Fluorescent Labeling Offers Flexibility Without Radioactivity

There is a war going on to win over the hearts and minds of molecular biologists: Radioactive isotopes-long the gold standard for tagging and later detecting RNA and DNA strands- are being challenged by a new generation of fluorescent labels that promise greater flexibility with fewer disposal problems. SEQUENCE DETECTION SYSTEM: Perkin-Elmer’s ABI Prism 7700 system uses a probe with a reporter and a quencher dye attached to it. With 14C, 32P, 125I, 3H, or some other radioactive atom built into the phosphorous backbone, ribose, or bases of DNA or RNA sequences (oligonucleotides), radiolabeled oligonucleotides are easily quantified using a Geiger counter or by the signature they produce on the grains of silver halide in a photographic emulsion. That endearing quality helped boost radiolabels to the forefront of molecular biology techniques. Labeled oligonucleotides will pair with complementary stretches according to the Watson-Crick model: Guanine on one chain links to a cytosine counterpart (G-C), and adenine closes on thymine (A-T). With a labeled strand in hand, a researcher can fish a sample for the complementary strand, then separate the complex and quantify it. DNA sequencing may begin with a 5®-labeled strand of DNA that is slowly degraded chemically and analyzed after each step. Enzymatic sequencing, on the other hand, relies on labeled primers that serve as a template for duplicating the original, unlabeled strand. Labeled probes may also be used to help scan chromosomes for damage or aberrations. As simple and efficient as radiolabeling is, nonradioactive labels seem poised to take over much of the labeling market. One reason: Sets of fluorescent labels of different colors give researchers a new tool to measure the presence of multiple sequences in cells. For example, one probe can be labeled with fluorescein, which is a green color, while the other probe can be labeled with rhodamine, which is orange, notes James McCughern-Carucci, a molecular biologist at Yale University School of Medicine. "On the same tissue sample, both probes can be visualized [independently]. This saves both time and sample," he explains. With such uses in mind, Amersham Life Science Inc. of Arlington Heights, Ill., sells its CyDye fluorescent dyes and pre-labeled reagents. These dyes have a maximum fluorescence at a variety of wavelengths: green (506 nm and 520 nm), orange (570 nm), scarlet (596 nm), far-red (670 nm), and near infrared (694 nm and 767 nm). They have narrow emission bands, allowing multiple color detection on a single sample. The fluors are highly sensitive, highly selective, quite stable, and water-soluble (making them easy to use in labeling reactions), according to the company. The dyes can handle a broad range of pH levels, maintaining fluorescence intensity at physiological pH, unlike fluorescein, which dampens below pH 8.0. The company stocks a series of pre-labeled products, including deoxynucleotides, whole human chromosome painting probes, human centromeric chromosome probes, avidin/streptavidin conjugates, and antibody conjugates. Perkin-Elmer Applied Biosystems of Foster City, Calif., carries fluorescently labeled deoxynucleotide triphosphates, which can be directly added to the polymerase chain reaction (PCR). The company's line consists of two bases-cytosine and uridine-that are available in three different colors: blue ([R110]dUTP and [R110]dCTP), green ([R6G]dUTP and [R6G]dCTP), and yellow ([TAMRA]dUTP and [TAMRA]dCTP). With the propargylamido linker arms separating the dyes from the bases, these products are the most efficiently incorporated dye nucleosides available, the company claims. OLIGONUCLEOTIDE LABELING SYSTEM: Oligos labeled with Promega’s FluoroAmp can be used as primers in a PCR or sequencing reaction. Promega Corp. of Madison, Wis., offers FluoroAmp oligonucleotide labeling systems that attach a green (fluorescein) label, an orange (rhodamine) label, or a biotin label (which binds tightly to avidin or streptavidin molecules) to the 5® end of a DNA sequence. This leaves the 3® terminus free for further extension or modification. "This means the FluoroAmp-labeled oligonucleotides can be used as primers in a PCR or a sequencing reaction-the most significant applications for these products," says Promega product manager Liz Marcus. She adds that the company has shown that "addition of a single fluorophore or biotin to the 5® end does not affect the behavior of the oligo; reaction conditions [such as the annealing temperature used in a PCR reaction] do not need to be modified." PAINT PROBE KIT: Stratgene's VanGlows allows users to detect chromosomal aberrations and examine effects of chemicals. Other products, like the VanGlows mouse chromosome paint probe kits offered by Stratagene Inc. of La Jolla, Calif., take a bigger view of DNA. The probes in these kits were amplified from sections of the mouse chromosomes 1, 2, 3, and 8, and then labeled with either fluorescein (green) or rhodamine (orange). These labeled probes hybridize to the entire length of chromosomes 1 and 3 or chromosomes 2 and 8- excluding the centromeric regions-in mouse metaphase cells. Staining the remaining chromosomes with an appropriate counterstain allows researchers to detect chromosomal aberrations and to examine the toxicological effects of chemicals and other agents. For users of automated DNA synthesizers, several companies offer labeled phosphoramidites. Glen Research Corp. of Sterling, Va., sells various phosphoramidite derivatives of biotin, fluorescein, acridine, and psoralen. Acridine is an intercalator, wedging itself between the stacked base pairs of a DNA or RNA duplex and stabilizing the complex. Psoralen can cross-link to thymidine bases on the opposing strand, forming a permanent bond between the complementary sequences. Different versions of these products may introduce a label at the 5' or 3' terminus of a sequence, at phosphate linkages anywhere along the sequence. Alternatively, in the case of Biotin dT, the label can actually replace any base in the sequence with a modified thymidine that links to a biotin. The classic method of adding tags to synthesized oligonucleotides is to modify the terminus of the strand with either a primary amine or thiol, and conjugate it to the succinimide or maleimide ester derivative of a label. "We've developed phosphoramidites that allow you to put a lot of those in directly during the synthesis-which is a lot simpler, and a lot easier in terms of working them up," says Bob Somers, senior applications chemist at Glen. FOR LABELING NUCLEIC ACIDS: Vector Laboratories" FastTag labeling kit includes labeled control DNA. Though convenient, that method limits the labels available to the researcher. Vector Laboratories Inc. of Burlingame, Calif., offers its FastTag labeling system, which incorporates a sulfide linkage into the target sequence, allowing the researcher to use any label that can bind chemically to that linkage. "FastTag is particularly well suited for labeling nucleic acids with detectable or reactive groups that are unavailable as nucleotide conjugates," maintains Steve Daniel, senior scientist at Vector. "In theory, any desired label that is coupled to a sulfhydryl-reactive group can be attached to any nucleic acid via the universal FastTag [system]." The company sells versions of the kit that label with fluorescein, biotin, dinitrophenyl, and fucose tags, with others on the way. BioGenex of San Ramon, Calif., builds on the base- replacement theme with its non-nucleosidic biotin and fluorescein phosphoramidites. Its Biotin-CX CED and Fluorescein-CX CED Phosphoramidites incorporate a six- membered ring in the space that the ribose ring of the nucleic acid would normally occupy, and these structures maintain the natural three-carbon atom distance between the phosphates in DNA/RNA. The 5® phosphate and the 3® end of the nucleic acid both branch off from the same carbon in the ring, while the biotin hangs off the opposite end of the ring, in the same general region of space that a nucleic acid's base would fill. The six-membered ring also improves the rigidity of the molecule, thus preventing the label from bumping into an opposing strand during hybridization. That results in greater hybridization frequency, according to the company. A researcher can instruct the DNA synthesizer to incorporate this base replacement at any point in a probe sequence. The new labels-already big in the automated sequencing market-avoid radioactivity's main pitfall: disposal problems. To meet safety standards, a lab using radiolabels must be tested for background radiation, usually monthly, and researchers must keep records of purchases and disposal of radioactive reagents. "It's very paperwork-intensive," says Peter Melera, director of the molecular and cellular biology program at the University of Maryland in Baltimore. Those measures help protect researchers from the dangers of long-term exposure, including heightened cancer risk and reproductive effects. Despite routine safety precautions, researchers in Melera's lab are hesitant about using radiolabels for fear of the potential effects of exposure on the human reproductive system. As useful and versatile as fluorescent probes are, radiolabeled probes tend to be more sensitive, and they hybridize more consistently to target sequences. And that means radiolabels will remain popular in applications in which target DNA or RNA is in low abundance, or when quantitation is essential. Many researchers report that the images provided by radiolabels are generally cleaner and more crisp than their fluorescent counterparts. Still, fluorescent labels are catching up, and "most radioactivity- based protocols can and have been adapted to use non- radioactive labels, and once the assay itself is working, the images can be quite nice," according to McCughern-Carucci. Radioactive labels never satisfactorily tackled the problem of how to quantitate the amount of RNA or DNA that is originally present in a sample, but their fluorescent challengers have proved to be up to the task. To detect a very small amount of contaminant-for example, Salmonella bacteria in foods-the researcher must use PCR to amplify any tiny amount of impurity to a concentration high enough to be detected. If the sample initially has 1,000 of the organisms, PCR might detect it at, say, the 22nd cycle, when the organism's target DNA strand has been amplified to a level at which it is detectable. If there are 2,000 organisms to begin with, it might show up one cycle earlier. "Knowing that difference is the important thing for quantitation," says Mike Lucero, senior product manager at Perkin-Elmer Applied Biosystems. But that's no easy task, Lucero adds. The complex protocols make it especially formidable to large quality-control operations. Perkin-Elmer's ABI Prism 7700 Sequence Detection System relies on a clever fluorogenic assay-dubbed TaqMan. The system uses a probe with two dyes attached to it-a reporter and a quencher. The quencher absorbs at the same wavelength that the reporter fluoresces, so while the probe is intact there is no detectable signal. For instance, suppose a Salmonella probe is introduced into a sample that contains a tiny amount of the organism. The probe will quickly hybridize to the complementary sequence, while a DNA polymerase initiates synthesis of a strand complementary to the entire DNA strand. During the course of strand synthesis, the polymerase eventually meets up with the probe and dismantles it as it continues to make the new strand. It soon snips the quencher dye away from the rest of the probe, and the unfettered reporter dye provides an instant fluorescent signal. From this signal the 7700 can calculate the initial concentration of the target sequence in the sample. Selected Suppliers of Labeling Systems: Ambion Inc. Amersham Life Science Inc. BioGenex Ericomp Inc. Glen Research Corp. Perkin-Elmer Applied Biosystems Promega Corp. Stratagene Inc. Vector Laboratories Inc. As a result of several deaths from food-poisoning outbreaks, President Clinton announced recently that meat and poultry processors will soon be required to test for Salmonella and E. coli. To tackle the Salmonella problem, Perkin-Elmer introduced the TaqMan Salmonella PCR Amplification/Detection System-the first automated PCR-based assay to identify the organism in food, according to the company. Ambion Inc. of Austin, Texas, has taken a different approach, offering a reagent that supplements an existing method-reverse transcription (synthesis of DNA from RNA) coupled with PCR. This is a very sensitive method of detecting the presence of the original RNA, but it can't quantify it without some standard against which to measure the final number of amplified strands. One good standard would be ribosomal RNA (rRNA), since it will be present in any sample. But to work well, a control should be expressed at about the same level as the target RNA, and rRNA is too abundant to be of much use. To counteract the problem, Ambion has developed its 18S rRNA Primers and Competimers. Both are included in the QuantumRNA kit, which allows the researcher to control the amplification of 18S rRNA, making it useful as a control for quantitation. Even as fluorescent labels are pushing radiolabels, new technologies are on the horizon. San Diego-based Ericomp Inc. has developed a magnetic messenger labeling system that exploits techniques developed in the disk drive magnetic recording industry. Company researchers have used biotin- labeled DNA and RNA in combination with a streptavidin molecule that is tagged with magnetizeable particles. Once separated out, the biotinylated oligonucleotides are treated with the streptavidin-magnetic particle complex. Exposed to a magnetic field, the accompanying particles emit a magnetic field of their own that is easily detectable with standard (and inexpensive) magnetic readers. "It's much more sensitive for the dollar [than other labeling techniques]. You can use very inexpensive instrumentation to detect DNA or proteins," says Lonnie W. Adelman, president of Ericomp. The process appears to be more sensitive than current labeling methods, according to Adelman, but the company is still in the early stages of developing the methods and can't yet put a specific number on the improvements. "We feel that this will replace a lot of the systems that are optically based [such as fluorescence and luminescence], only because it will become affordable without sacrificing any performance. It's [also] very safe," maintains Adelman. Whether or not nonradioactive labels will ever match the technical simplicity of the radiolabels-and thus replace these traditional tools in small biological laboratories- remains to be seen. "We still use [radiolabels] in the laboratory because we don't have [the efficient DNA sequencers]. We're still doing it with gels the old way," says Melera. Even if the nonradioactive technology comes along, though, it faces one more barrier: the need to convince traditional- minded scientists to change the tools they use. "I have struggled for the past few years trying to convince people in my own group to convert [to fluorescent probes]," reports McCughern-Carucci. Melera concurs: "You know how to do these techniques . . . they work for you and you know what to expect. [To switch], there is an inertia you have to overcome." James Kling, a science writer based in Bellingham, Wash., is online at jkling@nasw.org.

Fluorescent Labeling Offers Flexibility Without Radioactivity

Apr 13, 1997

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