Blotting Technology's Permanence Is Assured As Its Applications In The Laboratory Flourish

Nearly everyone within earshot of a television or radio has become familiar with the molecular biology term "Southern blotting," thanks to the publicity attached to some highly publicized forensic cases such as the O.J. Simpson murder trial. Southern blotting is the technique that may ultimately determine Simpson's fate, by matching--or not matching--his genetic profile obtained through DNA fingerprinting with the DNA pattern derived from blood found at the scene of the murder of his wife, Nicole, and at his Brentwood, Calif., home. Advanced Biotechnologies Inc. Columbia, MD Advanced Genetic Technologies Corp San Diego, CA Ahlstrom Filtration Inc. Mount Holly Springs, PA American Bioanalytical Inc. Natick, MA Boehringer Mannheim Corp. Indianapolis, IN P.J. Cobert Associates St. Louis, MO Crescent Chemical Inc. Hauppauge, NY DuPont NEN Boston, MA Fluka Chemical Corp. Ronkonkoma, NY Gallard-Schlesinger Industries Inc. Carle Place, NY Hoefer Scientific San Francisco, CA ICN Biomedicals Costa Mesa, CA Innotech Biosystems International Lansing, MI Intermountain Scientific Corp. Kaysville, UT Life Technologies Inc. Gaithersburg, MD Micron Separations Inc. Westboro, MA Midwest Scientific St. Louis, MO Millipore Bedford, MA Novex San Diego, CA Oncor Inc. Gaithersburg, MD Pierce Chemical Co. Rockford, IL Sartorius Corp. Bohemia, NY Schleicher & Schuell Inc. Keene, NH Thomas Scientific Swedesboro, NJ VWR Scientific West Chester, PA Whatman Lab Sales Inc. Hillsboro, OR In Southern blotting, DNA fragments separated by gel electrophoresis are transferred to a support membrane made of nitrocellulose, nylon, or some other type of support material. The process is named after the technique's inventor, biochemist Edward M. Southern, Oxford University, England. The paper describing this technique (E. M. Southern, Journal of Molecular Biology, 98:503, 1975) has been explicitly cited more than 25,000 times, according to the Institute for Scientific Information in Philadelphia. Northern blots (the transfer of RNA molecules from a gel to a membrane) and Western blots (the transfer of proteins) were later developments. Once blotted to a membrane, the bound macromoleculescan be analyzed in numerous ways--probed with labeled oligonucleotides to detect specific genes for example, or treated with antibodies to identify a single protein in a cellular extract from among several hundred. All three of these techniques quickly became workhorses in the molecular-biology laboratory and for several other life-sciencesdisciplines, such as developmental biology and evolution. "These types of techniques have basically reached commodity status," explains Keld Sorensen, senior research scientist at Pierce Chemical Corp. in Rockford, Ill. "Blots are generally no longer the focus of a scientist's research, but they are often the essential tools that allow researchers to get on with their studies." Molecular biologists rely on Southern blots in conjunction with Westerns and Northerns to aid in cloning genes and characterizing the gene products. Cell biologists and organismal scientists use blotting assays to study signal transduction or gene expression, for example, or to analyze the events that occur during development. Evolutionary biologists perform Southern blots to study the evolution of conserved genes. "Assays involving blotting are protected by history," comments George Dearden, a technical specialist with Schleicher and Schuell Inc., a company specializing in blotting membranes and equipment in Keene, N.H. "To get results published, you have to show the standard Southern and Northern blot results and prove that the assays are sensitive and repeatable." The scientific concepts governing all of the various forms of blotting are essentially the same, with only the starting material--DNA, RNA, or proteins--as the major difference. The macromolecules separated in a gel or colonies growing on culture media in a petri dish are placed in contact with a suitable support material along with vast quantities of buffer. Capillary action causes the buffer to flow through the gel toward the solid support. The macromolecules in the gel are transported along with the buffer to the support membrane where they bind, producing an exact replica of the gel or petri plate on the membrane. Using a labeled probe, researchers can detect their target and localize it to specific colonies on the plate medium, for instance, or to a specific molecular weight on the original gel. Southern, Western, and Northern blots are all performed in the same general way. In its original and still widely used form, blotting is achieved via capillary transfer. Gels are overlaid with the support membrane and then topped by huge stacks of paper towels or some other absorbent material. The stacks are crowned with a weighted object, usually the lab's largest and heaviest molecular biology handbook, and left overnight. Buffer from below wicks upward through the gel and the membrane, toward the absorbent paper towels above. Variations on this standard theme began to evolve shortly after blotting techniques were developed. Although capillary transfers, which require very little in the way of equipment, remain popular, additions and improvements to the standard blot are gaining ground with researchers who want to decrease the time required to complete an experiment by speeding the movement of macromolecules out of the gel and onto the membrane. Systems for vacuum blotting--in which vacuum pressure literally sucks the macromolecules out of the gel--and electroblotting equipment that operate on a principle similar to electrophoresis are available from a number of companies that specialize in electrophoresis, such as Bio-Rad Laboratories, based in Hercules, Calif., along with companies that supply life-sciences research tools, like Life Technologies of Gaithersburg, Md., and Stratagene Inc. of La Jolla, Calif. New designs based on the standard capillary-transfer technique also increase the efficiency of the method. The Turbo Blotter from Schleicher and Schuell, for example, is a capillary blot system in which blots proceed downward with gravity rather than upward. "The downward blot is a more natural progression of events and the nucleic acids move out of the gel quickly," says Judith Peter, marketing manager at Schleicher and Schuell. "Transferring DNA or protein to a support membrane is absolutely essential, because most applications such as nucleic acid hybridization cannot be carried out in gels," says Richard P. Cunningham, a professor of biology at the State University of New York, Albany. "Hybridization reactions in solution have been performed, but the results lack context. With a blot, the hard-copy results can be analyzed spatially in terms of molecular weight or relative position." "There will be a place for blotting membranes in research laboratories for the foreseeable future," agrees Michael Mansfield, a consulting scientist with Millipore Corp. in Bedford, Mass., which specializes in filtration and separation technologies. "Blotting allows researchers to conduct studies that simply can't be done any other way." One area of blotting that is attracting the attention of numerous scientists in clinical and forensic laboratories as well as in molecular biology labs is RFLP (restriction fragment length polymorphism) analysis. Known also by its more common designations of genetic or DNA fingerprinting, this application has advanced Southern blotting beyond the research lab and into new areas of forensic science and diagnostic testing. "There were inklings that Southerns were on their way out because of the development of new procedures along the lines of PCR that would enhance researchers' abilities to perform DNA analysis without first blotting to a membrane," says Mansfield. "However, the standard RFLP procedures currently in use are the most deeply entrenched right now from a legal standpoint. As other techniques develop they will have to go through the same sort of scrutiny before gaining widespread acceptance within the legal community." To produce a DNA fingerprint, DNA from a sample of blood or a forensic specimen is extracted and treated with a restriction enzyme, which cuts the genomic DNA derived from the cells in the specimen into millions of small fragments. The fragments are separated by gel electrophoresis, and then Southern-blotted to a support membrane. After the bound DNA is denatured by alkaline treatment, a labeled probe is added to the blot. The probe may be specific for regions in the DNA that occur frequently (multi-locus probes) or for sequences found singularly within specific genes (single-locus probes). The bands that appear following the detection steps are compared. Matches in the band patterns indicate that samples came from the same source. A DNA fingerprint made from a tissue sample left at the scene of a crime can identify the perpetrator with virtual certainty. Forensic scientists are not the only group benefiting from the use of Southern blots in genetic fingerprinting. Scientists can study the evolution of genes across species and ecologists can study the parentage of animals in selected niches using RFLP applications. Human geneticists use RFLPs to track diseases through families, with the potential for identifying sequences that can be used as genetic-disease markers. Add these to the large numbers of researchers currently involved in the Human Genome Project and you find lots of individuals interested in the Southern blot. Gene mappers who are sorting through the millions of nucleotide bases that make up the 23 pairs of human chromosomes are developing technologies aimed at speeding up and miniaturizing the DNA-sequencing process. DNA sequence data are already available for a sizable portion of the human genome. "Ultimately, however, all of the data obtained from sequencing will have to be related back to what's really going on in the tissues," Mansfield says. "And that will probably be accomplished using standard Southern blot technology." Developers and users of Western blot technology maintain that it, too, is here to stay. One area in particular that is keeping this market moving forward comes as an outgrowth of work done with HIV. "With HIV, suddenly there was a disease that was approachable only by Western blot," for mass screening purposes, according to Mansfield. "With that model out there, it seems likely that other diagnostic applications will be taking off in the near future." Mansfield notes the appearance of a Western blot screening test within weeks of the recent hantavirus outbreak in the Southwest (K.Y. Kreeger, The Scientist, July 11, 1994, page 14; July 25, 1994, page 15). "The real hurdle now appears to be improving the technology to make it applicable to viral outbreaks in Third World countries, where the worst diseases continue to prevail," he adds. In these and most types of Western blot assays, looking for a single protein in an extract of cells can be likened to a search for a needle in a haystack. Just as employing a magnet in the needle search would quickly localize the needle, using a specific antibody expedites the search for a target protein on a Western blot. Companies such as Pierce Chemical, Upstate Biotechnology Inc. in Saranac Lake, N.Y., and St. Louis-based Sigma Chemical Co. provide antibodies as well as the reagents required to detect antibody binding to scientists in search of a protein. Enzyme-antibody conjugates bearing an isotopic or enzymatic tag, buffers and blocking reagents, and detection systems for visualizing the antibody-bound target are available alone or together in kit form, depending on a user's individual preferences. "Researchers are asking more of manufacturers in terms of higher-quality biochemicals, such as cleaner enzyme-antibody conjugates and better-defined reagents," says Pierce Chemical's Sorensen. "They are also relying more on provided protocols. Rather than working up their own Western blot systems, the trend is toward purchasing kits that supply the reagents along with a protocol in a system that is guaranteed to work." One of the most commonly used applications of Western blotting is immunodetection of a target protein using a labeled antibody. Although there are other methods, such as ELISA (enzyme-linked immunosorbent assay) and immunoprecipitation, that can provide essential information about the presence of a protein in a mixed sample, these procedures lack a feature that many researchers find important. "A Western blot of electrophoretically separated proteins provides spatial resolution that no other method provides," says Sorensen of Pierce Chemicals. "A major reason for performing a Western blot analysis of a cell extract is to first confirm the presence of a protein and then characterize it by molecular weight," SUNY-Albany's Cunningham adds. Sequencing proteins by mass spectroscopy is a recent addition to the Western blot's repertoire of applications. Performing protein analysis by mass spectroscopy is a procedure that requires equipment that is not found in most laboratories because of the expense. Although scientists interested in protein chemistry may not have access to a mass spectrometer, they are likely to have standard blotting equipment available in their labs. "Once the proteins are blotted, the membrane can be transported to someone with a mass spectrometer who can perform the mass analysis," says Mansfield. "This looks like the wave of the future for protein sequencing." As in many other areas of science, blotting technology is moving away from radioactivity. "The trend away from radioactivity is nearly universal," says Sorensen. "Blotting is an area where it has been shown that you can get the same sensitivity without the use of radioactivity." For Southern and Western blot techniques, nonisotopic labeling and detection systems based on chromogenic or chemiluminescent substrates are widely available from suppliers like Boehringer Mannheim Biochemicals of Indianapolis, Madison, Wis.-based Promega Corp., Bio-Rad, Pierce, and many others. Labeling systems for RNA, such as Life Technologies' Nonradioactive RNA Labeling system used in conjunction with their BluGene or PhotoGene detection system, are emerging. Nonisotopic detection systems for Southern and Western blots are in many cases easier to use than isotope-based systems, and they virtually eliminate the complications associated with the disposal of radioactive waste. Sensitivity with nonisotopic detection systems is reportedly as good as the sensitivity achieved with radioisotopes. The backbone of the blot is the support membrane that holds the transferred molecules. Membranes made of nitrocellulose, nylon, polyvinylodine fluoride (PVDF), and even copier paper have occupied different niches within the blotting world. Membrane suppliers such as Scheicher and Schuell and Millipore generally have a selection of membranes available to meet the preferences of individual users whose choice of membrane hinges on the application. For proteins, nitrocellulose appears to be the membrane of choice for blotting because proteins are inherently attracted to the polymer and remain permanently attached. For nucleic acids, which carry a negative charge, PVDF and nylon membranes are more widely used. "There are a lot of different types of membranes available," says Sorensen. "Researchers may try one and find that it works in their application. If they like it, they stick with it." Developing new polymers or chemically treating old ones in new ways are areas under consideration within the blotting membrane industry. "As more polymer chemistries are searched out where the interactions between the biomolecule and the membrane are more than simply an affinity, it should be possible to do things that we could not have been done in membranes in the past," Mansfield observes. An example is studies of small molecules and peptides. These small substances do not stick well to the supports currently available. Changing the qualities of membranes to allow these molecules to attach more securely will open the door to a host of new research applications. According to the manufacturers and users of blotting equipment, there is no reason to view blotting membranes as simply an inert support to which proteins or DNA can be applied. In newer procedures the membrane is involved in the application. For many of the PVDF-based applications, the membrane is used first as a vehicle for purification. Once the macromolecules are anchored on the membrane, biochemical analysis can be performed right on it. This circumvents the need to elute the protein off of a membrane or out of a gel before the chemical analysis can take place. Using membranes, it is possible for researchers to look at reactions between different proteins or between a protein and some ligand such as GTP. To study DNA-binding proteins, for instance, proteins on a blotting membrane can be probed with a fragment of DNA containing a possible recognition sequence (a Southwestern blot). Other small biologically active substances such as signal molecules can also be studied this way. "Activity blots such as the Southwestern blot can be used to look at physiologically relevant reactions," Mansfield explains. "These types of assays are of interest to many researchers, especially in the area of development." Southwestern blots and the potential to perform specialized chemistries right on blotting membranes are indications of blotting's permanence in the lab. "It is unlikely that blotting technology will be completely superceded by another technology," says Mansfield. "Procedures will be changed and different technologies will be allied with it, but blotting will continue to have an important place in research and diagnostic laboratories." Predicts Sorensen: "The future of blotting is evolution, not revolution." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Blotting Technology's Permanence Is Assured As Its Applications In The Laboratory Flourish

Interested in reading more?

Become a Member of