Is a More Sensitive Detection System Always Better? Greater sensitivity can solve a problem or create one. The more sensitive the system, the less forgiving it is in terms of background a probe that generates an extremely strong signal may require an extremely short exposure time on film, making it difficult to capture signal at all or in a controlled fashion. Femtogram sensitivity is required to detect a single-copy gene and represents the lower detection limit for the most sensitive probes. Methods at or below femtogram sensitivity can detect 1 to 5 molecules, but this increases the difficulty in discerning true pos itive signals when screening low-copy targets(Klann et al., 1993 Rihn et aL, 1995). Single-molecule detection is better left to tech niques such as nuclear magnetic resonance or mass spectrometry The pursuit of hotter probes for greater sensitivity can be an unnecessary expense. Up to 56% of all available sites in a 486 nucleotide (nt) transcript could be labeled with biotinylated dUTP, but 8% was sufficient to achieve similar binding levels of Streptavidin than higher-density labeled probes(Fenn and Herman, 1990). Altering one or more steps of the hybridization process might correct some the above-mentioned problems. The key is to evaluate the true need, the benefits and the costs of increased sensitivity What Can You Conclude from Commercial Sensitivity Data? Manufacturers can accurately describe the relative sensitivities of their individual labeling systems. Comparisons between label- ing systems from different manufacturers are less reliable becaus each manufacturer utilizes optimal conditions for their system. Should you expect to reproduce commercial sensitivity claims? Relatively speaking, the answer is yes, provided that you optimize your strategy. However, with so many steps to a hybridization ex periment (electrophoresis, blotting, labeling, and detection) quantitative comparisons between two different systems are imperfect Side-by-side testing of different detection systems uti lizing the respective positive controls or a simple probe/target system of defined quantities (e. g, a dilution series of a house- keeping gene)is a good approach to evaluation. LABELING ISSUES Which Labeling Strategy Is Most Appropriate for Your situation? Each labeling strategy provides features, benefits, and limita- tions, and numerous criteria could be considered for selecting the Nucleic Acid Hybridization 403Is a More Sensitive Detection System Always Better? Greater sensitivity can solve a problem or create one. The more sensitive the system, the less forgiving it is in terms of background. A probe that generates an extremely strong signal may require an extremely short exposure time on film, making it difficult to capture signal at all or in a controlled fashion. Femtogram sensitivity is required to detect a single-copy gene and represents the lower detection limit for the most sensitive probes. Methods at or below femtogram sensitivity can detect 1 to 5 molecules, but this increases the difficulty in discerning true pos￾itive signals when screening low-copy targets (Klann et al., 1993; Rihn et al., 1995). Single-molecule detection is better left to tech￾niques such as nuclear magnetic resonance or mass spectrometry. The pursuit of hotter probes for greater sensitivity can be an unnecessary expense. Up to 56% of all available sites in a 486 nucleotide (nt) transcript could be labeled with biotinylated dUTP, but 8% was sufficient to achieve similar binding levels of Streptavidin than higher-density labeled probes (Fenn and Herman, 1990). Altering one or more steps of the hybridization process might correct some the above-mentioned problems. The key is to evaluate the true need, the benefits and the costs of increased sensitivity. What Can You Conclude from Commercial Sensitivity Data? Manufacturers can accurately describe the relative sensitivities of their individual labeling systems. Comparisons between label￾ing systems from different manufacturers are less reliable because each manufacturer utilizes optimal conditions for their system. Should you expect to reproduce commercial sensitivity claims? Relatively speaking, the answer is yes, provided that you optimize your strategy. However, with so many steps to a hybridization ex￾periment (electrophoresis, blotting, labeling, and detection), quantitative comparisons between two different systems are imperfect. Side-by-side testing of different detection systems uti￾lizing the respective positive controls or a simple probe/target system of defined quantities (e.g., a dilution series of a house￾keeping gene) is a good approach to evaluation. LABELING ISSUES Which Labeling Strategy Is Most Appropriate for Your Situation? Each labeling strategy provides features, benefits, and limita￾tions, and numerous criteria could be considered for selecting the Nucleic Acid Hybridization 403