DNA Probes Yield Expanded Research And Clinical Uses

Author: Ricki Lewis Date:January 10, 1994, pp.17 The following vendors develop and/or market DNA probes for a variety of research and diagnostic purposes. For information about specific services, products, and prices, please contact these companies directly. Lofstrand Laboratories 7961 Cessna Ave. Gaithersburg, Md. 20879 (310) 330-0111 Fax: (301) 948-9214 Microbiological Associates Inc. Life Sciences Center 9900 Blackwell Rd. Rockville, Md. 20850 (301) 738-1000 Fax: (301) 738-1036 Microprobe Corp. 1725 220th St., S.E. Bothell, Wash. 98021 (206) 485-8566 Midland Certified Reagent Co. 3112-A W. Cuthbert Ave. Midland, Texas 79701 (800) 247-8766 Fax: (915) 694-2387 National Biosciences Inc. 3650 Annapolis Lane Suite 140 Plymouth, Minn. 55447-5434 (800) 747-4362 Fax: (612) 550-9625 New England Biolabs Inc. 32 Tozer Rd. Beverly, Mass. 01915 (508) 927-5054, Ext. 309 Fax: (508) 921-1350 Oncogene Science 80 Rogers St. Cambridge, Mass. 02142 (617) 492-7289 Fax: (617) 492-8438 Oncor Inc. 209 Perry Parkway Gaithersburg, Md. 20877 (301) 963-3500 Fax: (301) 926-6129 Perkin-Elmer Corp. 761 Main Ave. Mail Station 105 Norwalk, Conn. 06859-0105 (800) 762-4000 Fax: (203) 762-6000 Promega Corp. 2800 Woods Hollow Rd. Madison, Wis. 53711 (608) 274-4330 Fax: (608) 273-6967 R & D Systems 614 McKinley Place, N.E. Minneapolis, Minn. 55413 (612) 379-2956 Fax: (800) 328-2400 Research Genetics 2130 Memorial Parkway South Huntsville, Ala. 35801 (800) 533-4363 Fax: (205) 536-9016 Roche Molecular Systems Div. of Hoffmann-La Roche 1145 Atlantic Ave. Alameda, Calif. 94507 (510) 814-2853 Sigma Chemical Co. 3050 Spruce St. St. Louis, Mo. 63103 (800) 325-3010 Fax: (314) 771-5750 Stratagene Corp. 11099 N. Torrey Pines Rd. La Jolla, Calif. 92037 (619) 535-5400 Fax: (619) 558-0947 Synthecell/Vega Biomolecules 7101 River Wood Dr. Columbia, Md. 21046 (800) 336-7455 Fax: (410) 381-4965 Synthetic Genetics 3347 Industrial Ct., Suite A San Diego, Calif. 92121 (619) 793-2661 Fax: (619) 793-2666 StressGen Biotechnologies Corp. 4243 Glandford Ave. Suite 120 Victoria, B.C. V8Z 4B9 Canada (604) 744-2811 Fax: (604) 744-2877 United States Biochemical P.O. Box 22400 Cleveland, Ohio 44122 (216) 765-5000 Fax: (800) 535-0898 Zymed Laboratories Inc. 458 Carlton Ct. South San Francisco, Calif. 94080 (415) 871-4494 Fax: (415) 871-4499 The following vendors develop and/or market DNA probes for a variety of research and diagnostic purposes. For information about specific services, products, and prices, please contact these companies directly. Accurate Chemical Scientific 300 Shames Dr. Westbury, N.Y. 11590 (800) 645-3569 Fax: (516) 997-4948 Advanced Biotechnologies Inc. 9108 Guilford Rd. Columbia, Md. 21046 (301) 470-3220 Fax: (301) 497-9773 American Type Culture Collection 10801 University Blvd. Manassas, VA 20110-2209 (USA) (703) 365-2700 www@atcc.org http://www.atcc.org Biomeda Corp. P.O. Box 8045 Foster City, Calif. 94404 (415) 341-8787 Fax: (415) 341-2299 BioServe Biotechnologies Ltd. 1050 West St. Laurel, Md. 20707 (301) 470-3362 Fax: (301) 470-2333 Bio-Synthesis Inc. 612 East Main St. Lewisville, Texas 75067-0028 (800) 227-0627 Fax: (214) 420-0442 Boehringer Mannheim Biochemicals 9115 Hague Rd. Indianapolis, Ind. 46250 (800) 428-5433 Fax: (317) 845-2000 Calbiochem-Novabiochem Corp. 10394 Pacific Center Ct. San Diego, Calif. 92121 (800) 854-3417 Fax: (619) 453-3552 Cambridge Research Biochemical Fairfax Research Center Wilmington, Del. 19897 (800) 327-0125 Fax: (302) 886-5937 Cayman Chemical Co. 690 KMS Place Ann Arbor, Mich. 48108 (313) 662-6756 Fax: (313) 662-6896 Cellular Products Inc. 872 Maine St. Buffalo, N.Y. 14202 (716) 882-0920 Fax: (716) 882-0959 DAKO Corp. 6392 Via Real Carpinteria, Calif. 93013 (800) 424-0021 Fax: (805) 566-6688 DuPont Biotechnology Systems Barley Mill Plaza P22-2278 Wilmington, Del. 19898 (302) 992-4785 FMC Bio Support Material Group 200 East Randolph Chicago, Ill. 60601 (312) 861-5900 Genemed Biotechnologies Inc. 458 Carlton Ct. Suite B South San Francisco, Calif. 94080 (800) 344-5337 Fax: (415) 952-0447 Gene-Trak Systems/ Betagen/Imagenetics 31 New York Ave. Framingham, Mass. 01701 (508) 872-3113 Fax: (508) 879-6462 Genosys Biotechnologies Inc. 8701-A New Trails Dr. The Woodlands, Texas 77381-4241 (713) 363-3693 Fax: (713) 363-2212 Gen-Probe Inc. 9880 Campus Point Dr. San Diego, Calif. 92121 (619) 546-8000 Fax: (619) 452-5848 Imagenetics 150 W. Warrenville Rd. Mail Code F-2 Naperville, Ill. 60563-8460 (708) 420-5875 Fax: (708) 420-3845 Integrated Genetics One Mountain Rd. Framingham, Mass. 01701 (800) 255-7357 Life Technologies 8451 Helgerman Ct. Gaithersburg, Md. 20884-9980 (301) 840-4150 As the growing numbers of molecular biologists well know, their art and laboratory craft depend on the extrusion of information residing in nucleic acid sequences. It follows that the tool that's come to be known as the DNA probe--a piece of single-stranded DNA that seeks out its complementary sequence in a biological sample--has become increasingly important to them. The capabilities of probes have expanded rapidly in the past few years, and they have become easier to use. These developments have benefited not only basic researchers, but also those engaged in specifically directed investigations-- in diagnostics, forensics, and epidemiology, for example. The technology's applications continue to increase, and DNA probes are now "well entrenched" in molecular biology labs, according to John Sninsky, senior director of research at Roche Molecular Systems, Alameda, Calif. In the research community, he says, investigators are being stimulated to use the probes in more and more "wild and wonderful ways." A DNA probe may be a chain of as few as 15 nucleotide bases, the molecular building blocks of DNA and RNA, or it may stretch to thousands of such bases. The usefulness of the probe technology, researchers say, comes from the inherent tendency of these single-stranded nucleic acids to seek and hybridize to their complementary sequences, following the base-pairing rules that guide the formation of the DNA double helix--adenine with thymine, and cytosine with guanine. A list of the methodologies dependent on DNA probe technology reads like a table of contents in a molecular biology text: Southern blotting (to detect other DNAs), Northern blotting (to detect RNA, measuring gene expression), colony and plaque hybridization (to spot bacteria and viruses), in situ hybridization (to highlight DNA sequences directly in cells), footprinting (identifying the sites where certain proteins bind DNA), mapping genes, and sequencing genes. Even in the case of the polymerase chain reaction (PCR), the widely used lab process that won its inventor Kary Mullis a 1993 Nobel Prize, the primers used--short pieces of DNA--are essentially DNA probes bracketing sequences to be amplified. "DNA probes and PCR are inextricably linked at the hip," says Sninsky. Clinical Success, Research Staple DNA probes are farther from the public eye than some other biotechnologies, such as cloning human embryos or genetically engineering tomatoes. They are nonetheless very much in the news, especially as diagnostic tools. They were used, for instance, to help track down the sources of bacterial contamination in hamburgers served by a fast- food chain that caused illness last spring, and they have been used to analyze contaminated municipal water supplies. DNA probes also helped researchers investigating the hantavirus pulmonary disease that struck the southwestern United States last year. "They identified the virus initially using serology, but they quickly used DNA probes to identify the group of viruses to which it belonged," says Sninsky. The information showed that the condition had indeed been seen before. Sninsky says that this capability of DNA probes to precisely identify a pathogen, even the particular strain of that pathogen present in a given patient, is a critical one. With both reemergent tuberculosis and AIDS, for example, the disease-causing targets of potential therapies are constantly changing, mutating to present new characteristics to researchers and physicians. "Mutations are responsible for microbial resistance to drugs," Sninsky explains. "If you don't detect a drug- resistant pathogen before you treat the patient, you could be exposing him or her to side effects without [a corresponding] benefit." DNA probes were one of the first biotechnologies to successfully negotiate the passage from the laboratory to the clinic. One reason is that, compared with traditional microbiological detection tests, DNA probes generally offer both greater specificity and higher sensitivity. Also, the costs of most commercially available probe systems for specific targets range between about $150 and $400, making them affordable for many laboratories. With standard tests, "there's a limitation in certain patients, where there is not enough antigen or antibody detected," according to Larry A. Risen, marketing manager at San Diego-based Gen-Probe Inc. "But with DNA probes, you can amplify material or use kinetics" to yield a diagnosis, Risen says. Gen-Probe has focused its efforts on developing and marketing DNA probes targeted to common infectious disorders, such as those caused by streptococcus, enterococcus, Hemophilus influenzae, and several sexually transmitted diseases. 'A new probe-based test from Gen-Probe may help clinicians in their efforts to confront reemergent, drug-resistant tuberculosis. The probe is just entering trials in the U.S., having shown in European trials that it can greatly speed diagnosis over traditional culturing methods. Called the Amplified Mycobacterium tuberculosis Direct Test (MTD), the probe is capable of identifying the strain of tuberculosis present in about five hours, instead of up to eight weeks with the culturing methods, helping physicians prescribe appropriate treatments sooner. "The test represents a breakthrough in adapting [this] technology to routine clinical use," said Mathew Longiaru, Gen-Probe's director of research and development, in a statement. "Designed specifically for the clinical laboratory, the test is not as burdensome as some complicated research methods." Some companies focus on probes for research purposes, although they also may offer one or two for diagnostic uses. For example, Oncogene Science of Cambridge, Mass., offers research probes for a wide range of human genes, including interleukins, interferons, colony stimulating factors, myosin, tubulin, and oncogenes. The Food and Drug Administration also has cleared for marketing the company's Trans-Probe-1, which targets the site where pieces of chromosomes 9 and 22 join in 90 percent of patients who have chronic myelogenous leukemia. The classic test to spot this so-called Philadelphia chromosome, discovered in 1959 by Peter C. Nowell and David A. Hungerford (Science, 132:1497, 1960), is to prepare a chart of all of the chromosomes, then search for the unusual joining--a much more painstaking task. Similarly, most products from Oncor Inc. of Gaithersburg, Md., whose specialty is in situ hybridization, are for research use only, but the company does have FDA approval for an in vitro diagnostic kit to detect rearranged chromosomes in certain white blood cell cancers. Many DNA probes are available for research or investigational uses, but with clinical applications clearly in mind. GeneMed Biotechnologies, for example, offers probes to the human genes for beta globin, the cystic fibrosis transmembrane regulator, and key immune system proteins. As a group, these probes target inherited disorders. The company also markets infectious disease probes to the viruses causing hepatitis, herpes, and rubella, as well as the microbe that causes chlamydia. Straddling the research-clinical line even more closely are the companies using FISH (fluorescence in situ hybridization) technology to speed fetal chromosome checks. The In-Sight system from Integrated Genetics of Framingham, Mass., highlights chromosomes 13, 18, 21, X, and Y--those most likely to be present in extra copies in fetuses--by using three to five DNA probes per chromosome. They offer this service on an investigational basis, as an adjunct to the conventional approach, which requires a week to culture cells from amniotic fluid. Brian Ward, director of the cytogenetics lab at Integrated Genetics, says that FDA approval for In-Sight is not required. FISH, he says, "is called `home brew technology.' FDA will only certify drugs and medical devices that are marketed and sold. It doesn't regulate services." He notes that states, however, may regulate such services. Integrated Genetics' concentration on the chromosomal anomalies most commonly seen in fetuses reflects its clinical bent. But FISH technology also can distinguish among all 24 types of human chromosomes, making it useful in many research areas and in detecting rare or highly specific abnormalities. "It is the same technology, but using different sequences and different applications," says Ward. Researchers at Lawrence Livermore Laboratory in Livermore, Calif., for example, have developed what are called "whole-chromosome paints," which target repetitive DNA sequences. "These light up each chromosome from one end to the other. In contrast, ours are specific, brilliant, and intense, a small pinpoint of light in a nucleus," Ward says. Oncor's FISH system targets unique DNA sequences and currently identifies 19 human chromosomes. Oncor's newest probes detect genes currently at the forefront of genetic research, including those behind the Prader-Willi/Angelman syndromes, in which a small deletion in chromosome 15 causes differing symptoms depending upon which parent transmits it; and XIST, the site on the X chromosome that triggers one X to be shut off in cells of female mammals. They also have probes to telomeres, the tips of chromosomes that are important to their stability and are being intensively studied. As a research tool, DNA probes are the latest in a long series of techniques to isolate and describe biological functions by labelling and tracking various biological molecules. Probes can carry different kinds of additional chemical marker units that allow researchers to locate and quantify them and their targets. Although biologists and chemists have attempted to unravel various metabolic reactions since early this century, it wasn't until World War II that research in nuclear physics made radioactive materials readily available. This, in turn, allowed radiolabeling of biomolecules, and this led to new kinds of biological experiments. Radioactively labeled isotopes can be detected through their ability to expose a photographic emulsion on film, or by the alteration of the density of DNA incorporating them. Many of the key experiments that laid the groundwork for molecular biology followed labeled DNA, for example. Often an isotope of phosphorus, 32P, was used, which labels the sugar-phosphate backbone of the molecule. The fact that the genetic material in a cell is DNA and not protein was discerned in the so-called blender experiments of Alfred Hershey and Martha Chase in 1950. They tracked and distinguished DNA by labeling phosphorus, and protein by labeling sulfur (A. Hershey, et al., Journal of General Physiology, 36:39-56, 1952). Matthew Meselson and Franklin Stahl later revealed the semi-conservative mode of DNA replication by differentially labeling nitrogen and following the densities of daughter DNA strands (M. Meselson, et al., Proceedings of the National Academy of Sciences, 44:671-682, 1958). Using radioactive isotopes has obvious drawbacks, however. The beta particles emitted by 32P are so energetic that the signal on photographic film can be too fuzzy to be meaningful. Richard Rhodes, technical service representative at Promega Corp. of Madison, Wis., claims that his company's LIGHTSMITH I System, which uses chemiluminescence to label DNA, is much easier to use than traditional 32P labeling. LIGHTSMITH is a direct, nonisotopic alternative to radioactive labeling, which means that the chemical that generates the signal is conjugated directly to the probe. Specifically, alkaline phosphatase is hooked to the probe. This enzyme catalyzes removal of a phosphate from a proprietary chemiluminescent compound. The resulting light emission marks the probe's presence. "You don't have to deal with handling or disposal, as you do with radioactive labels," Rhodes says. "And we've found that our probes display a higher level of sensitivity than radioactively labeled probes." Another direct approach to tagging DNA probes is the Prime- It Fluor Random Priming kit offered by Stratagene Corp. of La Jolla, Calif., which incorporates a fluorescently labeled nucleotide into the DNA. Commonly used fluorescent markers include fluorescein, which emits yellow-green when excited with blue light, and rhodamine, which emits a deep red. Most nonisotopic labeling systems, however, are indirect, with the probe bound to a molecule that is attracted to a second molecule that is itself bound to the signaling enzyme, usually alkaline phosphatase. In the most common of these strategies, the DNA probe is immobilized on a support and bound to biotin (a small, water-soluble vitamin). Biotin binds strongly to the bacterial protein streptavidin, which is linked to alkaline phosphatase. Such streptavidin- biotinylated DNA probes are offered by Genemed Biotechnologies Inc. of South San Francisco, Calif.; United States Biochemical of Cleveland; New England BioLabs Inc. of Beverly, Mass.; and others. As popular as DNA labeling with biotin-streptavidin is, it is gradually being replaced by what some researchers say is a better alternative--digoxigenin, a derivative of the foxglove plant. The digoxigenin molecule binds DNA at one site and a highly specific antibody at another. The antibody is the part that is detected. Charles Schroeder, technical service specialist at Indianapolis-based Boehringer Mannheim Biochemicals, which produces digoxigenin, explains that its success is due to its uniqueness. "Many of our customers had noted that because biotin is a vitamin, it is endogenous to many [animal] systems," Schroeder says. "But digoxigenin is found only in the foxglove plant." As a result, there is less background interference caused by the antibody binding here than to the DNA sequence labeled with digoxigenin. Ricki Lewis is a freelance science writer based in Scotia, N.Y., and is the author of Human Genetics: Concepts and Applications (Dubuque, Iowa, Wm. C. Brown Communications, 1994).

DNA Probes Yield Expanded Research And Clinical Uses

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