Will you need to detect a signal from your blot several times over a period of hours or days? Are you pursuing a low-copy target that requires an exposure time of days or weeks? Would ou prefer a short-lived signal t id stripping a blot pri re-probing? Some nonradioactive detection systems allow for the quick inactivation of the enzyme that generates the signal, eliminating the stripping step prior to re-probing(Peterhaensel, Obermaier, and Rueger, 1998). The effects and implications of stripping are discussed in greater detail later in the chapter The practical lifetime of common radiolabels is several days to weeks, and is dependent on the label, the ligand, and its environ ment, as discussed in Chapter 6, Working Safely with Radioactive Materials. Some nonradioactive systems based on alkaline phos phatase can generate signals lasting 10 days without marked reduction(personal observation). Some chemifluorescent systems generate a fluorescent precipitate capable of producing a cumu lative signal, much like isotopes. The functional lifetime of fuorescent labels will vith the chemical nature of the fluorescent tag and the methodology of the application. For example, signal duration of a fluorescent tag could be defined by the number of times the chromophore can be excited to produce a fluorescent emission. Some tags can only be excited/scanned once or a few times, while others are much more stable. Consult the manufacturer of the labeled product for this type of stability information. In systems where an enyzme cat alyzes the production of a reagent required for fluorescence, the enzyme's half-life and sufficient presence of fresh substrate can limit the duration of the sign Will the Label Be efficiently Incorporated into the Probe? The effects of label size, location, and linkage method on the corporation of nucleotides into DNA or RNA are enzyme dependent and can be difficult to predict. Small side chains can inhibit nucleic acid synthesis(Racine, Zhu, and Mamet-Bratley, 993), while larger groups such as biotin might have little or no effect(Duplaa et al., 1993; Richard et al., 1994).In general, nucleotides labeled with isotopes of atoms normally present in nucleotides(P, P,H, C)will be incorporated by DNA and RNa polymerases more efficiently than nucleotides labeled with isotopes of nonnative atoms. Commercial polyermases are frequently engineered to overcome such incorporation bias Nucleic Acid Hybridization 407Signal Duration Will you need to detect a signal from your blot several times over a period of hours or days? Are you pursuing a low-copy target that requires an exposure time of days or weeks? Would you prefer a short-lived signal to avoid stripping a blot prior to re-probing? Some nonradioactive detection systems allow for the quick inactivation of the enzyme that generates the signal, eliminating the stripping step prior to re-probing (Peterhaensel, Obermaier, and Rueger, 1998). The effects and implications of stripping are discussed in greater detail later in the chapter. The practical lifetime of common radiolabels is several days to weeks, and is dependent on the label, the ligand, and its environ￾ment, as discussed in Chapter 6,“Working Safely with Radioactive Materials.” Some nonradioactive systems based on alkaline phos￾phatase can generate signals lasting 10 days without marked reduction (personal observation). Some chemifluorescent systems generate a fluorescent precipitate capable of producing a cumu￾lative signal, much like isotopes. The functional lifetime of fluorescent labels will vary with the chemical nature of the fluorescent tag and the methodology of the application. For example, signal duration of a fluorescent tag could be defined by the number of times the chromophore can be excited to produce a fluorescent emission. Some tags can only be excited/scanned once or a few times, while others are much more stable. Consult the manufacturer of the labeled product for this type of stability information. In systems where an enyzme cat￾alyzes the production of a reagent required for fluorescence, the enzyme’s half-life and sufficient presence of fresh substrate can limit the duration of the signal. Will the Label Be Efficiently Incorporated into the Probe? The effects of label size, location, and linkage method on the incorporation of nucleotides into DNA or RNA are enzyme￾dependent and can be difficult to predict. Small side chains can inhibit nucleic acid synthesis (Racine, Zhu, and Mamet-Bratley, 1993), while larger groups such as biotin might have little or no effect (Duplaa et al., 1993; Richard et al., 1994). In general, nucleotides labeled with isotopes of atoms normally present in nucleotides (32P, 33P, 3 H, 14C) will be incorporated by DNA and RNA polymerases more efficiently than nucleotides labeled with isotopes of nonnative atoms. Commercial polyermases are frequently engineered to overcome such incorporation bias. Nucleic Acid Hybridization 407