Radioactive Isotope signal strength diminishes in the order: P>>P>S> 3H. When sensitivity is the primary concern, as when searching for a low-copy gene, P is the preferred isotope Tritium is too weak for most blotting applications, but a nucleic acid probe labeled with multiple tritiated nucleotides can produce a useful, highly resolved signal without fear of radiolytic degradation of the probe H and"S are used for applications such as in situ hybridiza tion(ish) where resolution is more essential than sensitivity. The resolution of P is similar to 3S, but Ausubel et al. ( 1993)cites an improved signal-to-noise ratio when"P is applied in ISH Signal strengths of nonradioactive labels are difficult to compare. It is more practical to assess sensitivity instead of signal strength. The resolution of nonradioactive signals is also more omplicated to quantify because resolution is a function of signal strength at the time of detection and most nonradioactive signals weaken significantly over time. Therefore the length of exposure to film must be considered within any resolution discussion Background fluorescence or luminescence from the hybridi zation membrane has to be considered as well. Near-infrared dyes are superior due to low natural near-infrared occurrence (Middendorf, 1992). Some dyes emit in the far red 2700nm (Cy7, Alexa Fluor 549, allophycocyanin) Older nonradioactive, colorimetric labeling methods suffered from resolution problems because the label diffused within the membrane. Newer substrates, especially some of the precipitating chemifluorescent substrates, alleviate this problem. Viscous components such as glycerol are often added to substrates to limit diffusion effects. Colorimetric substrates and some chemilumi nescent substrates will impair resolution if the reaction proceeds beyond the recommended time or when the signal is too strong Hence background can increase dramatically due to substrate diffusion Mohandas ghandi said that there is more to life than increas- its speed(John-Roger and McWilliams, 1994), and the sam olds true for detection systems. Most nonradioactive systems deliver a signal within minutes or hours, but this speed is useless if the system cant detect a low-copy target Searching for a single copy gene with a P labeled probe might require an exposure of several weeks, but at least the target is ultimately identified 406 Herzer and EnglertRadioactive Isotope signal strength diminishes in the order: 32P > 33P > 35S > 3 H. When sensitivity is the primary concern, as when searching for a low-copy gene, 32P is the preferred isotope. Tritium is too weak for most blotting applications, but a nucleic acid probe labeled with multiple tritiated nucleotides can produce a useful, highly resolved signal without fear of radiolytic degradation of the probe. 3 H and 35S are used for applications such as in situ hybridiza￾tion (ISH) where resolution is more essential than sensitivity. The resolution of 33P is similar to 35S, but Ausubel et al. (1993) cites an improved signal-to-noise ratio when 33P is applied in ISH. Nonradioactive Signal strengths of nonradioactive labels are difficult to compare. It is more practical to assess sensitivity instead of signal strength. The resolution of nonradioactive signals is also more complicated to quantify because resolution is a function of signal strength at the time of detection, and most nonradioactive signals weaken significantly over time. Therefore the length of exposure to film must be considered within any resolution discussion. Background fluorescence or luminescence from the hybridi￾zation membrane has to be considered as well. Near-infrared dyes are superior due to low natural near-infrared occurrence (Middendorf, 1992). Some dyes emit in the far red ≥700nm (Cy7, Alexa Fluor 549, allophycocyanin). Older nonradioactive, colorimetric labeling methods suffered from resolution problems because the label diffused within the membrane. Newer substrates, especially some of the precipitating chemifluorescent substrates, alleviate this problem. Viscous components such as glycerol are often added to substrates to limit diffusion effects. Colorimetric substrates and some chemilumi￾nescent substrates will impair resolution if the reaction proceeds beyond the recommended time or when the signal is too strong. Hence background can increase dramatically due to substrate diffusion. Detection Speed Mohandas Ghandi said that there is more to life than increas￾ing its speed (John-Roger and McWilliams, 1994), and the same holds true for detection systems. Most nonradioactive systems deliver a signal within minutes or hours, but this speed is useless if the system can’t detect a low-copy target. Searching for a single￾copy gene with a 32P labeled probe might require an exposure of several weeks, but at least the target is ultimately identified. 406 Herzer and Englert