Nonradioactive Probes Protect Scientists And Environment

For many years, geneticists determined the genetic makeup of organisms by examining the physical characteristics of their offspring. But with the discovery of the structure of the DNA double helix, first published by James D. Watson and Francis H.C. Crick in 1953 (Nature, 171:964-7), the science of genetics was forever changed. Scientists soon developed techniques to study the genetic message found in all living cells at the molecular level. One of most important of these methods was the use of nucleic acid hybridization in the isolation and characterization of specific DNA sequences. Nucleic acid hybridization, first described in a classic paper by Julius Marmur and Paul Doty in 1961 (Journal of Molecular Biology, 3:595-617), is based on the finding that two separate but complementary strands of DNA will reassociate under specific conditions to form a double helix. By the late 1960s, the principles of nucleic acid hybridization had been used to pinpoint the location of specific sequences of DNA within the nucleus of cells by fixing the cells to a microscope slide and "probing" the cells with a small fragment of DNA. The discovery of restriction enzymes and the advent of recombinant DNA technology in the 1970s paved the way for new hybridization applications, including colony hybridization, analysis of DNA sequences from genomic DNA, and, most recently, DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis. All of these different applications center on the simple principle that two DNA strands with complementary nucleotide sequences will combine with one another under the appropriate physical conditions, such as temperature and pH. For the strands to combine, or hybridize, they must share a common, but not necessarily identical, DNA sequence. The degree of complementarity between the two strands determines the stability of the newly formed hybrid. Traditionally, scientists perform nucleic acid hybridization experiments with a "probe" labeled by the enzymatic incorporation of a radioisotope into a newly synthesized strand of DNA (or RNA). Researchers use the labeled probe to search for homologous nucleotide sequences in nylon or nitrocellulose filter-bound "target" DNA. Investigators detect any probe-target DNA hybrids by allowing the radioactive hybrids to expose X-ray film (autoradiography), or by directly quantitating the radioactivity using a scintillation counter. The radioisotope 32P is often used in such experiments, because the high-energy á particles emitted by this isotope provide a very strong, easily detected signal. Yet the advantages of using 32P or other radioisotopes in these types of experiments are sometimes overshadowed by the hazards associated with working with radioactivity - especially the risk of exposure to radiation at mutagenic levels. As a result, nucleic acid hybridization experiments using a radiolabeled probe must be carried out under strictly controlled conditions. These conditions require the use of expensive shielding equipment and stringent quality control methods. The radioactive waste generated from these types of experiments has also presented a problem to researchers. For example, the half-life, or the time it takes a radioisotope to decay, of 35S is 87.4 days. It takes approximately eight half-lives, which translates into many years, before waste generated from experiments using 35S can be safely discarded. In the meantime, the radioactive waste must be stored, an expensive and potentially dangerous practice. During the early 1980s, nonradioactive DNA labeling and detection systems emerged as an alternative to the use of radioactivity in nucleic acid hybridization experiments. There are many advantages associated with the use of these systems. The most obvious is the complete absence of radiation biohazard. The reagents used to label DNA enzymatically or otherwise are nonmutagenic and require no special expertise or equipment. Probes tagged with a nonradioactive label also tend to be much more stable than those labeled with radioisotopes, which decay over time. Since autoradiography is not required, the detection steps are simplified, and require only inexpensive reagents, resulting in a savings of money and time for the researcher. Recent improvements in nonradioactive technology have given this new method a sensitivity equal to that of more conventional techniques that use radioisotopes. With the increased emphasis in the 1990s on the need to protect both the environment and the health of researchers, the demand for nonradioactive systems has grown. Many companies have recognized and responded to these concerns. The Indianapolis-based Boehringer Mannheim Biochemicals, for example, markets the Genius nonradioactive DNA labeling and detection system. The Genius system uses a digoxigenin-labeled nucleotide, digoxigenin-dUTP, enzymatically incorporated into probe DNA. After hybridization to target DNA sequences, researchers can detect the labeled hybrids by using antidigoxigenin antibodies linked to the enzyme alkaline phosphatase. When substrate is added, bound enzyme catalyzes a color reaction, resulting in the formation of a colored precipitate. The specificity of the antibody for digoxigenin virtually eliminates the problem of nonspecific background on the membrane. According to the company, the Genius system is sensitive enough to detect as little as 0.1 picogram of target DNA, and can be used in place of radiolabeled probes in applications that previously required radioactivity and autoradiography. This kit, which contains all of the reagents necessary to label and detect probe DNA, costs $330. Two companies, FMC BioProd-ucts (based in Rockland, Maine) and Sigma Chemical Company (in St. Louis), have developed systems in which probe DNA is labeled by sulfonating specific cytosine residues following a simple chemical reaction. A mouse monoclonal antibody, specific for sulfonated DNA, binds to the modified probe. A second, enzyme-conjugated antimouse antibody then tags the hybrids with enzyme. Researchers detect hybri- dization by following an enzyme-catalyzed colorimetric reaction. The antibody binds directly to the sulfonated DNA, and not to the membrane; therefore, nonspecific background is eliminated. The ChemiProbe kit from FMC BioProducts is available as a complete labeling and detection system for $265. According to the company, this kit can detect less than 1 picogram of DNA. The SulfoProbe system, Sigma's offering, is also said to have sensitivities in the picogram range. This kit can be purchased as a complete system for $246.50. One of the problems associated with immuno-enzymatic detection systems has been that of obtaining a stable, hard-copy record of the experiment. Difficulties in recording investigations arise because the colored precipitate left on membranes after enzymatic detection can fade with time. A novel approach to this problem has been the development of chemiluminescent substrates for use with enzyme-labeled probes. Tropix Inc., based in Bedford, Mass., markets the Southern-Light system, which uses adamantyl-1,2-dioxetane phosphate (AMPPD) as substrate for the enzyme alkaline phosphatase. AMPPD spontaneously chemiluminesces upon reaction with alkaline phosphatase. The light emissions from this reaction can be used to expose X-ray film in a manner similar to autoradiography. This provides researchers with a permanent hard-copy result of their nucleic acid hybridization experiments. The use of chemiluminescent substrates can be coupled to any number of labeling systems, including bio-tinylated or digoxigenin-labeled probes. The Southern-Light Plus kit, a complete labeling and detection system, uses a biotin label with a streptavidin-alkaline phosphatase conjugate, along with the AMPPD substrate for detection of hybrids. The complete kit can be purchased for $325. Nonradioactive DNA labeling and detection systems offer scientists an affordable, sensitive, and safe approach to performing a wide range of nucleic acid hybridization experiments. With radioactive waste disposal rapidly becoming a major environmental issue, nonradioisotopic systems are expected to increase in popularity in the future. Holly Ahern teaches immunology and cell and molecular biology at the State University of New York, Albany.

Nonradioactive Probes Protect Scientists And Environment

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