New DNA/RNA Probe Applications Help To Unlock Secrets Of Genetics

SIDEBAR: Selected Suppliers of DNA and RNA Probes In the doctor's office or the research lab, the supreme value of DNA and RNA as diagnostic tools and the high potential of genetic therapy are putting a premium on DNA and RNA probes. Probes are short, single-stranded chains of nucleic acid that seek out and latch themselves to a complementary sequence of nucleic acids, often buried anonymously in a much larger section of DNA/RNA. Guanine on one chain links to a cytosine counterpart (G-C), and adenine closes on thymine (G-T). If the sequence on the probe exactly matches a counterpart sequence on the target DNA/RNA, the probe latches on. The probe is usually chemically bound to a "reporter" molecule, which produces a signal -- often fluorescence -- and advertises the presence of the target sequence. There is no shortage of applications for DNA probes. Southern blotting detects other DNAs; northern blotting pinpoints the presence of RNA by detecting gene expression. Colony and plaque hybridization screen for particular bacteria and viruses. Other applications include in situ hybridization, in which DNA hybridization is carried out on the cells themselves; footprinting, which identifies the sites where given proteins bind to DNA; mapping the locations of genes on chromosomes; and sequencing individual genes. "In my field of cytogenetics [the examination of the cellular components of genetics], we use probes to supplement conventional imaging techniques such as chromosome banding, where you count the chromosomes and see if there are any deletions or duplications," says David H. Ledbetter, director of the Center for Medical Genetics at the University of Chicago. "Molecular probes offer much higher sensitivity to detect small deletions or duplications." Clinically, probes could alter the diagnosis and treatment of diseases. Take telomeres, for example. These repeating sequences of DNA, which hang on the coattails of chromosomes, remained mysterious and apparently nonfunctional until recent research linked them with the programmed cell death that keeps cells from becoming immortal and cancerous (R. Lewis, The Scientist, Feb. 19, 1996, page 12). Most cells have a limit of 50 or so divisions. Each time a cell divides, the last telomere sequence is not copied, so the parent chromosome ends up with one less telomere repeat. After enough divisions, the shortened telomere destabilizes the chromosome and prevents the cell from dividing further, producing a state referred to as senescence. But cells that produce telomerase-an enzyme that replaces that last telomere sequence after a cell division-are spared the slow bleeding of telomeres, and can continue to divide long after the 50-cycle limit imposed on normal cells. Most cancer cells show telomerase activity, with the most aggressive cancers showing the highest levels. The level of activity may be a predictor of the patient's chances for survival. Ledbetter's group recently developed a collection of fluorescently labeled probes corresponding to each individual human telomere region (D.H. Ledbetter et al., Nature Genetics, 13:86-90, 1996). "This will allow us to look for subtle chromosome translocations in situations like kids with mental retardation," he explains. "Here the chromosomes might look normal, but they may have a more subtle deletion at the ends of the chromosome." And most cancers, he notes, have multiple chromosomal rearrangements that may be involved in the initiation and progression of the disease. By visual inspection, a stained chromosome end may appear suspicious, but probes can give an unambiguous answer. "With telomere probes you can be more certain-with objective hybridization whether the correct chromosome end is present," he says. Probes are not a new tool. "This is relatively old technology with new methods of probe detection becoming available," states Bert Ely, director of the Institute for Biological Research and Technology at the University of South Carolina in Columbia. The classic method of attaching radiolabels to probes is fast fading as an imaging technique. The highpowered intensity of some radionuclides often causes "fuzzy" pictures on the photographic film used for detection, and these reagents are expensive to handle and dispose of. Increasing sensitivity and far simpler disposal are boosting nonradioactive imaging agents to the forefront of probe imaging. Amersham Life Science Inc. Clontech Laboratories Inc. Epicentre Technologies FMC Corporation Genosys Biotechnologies Inc. IDEXX Laboratories Inc. Kamiya Biomedical Co. Oncor Inc. Operon Inc. Signet Laboratories Inc. Vysis Inc. Probes can be directly or indirectly labeled. A direct-labeled probe is similar to the venerable radionuclide-labeled probes, in that the reporter molecule-typically a fluorochrome-is directly attached to the probe. Visualization is simple and can be accomplished with light-sensitive film or a fluorescent microscope. Indirect-labeled probes are linked to a hapten such as biotin or digoxigenin. In a separate step, anti-hapten antibody conjugated to a reporter molecule is introduced. What the method sacrifices in technical simplicity, it makes up for in the potential for increased sensitivity: The reporter molecule linked to the antibody is often an enzyme-such as alkaline phosphatase-that can produce a signal via chemiluminescent, colorimetric, or fluorometric enzyme substrates. This allows increased exposure time and sensitivity. Indirect labeling can suffer from higher background noise as a result of nonspecific binding of the antibodies. Alternatively, products such as E-Link-produced by Genosys Biotechnologies Inc. of The Woodlands, Texas-allow researchers to directly attach a reporter molecule (in this case, alkaline phosphatase) to their probes, a direct-labeling technique that also offers the signal amplification of an enzyme. The conjugates are stable for up to six months, and two conjugates can be prepared in two hours, according to the company. Similarly, the ECL system-offered by Amersham Life Science Inc. of Arlington Heights, Ill.-allows researchers to label their probes with horseradish peroxidase, which in turn provides chemiluminescence by oxidizing luminol in the detection solution provided. "The probe-labeling procedure takes 20 minutes, and no secondary immunodetection steps [are] required as with indirectly labeled systems, which saves at least two hours of protocol time," says Mike Deines, marketing manager at Amersham. In the past, most probes have been indirectly labeled, according to Ledbetter. Directly labeled versions of the indirectly labeled products are beginning to appear. Sensitivity is a problem, however. "You have to have a bigger probe, or a more robust probe-hybridization system for high sensitivity. [But] if the sensitivity is equivalent, I think everybody will prefer the direct-labeled version," says Ledbetter. For those labs in which simplicity and a minimal learning curve are required, Signet Laboratories Inc. of Dedham, Mass., offers its line of DNA In Situ Hybridization (DISH) kits, which include everything needed for testing-digestion and pretreatment reagents, target-specific labeled probes, controls, buffers, and imaging reagents. Signet's kits, like those made by other companies, detect a range of disease causing organisms. Signet offers a choice of three probe linkers: biotin, fluorescein, and digoxigenin. For all three methods, the second step is addition of an anti-linker antibody fragment (Fab2) conjugated to alkaline phosphatase. Once the alkaline phosphatase enzyme is in place, either the sample can be directly imaged-if the label is fluorescein-or the researcher can add a reporter substrate that is converted to a compound that can be detected with light or electron microscopy. Of the three linkers, according to Ron Van Eysden, product manager at Signet, digoxigenin is the most sensitive because it has the highest affinity for its antibody fragment. Since it is not present in human tissues, it also gives the cleanest stain. The Anti-thymine Dimer MAb antibody, offered by Kamiya Biomedical Co. of Seattle, bypasses the labeling process altogether, requiring only "naked," unlabeled probes. It targets the thymidine-thymidine dimer that is formed during ultraviolet irradiation of the probe. The method circumvents one fundamental problem with DNA probe technology, which is that a label attached to the probe increases its molecular weight. This can hinder the probe from entering cells and tissues to hybridize with the target strands. "The method has high sensitivity-detecting less than 10 picograms of DNA in a dot blot-and good reproducibility," claims Robert Aline, Jr., scientific director at Kamiya. Oncor Inc. of Gaithersburg, Md., offers a system that allows fluorescein signals to be amplified, whether they are directly or indirectly labeled. The company's Fluor-Amp system uses antibody-fluorescein conjugates to "sandwich" antibodies. For instance, a researcher may use a biotin-labeled probe and image it with an avidin-fluorescein conjugate. If the signal is too weak, the Fluor-Amp kit offers a mouse-derived anti fluorescein antibody that can be applied, followed by an anti mouse antibody that is itself conjugated to fluorescein, increasing the overall signal. According to Bob Chapman, a marine scientist at the Marine Resources Research Institute in Charleston, S.C., one problem facing companies that make probes is that the technology is almost too useful: Probes are advancing research so quickly that companies have trouble keeping up with specific demands. "What we need today is different than what we'll need tomorrow, and in many cases it is prohibitively expensive for companies to develop and mass-produce probes when the researchers can do it themselves for far less," Chapman points out. FOR RNA PRODUCTION: The AmpliScribe High-Yield Transcription Kit from Epicentre Technologies With that in mind, Epicentre Technologies of Madison, Wis., offers a kit-called AmpliScribe-that allows in vitro synthesis of biotin- or digoxygenin-labeled RNA probes. "A researcher can use one AmpliScribe Kit to synthesize short or long transcripts, capped RNAs, or probes, rather than having to purchase many specialized kits to synthesize each type of transcript," says Harry Burrell, vice president of marketing and sales at Epicentre. A few time-honored, and clinically significant, probes continue to sell well. Oncor, which stresses that all of its products are for research use only, offers a number of probes that detect deletions or translocations in a number of human disorders. One probe detects a chromosomal microdeletion that is associated with Miller-Dieker syndrome, a malformation in which normal brain convulsions are absent and the brain surface is smooth. Another probe detects a deletion that usually accompanies Smith-Magenis syndrome. This condition encompasses varying degrees of mental retardation and behavioral problems, including self-destructive behavior. The company also offers a probe that detects translocations in acute leukemia patients. E-LINK: The product from Genosys labels up to 10 5'-amine modified oligonucleotides with alkaline phosphatase. Other products with clinical applications include a probe that detects mutations in the human ras oncogenes from Clontech Laboratories Inc. of Palo Alto, Calif. Additionally, both Oncor and Vysis Inc. of Downers Grove, Ill., offer probes that detect the presence of the female XX and the male XY chromosome combinations. One potential use of such probes is a check to see if a bone marrow transplant has taken hold. If the recipient and donor are of the opposite sex, the probe can identify the gender of the surviving marrow of the recipientif it's mismatched, it is evidence that the transplant may have been successful. The application demonstrates the simplicity and attraction of DNA/RNA probes as a potential diagnostic tool. "The CEP X/Y allows a pathologist to examine the genetics of non-dividing cells on a cell-by-cell basis. Other molecular techniques require complex DNA extraction protocols, which also mix normal and abnormal cells together, complicating interpretation of results," maintains Steven Seelig, vice president of research and development at Vysis. Other probes targeting specific diseases or organisms include the Human ras Muta-Lyzer line, offered by Clontech Laboratories, which screen mutations of the Ha-ras, Ki-ras, and N-ras oncogenes at three positions in the genes; probes for two common poultry diseases offered by IDEXX Laboratories Inc. of Westbrook, Maine; and a probe from Vysis that screens for trisomy 8, a condition associated with leukemia in which cells have three copies of chromosome 8 instead of the usual two. Ongoing genetic mapping initiatives such as the Human Genome Project, combined with the growing use of DNA fingerprinting evidence in the courts, are creating a stable market for products like the randomly generated 10-mers sold by Operon Inc. of Alameda, Calif. The 1,200 or so probes that Operon keeps in stock were screened so that they have no complementary ends, ensuring that the probes won't recognize each other and self-hybridize. "They probably remain the most frequently cited 10-mers in the literature," says Ralph Sinibaldi, technical services manager at Operon. The Quick-Light line of mapping probes, from FMC Corp. of Rockland, Maine, is designed to help map the genomes of eukaryotic organisms-a broad group that includes every organism whose cells bear a distinct nucleus. The probes hybridize with specific repetitive sequences that occur at a high frequency throughout eukaryote genomes. They come in two classes: One group includes family-specific probes culled from Genbank, the National Institutes of Health database that contains all known nucleic acid and protein sequences. The other group, called nucleotide repeat probes, hybridizes with DNA sequences containing microsatellites, which are distributed randomly in all eukaryotes outside of yeast. Back in 1950, when Alfred Hershey and Martha Chase demonstrated that DNA is the carrier of genetic information within a cell, they used radioactive phosphorus, built into the backbone of the probe, to image DNA samples. DNA/RNA probe imaging has come a long way since then. And scientists and company officials alike predict that imaging methods and probe kits will continue to evolve with the field, providing researchers with greater sensitivity and flexibility in their drive to unravel the secrets of genetics. James Kling is a science writer based in Bellingham, Wash.

New DNA/RNA Probe Applications Help To Unlock Secrets Of Genetics

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