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.
The blotting sandwich 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.
Southern blotting technique is used to identify the DNA sequence (gene) of interest in a biological sample. It allows 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. It allows 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. It allows investigators to determine the molecular weight of a protein molecule and to measure the relative amounts of the protein present in different samples.
SOUTHERN BLOTTING TECHNIQUE
Southern blotting technique is a molecular biology technique in which deoxyribonucleic acid (DNA) is transferred from a gel matrix onto a nitrocellulose matrix or paper. Southern blotting technique was first described by Sir Edward M. Southern in 1975 as a molecular biology technique for identifying specific nucleotide sequence of a piece of DNA. It is generally used to detect specific sequences of DNA molecules from a sample. Southern blotting technique is used to identify structural differences between genomes as well as to study related DNA sequences of a particular genome. It is applied in a variety of molecular biology technique, especially for the detection of gene fragments from a whole DNA band on a gel matrix.
With southern blotting, the actual genetic information that a piece of DNA molecule is carrying can be decoded. Southern blotting incorporates a hybridization process in which specific sequences of nucleotides among many fragments of DNA already separated by gel electrophoresis can be identified specifically. With the help of radioactive or radiolabelled oligonucleotides (i.e. gene probes), specific sequences of DNA can be marked or labeled and identified. Southern blotting technique can be used for paternity testing, in forensic sciences to solve crimes and for personal identification procedures.
STEPS FOR SOUTHERN BLOTTING
Southern blotting is a molecular biology technique that is used to identify structural differences between genomes as well as to study related DNA sequences of a particular genome. The steps of performing southern blotting technique are highlighted in this unit as follows:
- Digest the DNA to be analyzed with specific restriction endonuclease.
- Perform agarose gel electrophoresis (as earlier described) on the digested DNA fragments.
- Neutralize the separated DNA fragments by soaking the gel slab in an alkaline solution such as sodium hydroxide (NaOH). DNA is denatured at this stage.
- The nitrocellulose membrane is exposed to an X-ray film and photographed. This process is known as autoradiography. The resulting X-ray film is known as the autoradiograph (Figure 2) shows hybridized DNA fragments. The radiolabelled DNA probe binds to complementary DNA (cDNA) by forming base pairs with it; and this result to a dsDNA.
- Transfer the gel slab (carrying the fractionated DNA fragments) onto a nitrocellulose paper or nylon membrane by blotting. Practically, the single-stranded DNA are transferred by capillary action to a stack of nitrocellulose paper or membrane
- Place a weight on the stack of nitrocellulose membrane in order to hold the blot in place. The nitrocellulose membrane is an absorbent paper. The patterns of the DNA fragments are transferred onto the nitrocellulose paper via capillary action. DNA fragments are bound to the nitrocellulose paper in positions identical to those on the gel slab.
- Incubate the nitrocellulose paper in a specific gene probe or radiolabelled oligonucleotides for hybridization to take place. Hybridization is the process of forming a double stranded DNA molecule between an ssDNA probe and a target single stranded DNA fragment.
- Unbound probes are removed by washing with distilled water.
NORTHERN BLOTTING TECHNIQUE
Northern blotting technique is used to detect specific sequences of ribonucleic acid (RNA). The protocol for performing northern blotting technique is similar to that of southern blotting technique. Northern blotting technique was first described by James Alwine and colleagues in 1977 as a molecular biology technique for identifying specific nucleotide sequence of a piece of RNA. While southern blotting technique detect specific sequences of DNA, northern blotting technique exclusively detect specific sequences of RNA (particularly mRNA).
STEPS FOR NORTHERN BLOTTING
- The isolated RNA is digested with restriction endonuclease.
- Perform agarose gel electrophoresis (as earlier described) on the digested RNA.
- The RNA samples are separated according to their different sizes on agarose gel electrophoresis technique.
- Hybridize separated RNA in a neutralizing buffer by soaking the gel slab in an alkaline solution (e.g. NaOH).
- Transfer the gel slab (carrying the fractionated RNA fragments) onto a nitrocellulose membrane by blotting. Practically, the single-stranded RNA molecules are transferred by capillary action to a stack of nitrocellulose paper or membrane from the gel slab.
- Place a weight on the stack of nitrocellulose membrane in order to hold the blot in place. The nitrocellulose membrane is an absorbent paper; and thus, the patterns of the RNA fragments are transferred onto the nitrocellulose paper via capillary action. RNA fragments are bound to the nitrocellulose paper in positions identical to those on the gel slab.
- Place the nitrocellulose membrane in a dish containing hybridization buffer with a radioactively labeled probe (specifically designed for the RNA sequence of interest).
- Wash the membrane with distilled water to remove unbound probe.
- Expose the nitrocellulose membrane to an X-ray film, and photograph it by autoradiography. Expression patterns of the RNA sequence of interest in the different samples are compared and analyzed.
WESTERN BLOTTING TECHNIQUE
Western blotting technique (protein immunoblot) is used to identify specific proteins separated according to their sizes in an electrophoresis experiment. Protein immunoblot unlike other blotting techniques such as southern and northern blotting utilizes specific antibodies to identify the protein of interest. It is the transfer of proteins from gel to a matrix or nitrocellulose membrane. Western blotting allows molecular biologists to determine the actual molecular weight of a protein molecule and to measure its relative amounts in different samples. Instead of agarose gel electrophoresis which is usually used for the separation of nucleic acid fragments (particularly those of RNA and DNA), polyacrylamide gel in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique is used to separate the protein fragments prior to the blotting technique. The steps involved in carrying out western blotting or protein immunoblot is highlighted in this unit.
STEPS FOR WESTERN BLOTTING
- Separate the protein sample according to their sizes using SDS-PAGE.
- Transfer the separated proteins from the SDS-PAGE gel to the surface of a nitrocellulose membrane by electroblotting. In electroblotting, electric current is passed through the SDS-PAGE gel, and this transfers the separated proteins onto the nitrocellulose membrane.
- Incubate the separated proteins with a generic protein molecule so that unbound sites of the nitrocellulose paper shall be covered. Addition of generic protein (i.e. protein molecules of known sizes) helps to minimize background signals on the membrane.
- Add specific antibody that is unique to the protein of interest to the protein solution. The antibody (which is usually radioactively labeled) binds to its specific protein molecules on the nitrocellulose membrane. Only the band containing the protein of interest binds to the antibody, thus forming a layer of antibody molecules on the membrane.
- Expose the nitrocellulose membrane to an X-ray film, and photograph it by autoradiography in order to reveal the separated protein bands.
Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.
Das H.K (2010). Textbook of Biotechnology. Fourth edition. Wiley edition. Wiley India Pvt, Ltd, New Delhi, India.
Davis J.M (2002). Basic Cell Culture, A Practical Approach. Oxford University Press, Oxford, UK.
Mather J and Barnes D (1998). Animal cell culture methods, Methods in cell biology. 2rd eds, Academic press, San Diego.
Noguchi P (2003). Risks and benefits of gene therapy. N Engl J Med, 348:193-194.
Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.
Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.