, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12^th edition. Pearson Benjamin Cummings Inc, USA.

McPherson M and Moller S (2002). PCR: The Basics. 2^nd edition. Taylor and Francis Group. New York, USA.

Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.

Synder L, Peters J.E, Henkin T.M and Champness W (2013). Molecular Genetics of Bacteria. Fourth edition. American Society of Microbiology Press, USA.

Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.