Blotting: Southern, Northern, and Western

SIGNIFICANCE: Blotting is a technique that allows identification of a specific nucleic acid or amino acid sequence even when it is mixed in with all of the other material from a cell. This allows the rapid identification of the changes associated with mutant alleles.

Limitations of Gel Electrophoresis

Using gel electrophoresis to separate proteins and nucleic acids has been an invaluable tool in analyzing living systems. Changes in these molecules—such as a mobility shift in a mutant protein or the change in the size of a plasmid that has received a DNA insert—can be easily detected using this technique. However, the ability to differentiate between types of molecules is quite limited. An extract of red blood cell proteins run through an acrylamide gel might show one major band for that can be discerned from the many other proteins in the cell. However, the hundreds of different proteins that might be produced in a liver extract will produce a tight ladder of bands that are impossible to tell apart.

The situation can be even worse with DNA. A restriction enzyme digest of a plasmid or simple virus might yield fewer than six pieces of DNA that could be easily separated on an gel. If one were to digest the total genomic DNA of even a simple organism, such as Escherichia coli, with a typical such as EcoRI, the result would be a thousand bands of numerous sizes (4 × 10⁶ base pairs of DNA, since EcoRI recognizes a six-base-pair site, which should occur, on average, every 4⁶ or 4,096 bp). After separation on a gel, the result would be a smear with no individual bands visible. Working with an even more complex genome, such as the human genome, would result in millions of bands. The only way to study a specific protein or sequence using gel electrophoresis, therefore, would be to find a way to label it specifically so that it could be differentiated from the general background.

Basic Blotting Techniques

In 1975, Ed Southern developed a method that allowed the detection of specific DNA sequences after they had been separated by agarose gel electrophoresis. What makes a piece of DNA unique is the sequence of the nucleotides. This is most efficiently detected by the of the strand. This can only occur if the two strands are separated into single strands. Therefore, the first step is to soak the agarose gel in a strong base, such as 1 molar sodium hydroxide, and high salt, which stabilizes the single-stranded form. The base is then neutralized with a strong buffer, such as tris-hydrochloride, again in high salt. The DNA can now be analyzed by its ability to hybridize to a radioactive piece of single-stranded DNA. Since this radioactive DNA can “explore” the different sequences to find the one matching sequence, it is also known as a (an instrument or device that can be used to explore and send back information).

Although the agarose is porous, it would be very slow and inefficient to try to perfuse the gel with radioactive probe and then remove the pieces that did not hybridize. Southern realized that he needed to move the DNA to a thin material to be able to probe it efficiently. The material chosen was nitrocellulose, consisting of a variant of paper (cellulose) with reactive nitro groups attached. The treated gel is placed on a sponge soaked with a high-salt solution. The nitrocellulose sheet is placed onto the gel and then a stack of dry paper towels is laid on top. The salt solution is drawn through the gel to the dry towels and carries the DNA from the gel up into the paper. The positively charged nitro groups on the nitrocellulose stick to the negatively charged DNA, thereby holding the DNA in a pattern matching the band locations in the gel. The nitrocellulose is removed from the gel and baked at 80 degrees Celsius (176 degrees Fahrenheit) or treated with ultraviolet light, both of which covalently cross-link the DNA to the paper, locking it in its position. The filter is soaked in a solution that promotes reassociation of single-stranded DNA, and radioactive, single-stranded DNA is added. Since the added DNA could stick nonspecifically to the nitrocellulose, the paper is pretreated with unrelated DNA, such as sheared salmon DNA, which will bind the available nitro groups but not react with the probe.

A large molar excess of probe must be used to drive the hybridization reaction (reforming the “hybrid” of two matching antiparallel strands together), which means that it is necessary to make sure that enough probe is available in the solution to randomly run into the correct sequence on the paper and reanneal to it. The hybridization is done at an elevated temperature—often 50–65 degrees Celsius (122–149 degrees Fahrenheit), so that only strands that match exactly will stay together and those with short, random matches will come apart. After overnight hybridization, the paper is washed multiple times with a detergent-salt solution, which removes the DNA that did not hybridize. The paper is placed against a piece of X-ray film, and the radioactive emissions from the probe darken the film next to them. When the film is developed, a pattern of bands appears that corresponds to the position in the original gel of the DNA piece for which the researcher was probing.

Expanded Techniques to Study RNA and Proteins

The basic method of blotting has been expanded to include the study of RNA and proteins. James Alwine developed a very similar method to transfer messenger RNA (mRNA) that had been separated on an agarose gel. Since the started as single-stranded, there was no need to treat the gel with denaturant. However, to block the formation of internal double-stranded regions, which could alter the migration during electrophoresis, the gel contained an organic solvent. Other than that, the two methods are very similar. Although the DNA transfer system was named the in honor of Ed Southern, Alwine decided to defer the credit and called his system the to indicate that it was related but in a different direction.

Similarly, when W. N. Burnette developed a system for transferring and detecting specific proteins, he named the system Western blotting. This system of naming has been expanded: A technique for detecting viral DNA in tree leaves was named the Midwestern blot and a variant of the Northern blot developed in Israel was named the Middle Eastern blot.

Since proteins are generally smaller than DNA fragments, they are usually separated on polyacrylamide gels, which have a much smaller pore size than agarose gels. It is therefore necessary to use electrical current to pull the proteins out of the gel. The nitrocellulose is pressed onto the gel with a porous plastic pad. The gel is then placed in a buffer tank and electrodes are placed on either side. When a voltage is applied, the current that flows through the gel carries the proteins onto the nitrocellulose. The reactive side chains of the nitrocellulose also bind proteins very effectively, so they are all retained on the paper. The specific probe used to detect a protein is an that either can be radioactively labeled or can have an enzymatic side chain attached, which will produce light or a colored dye when the appropriate chemicals are added. Since the antibody is a protein, it could also stick nonspecifically to the paper, so the blot is pretreated with a general protein such as serum albumin before the antibody is added.

Blotting in Genetic Analysis

The ability to detect individual molecules in a large background has been very important for genetic analyses. For instance, restriction fragment length polymorphism (RFLP) analysis is a method that uses the change in the size of a DNA fragment in the genome, generated by restriction enzyme digestion as a genetic marker. The isolation of many disease genes, including the one causing Huntington’s disease, depended on RFLP mapping to localize the gene. It would not be possible to detect the changes in a single DNA fragment out of the millions generated by digesting the human genome without having the Southern blot to pick out the correct piece. Many other mutations that change a specific region of DNA—such as deletions, inversions, and duplications—are often detected by changes in a Southern blot pattern. The sensitivity of hybridization can be tuned to a level where probes that differ by only a single will not attach efficiently. This allows the rapid identification of the positions of point mutations. When polymerase chain reaction (PCR) is used to amplify DNA from a crime scene or to detect human immunodeficiency virus (HIV) in the bloodstream, the presence of DNA pieces on a gel is not sufficient proof that the correct DNA has been found. The DNA must be blotted and probed with the expected sequence to confirm that it is the correct piece.

Northern blot analysis allows scientists to see how mRNA is altered in different mutants. Northern blots can indicate if a mutant allele is no longer transcribed or if the level of mRNA produced has been dramatically decreased or increased. Deletions or insertions will also show up as shortened or lengthened messages. Alternative splicing can be seen as multiple bands on a Northern blot that hybridize to the same probe. As of 2022, researchers still used Northern blotting to validate data obtained from high-throughput whole transcriptome-based methods. However, researchers had begun modifying the technique to make it better able to detect mRNAs in total RNA. As of 2023, Western blotting was no longer recommended by the Centers for Disease Control and Prevention (CDC) because other tests were more reliable and gave a faster response.

Future Directions

Blotting techniques are the most generally efficient methods for detecting specific proteins or nucleic acids. Most improvements in the past years have been aimed at speeding up the transfer process using vacuums or pressure or the hybridization process by changing the conditions. The next step will be developing silicon chips that can interact with specific nucleic acid or amino acid sequences and produce an electrical output when they “hybridize” with the correct sequence. This will diminish the time required to confirm a sequence from several hours to minutes.

Key terms

• blottingthe transfer of nucleic acids or proteins separated by gel electrophoresis onto a filter paper, which allows access by molecules that will interact with only one specific sequence
• hybridizationincubation of a target sequence with an identifying probe, which allows the formation of annealed hybrids
• Northern blota blot designed to detect messenger RNA
• probea nucleic acid sequence or antibody that can attach to a specific DNA or RNA sequence or protein; the probes are often labeled with radioactive compounds or enzymes so their position can be determined
• Southern blota blot designed to detect specific DNA fragments
• Western blota blot that uses antibodies to detect specific proteins

Bibliography

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"A Guide to 10 Blotting Types." Boster Bio, 15 Feb. 2024, www.bosterbio.com/blog/post/a-guide-to-10-blotting-types. Accessed 9 Sept. 2024.

Hughes, Alex J., et al. "Single-Cell Western Blotting." Nature Methods 11.7 (2014): 749–55. Print.

Kurien, Biji T., and R. Hal Scofield. Protein Blotting and Detection: Methods and Protocols. New York: Humana, 2009. Print.

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Mishra, Manish, Shuchita Tiwari, and Aldrin V. Gomes. "Protein Purification and Analysis: Next Generation Western Blotting Techniques." Expert Review of Proteomics, vol. 14, no. 11, 13 Oct. 2017, pp. 1037–1053, doi: 10.1080/14789450.2017.1388167. Accessed 9 Sept. 2024.

Nicholas, M. W., and Kelly Nelson. "North, South, or East? Blotting Techniques." Journal of Investigative Dermatology 133.7 (2013): 10. Print.

Southern, E. M. “Detection of Specific Sequences among DNA Fragments Separated by Gel Electrophoresis.” Journal of Molecular Biology 98.3 (1975): 503–17. Print.

Walker, John M., and Ralph Rapley, eds. Molecular Biomethods Handbook. 2nd ed. Totowa: Humana, 2008. Print.