A Shot In The Dark: Products For The Chemiluminescent Detection Of DNA

Date: May 11, 1998 Author: Deborah A. Wilkinson Products for the Chemiluminescent Detection of DNA t's late at night. You've added radiolabeled probe to your Southern blot and just placed it in the incubator. Time to go home! But not quite. Now you have to clean up the radioactive waste that oozed out while you were sealing the bag. "There has to be a better way," you think. Well, you might want to consider one of the nonradioactive, chemiluminescent detection systems. The high backgrounds and low sensitivities associated with earlier generations of nonisotopic detection systems left many researchers frustrated with these techniques. However, significant improvements have been made in chemiluminescent detection methods during the past few years. These systems now merit a closer look. Recently developed chemiluminescent substrates have detection sensitivities that rival those of radioactive methods. Another advantage of chemiluminescent detection systems is that the probes are stable, at least in the sense that they do not undergo radiolytic decay. Nonradioactive probes can generally be stored frozen, then used long after their radioactive counterparts would have decayed. Chemiluminescent detection methods are now being exploited for a vast array of applications, including immunochemistry, reporter gene analyses, in situ hybridization studies, RNA labeling and detection, quantitative polymerase chain reaction (PCR), DNA sequence analyses, and nucleic acid blotting techniques. In this issue, we'll shine the spotlight on commercially available products for generating nonradioactive DNA probes that can be readily detected via chemiluminescent methods, and subsequently detecting those probes with blotting applications in mind. But be aware that chemiluminescent labeling and detection systems geared toward other applications also are available and are offered by many of the same companies noted in this article. Figure 1. Light Emission Kinetics of CDP-StarTM and CSPD® on a Nylon Membrane. Time course of relative light emissions produced by AP-catalyzed luminescent reactions utilizing CDP-Star versus CSPD. Reprinted with permission from Tropix. Chemiluminescent detection systems rely on the use of reporter enzymes that catalyze luminescent reactions. The last step in these systems will always be the detection of a luminescent reaction. Depending on which reporter enzyme your detection system uses, one of several basic chemiluminescent substrates can be employed. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are widely used as reporter enzymes in blotting techniques. In systems utilizing HRP, the substrate usually consists of luminol; in the presence of hydrogen peroxide, HRP oxidizes this substrate in a light-producing reaction. With the inclusion of enhancers, this substrate generates signals that peak quickly. Half-lives of these signals will vary depending on conditions, but they are relatively short, generally in the neighborhood of about an hour. Kits utilizing AP as a reporter enzyme will usually use either Lumi-Phos 530 or a 1,2-dioxetane-based substrate. Although the luminescent reaction is slower with these AP substrates, the resultant signals remain quite high for a relatively long time. Furthermore, even after several days, addition of fresh substrate sometimes allows for the ability to re-expose the original membrane. CDP-Star™, a 1,2-dioxetane chemiluminescent substrate, is the latest in a series of AP-catalyzed substrates that includes AMPPD® and CSPD®. CDP-Star is many times brighter than its predecessors, and its chemiluminescence is also longer-lived. Its fast light kinetics and superior light production capabilities make it ideal for applications requiring long exposures (such as DNA fingerprinting). Light emission peaks in less than 4 hours after the addition of the substrate, after which the signal reaches a plateau that lasts for hours to several days (See Figure 1). A major difference in the systems for the chemiluminescent detection of DNA is whether the detection is accomplished directly, through the use of just a probe, or indirectly with the use of a probe and at least one detection conjugate. Most kits use indirect methods employing a DNA probe and one secondary detection reagent. In these kits, the DNA probe is often labeled with an antigen, such as fluorescein or digoxigenin (DIG). Alternatively, the probe can be labeled with a molecule, such as biotin, that has a well-characterized specific binding partner. Although it's not a common strategy, more than one detection reagent can be used in indirect chemiluminescent detection systems. For example, one currently available detection kit employs a biotinylated DNA probe, which is then detected via subsequent incubations with streptavidin and a biotin-reporter enzyme conjugate. Incorporation of antigens or haptens into DNA probes is frequently achieved by enzymatic methods, such as random primer reactions, nick translation, PCR, or 3'-end labeling with terminal transferase. Also popular are chemical methods, including chemical- and photo- crosslinking, as well as the specific modification of oligonucleotides at their 5' ends. Once generated, the nonradioactive probes are ready to be used for blotting techniques. Blots are hybridized under conditions similar to those used for radioactive protocols. Afterward, the protocols become much like those for Western blots. Protein-binding sites on the membranes are blocked, and the probes are detected by the specific binding of appropriate secondary conjugates. The choice of secondary conjugate will depend on the application. In general, a secondary reagent consists of a reporter enzyme conjugated to a biospecific partner molecule. This partner molecule will be an appropriate antibody (such as antifluorescein or anti-DIG), or a specific binding protein (streptavidin for biotin, for example). As discussed in the previous section, the two most commonly used reporter enzymes for blotting techniques are AP and HRP. After reaction with the secondary conjugate, membranes are washed, and then exposed to a chemiluminescent substrate for subsequent visualization of signal. Chemiluminescent signals can be detected and recorded using X-ray film. More sophisticated imaging instruments can also be used for chemiluminescent signal visualization. These imaging systems, reviewed in a recent issue of LabConsumer (L. DeFrancesco, The Scientist, 12(4):16, Feb. 16, 1998), are becoming increasingly popular in today's laboratories. When using direct detection systems, the DNA probe is labeled with the reporter enzyme itself. This process eliminates secondary incubations, which cuts down on the time it takes to perform these studies. Eliminating the secondary reaction also can result in lower backgrounds. On the down side, conjugation of the enzyme to the DNA probe could decrease hybridization efficiency due to steric hindrance. When using this approach, you must also keep in mind that you have conjugated the reporter enzyme to the DNA probe, and successful chemiluminescent detection of your probe is dependent upon keeping that reporter enzyme active. The elevated temperatures commonly used for hybridizations and washes can have detrimental effects on the activities of some enzymes, as can reagents found in some hybridization cocktails. In some kits, thermostable enzymes (AP in particular) are used to overcome these difficulties. In addition, detailed protocols are available describing optimal buffers and conditions, and, in particular, the use of salt concentrations (in lieu of temperature) to control stringency levels. Several companies now market kits that combine different choices of DNA labels, labeling methods, reporter enzymes, and detection systems (summarized in the table). Although not in the table, many detection system kits include hybridization and wash buffers along with membranes that have been specifically optimized for use with each other. For brevity, we have listed only the chemiluminescent substrate and secondary conjugate for each detection kit. Keep in mind that some kits include both DNA labeling and chemiluminescent detection systems. Others include only labeling or detection systems that can be readily "mixed and matched" with complimentary products. Let's take a closer look at some of these products. Figure 2. Ambion's BrightStar Psoralen-Biotin kit. Psoralen-Biotin harnesses the intercalating property of psoralens to conjugate biotin to nucleotides. Psoralens are planar tricyclic compounds that intercalate between bases in a manner similar to ethidium bromide. Upon irradiation with long wave UV light, psoralens become covalently attached to nucleic acids, preferentially to thymidines, but also to uridines and cytidines. The resultant probes are generally stable for at least a year when stored at -80°C. Figure provided by Ambion, Inc. Figure 3. Sensitivities of Ambion's Psoralen-Biotin and 32P-labeled probes. Duplicate blots prepared using dilutions of mouse liver total RNA. Blots hybridized with GAPDH DNA probes labeled with either Psoralen-Biotin or 32P. Binding of the radiolabeled was visualized by autoradiography using an intensifying screen. The biotinylated probe was detected by use of the BrightStarTM BiodetectTM kit. Figure provided by Ambion, Inc. Ambion, Inc. uses psoralen-biotin as a sensitive and flexible method for generating nonisotopic probes. In their BrightStarTM Psoralen-Biotin Kit, biotin is chemically attached to RNA, DNA plasmids, inserts or PCR products directly. (Figure 2). The levels of biotin labeling obtained using this method do not interfere with nucleic acid hybridization, and according to Ambion, this process may even produce probes that are more sensitive than those prepared by enzymatically incorporating biotin. One advantage of this system, as well as other nonenzymatic methods, is that the reaction is readily scalable; large amounts of probe may be labeled at once. But what about obtaining high sensitivity and low background in blots? With regard to these issues, researchers at Ambion maintain that the method used for detecting the probe after hybridization is far more critical than the method chosen to label the probe molecule. In test blots, biotin-labeled probes generated using BrightStar Psoralen-Biotin compared favorably with radiolabeled probes (Figure 3). The biotinylated probes were visualized after development using Ambion's CDP-Star-based BrightStarTM BioDetectTM kit. In this kit, biotin is detected by the secondary conjugate streptavidin-AP. Schleicher & Schuell Inc. also markets a psoralen-biotin reagent for labeling nucleic acids. Roche Molecular Biochemicals offers the Biotin Chem-Link kit, which uses a different strategy to label nucleic acids with biotin. The cis-platinum Biotin Chem-Link reagent binds guanosine and adenosine bases to form non-covalent, coordinative complexes. Roche also sells a streptavidin-AP/CSPD®-based detection kit. NEN Life Science Products' Renaissance line of kits enables the incorporation of biotinylated nucleotides into probes via random primer labeling, end-labeling, or nick translation. Kits come with your choice of CDP-Star or a luminol-based substrate, along with the appropriate streptavidin-reporter enzyme conjugate. Figure 4. Kirkegaard & Perry Laboratories' DNADetector. Titration of PCR-amplified TIMP2 gene detected using a 611 base TIMP2 probe. DNA concentrations ranged from 0.3 pg to 83 pg. (Left) Blot detecting using a biotinylated probe and the DNADetector kit. (Right) Blot detected using radiolabeled probe. Figure provided by Kirkegaard & Perry Laboratories. Several companies market products that label DNA probes with biotin and a 14 to 21 atom spacer arm. The spacer arm can reduce steric hindrance during labeling, hybridization with probe, and detection using secondary conjugates. Kirkegaard & Perry Laboratories Inc. (KPL), markets a kit for the introduction of biotin-N4-dCTP into DNA via a random primer reaction. Probe binding can be visualized using KPL's streptavidin-HRP/LumiGLOTM based detection system, which is sold separately (Figure 4). Life Technologies Inc. vends kits for incorporating biotin-14 labeled nucleotides into DNA probes by either nick translation or random primer reactions. The company's streptavidin-AP/Lumi-Phos 530-based detection kit is available separately. New England BioLabs Inc's NEBlot Phototope kit uses random primer labeling to incorporate biotin-16-dATP into probes. The Phototope-Star Detection Kit employs two secondary detection reagents. The biotinylated DNA probe is detected by subsequent incubations with unlabeled streptavidin and biotin-AP. The supplied substrate is CDP-Star. Detection kits containing streptavidin-AP and either CDP-Star or CSPD can be purchased directly from Tropix Inc., a company that has developed a number of non-isotopic chemiluminescent technologies for detecting AP. Streptavidin-AP/CSPD®-based detection kits are also available from Sigma Chemical Co. and ICN Pharmaceuticals, Inc. Several enhanced luminol-based detection kits are available from Pierce Chemical Co. The SuperSignal® Nucleic line of products, which contains NeutrAvidin™, a deglycosylated avidin as a secondary conjugate, can be used for detecting biotinylated DNA probes hybridized to Southern or Northern blots. The company claims that nonspecific absorption is minimized with NeutrAvidin because of its neutral isoelectric point. Figure 5. Amersham's ECL 3'-oligolabelling and detection system. Oligonucleotide probes are tailed with 6-8 fluorescein-dUTP (Fl) nucleotides in a reaction catalyzed by terminal deoxynucleotidyl transferase. After hybridization, bound probe is indirectly detected by subsequent incubations with a-fluorescein-HRP and ECL reagents. Figure provided by Amersham Pharmacia Biotech. Amersham Pharmacia Biotech Inc. offers several systems for labeling DNA with flourescein-11-dUTP, either via random primer or end-labeling reactions. The labeling kits can be purchased with or without accompanying chemiluminescent detection systems. The company markets random primer labeling kits, which can be purchased with either CDP-Star or ECL detection reagents along with the appropriate antifluorescein conjugate. Amersham Pharmacia's 3'-oligolabeling and Detection System contains reagents for the tailing of oligonucleotide probes with six to eight fluorescein-11-dUTP molecules, as well as an ECL-based detection system (Figure 5). NEN Life Science Products' Renaissance product line enables the incorporation of fluorescein-labeled nucleotides into probes via either of three different enzymatic methods. This flexible collection of products allows you to choose either an anti-fluorescein-AP/CDP-Star or an antifluorescein-HRP/enhanced luminol detection system with any kit. A kit for incorporating fluorescein-12-dUTP via random primer labeling is available from Stratagene. The company also sells a detection kit with an antifluorescein-AP conjugate and CSPD. These kits, as well as some of the kit components, may be purchased together or separately. Roche Molecular Biochemicals offers an extensive line of products for labeling probes with DIG-dUTP via different techniques. A number of these kits feature an alkali-labile form of DIG-dUTP, in which the digoxigenin is connected to a spacer arm via an alkali-labile ester bond. This enables easier stripping and reprobing of blots. Roche also sells an anti-DIG-AP/ CSPD-based detection system. Tropix Inc. markets detection systems containing antifluorescein-AP and your choice of CSPD® or CDP-Star. "Save time and increase sensitivity of your hybridization reaction by directly labeling your oligonucleotide with alkaline phosphatase," says Will Scovill, a project manager at Genosys Biotechnologies, Inc. This company's E-LINK kits can be used to conjugate AP to the 5' end of oligonucleotide probes. The chemistry requires a 5' amino linked oligonucleotide. Briefly, the 5' aminoalkyl oligonucleotide is incubated with an activating reagent, purified, and conjugated to AP. The oligonucleotide-AP probe is then purified via gel filtration. Genosys' kits are available with or without the chemiluminescent reagent CSPD. Probes are generally 10 bases to 45 bases in length, but longer probes can be successfully labeled. Suitable oligonucleotides can be ordered from some custom oligo houses. However, should you want to synthesize your own modified oligo, the phosphoramidite reagents required are commercially available. (A list of recommended suppliers is available from Genosys.) Roche Molecular Biochemicals also sells a kit for conjugating AP to the 5' end of oligonucleotides. Amersham Pharmacia Biotech Inc. offers kits for labeling probes (DNA or RNA) with HRP and AP by chemical crosslinking. The Gene Images AlkPhos DirectTM system is based on the direct labeling of nucleotide probes with a thermostable AP enzyme, and detection with CDP-Star. ECL DirectTM involves the direct labeling of nucleotide probe with HRP, and subsequent detection via use of ECL reagents. The CDP-Star-based Quantum Yield® Chemiluminescent Detection System from Promega does not contain secondary detection reagents. Promega cautions that, "Blocking, hybridization and membrane combinations are known to have profound effects on the detection of nucleic acid sequences." Their Quantum Yield® products were developed to optimize the chemiluminescent detection of AP-labeled nucleic acid probes. There are an incredible number of choices for the nonisotopic labeling and chemiluminescent detection of nucleic acids. This review is designed to serve as a starting point in your quest to find the right products for your laboratory. It is likely that no one system will be ideal for every single line of experiments you do. You will probably need to select several different kits, depending on the nature of your probes and applications. Products for the Chemiluminescent Detection of DNA The author is Deborah Wilkinson, a freelance writer based in Memphis, Tenn. She may be contacted at deborahwilkinson@sprynet.com

A Shot In The Dark: Products For The Chemiluminescent Detection Of DNA

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