What Do You know About Your Target? The sensitivity needs of your system are primarily determined by the abundance of your target, which can be approximated according to its origin. Plasmids, cosmids, phagemids as colony lifts or dot blots, and PCr products are usually intermediate to high-abundance targets. Genomic DNa is considered an intermediate to low-abundance target. Most prokaryotic genes are present as single copies, while genes from higher eukaryotes can be highly repetitive, of intermediate abundance, or single copy(Anderson, 1999). However, sensitivity requirements for single-copy genes should be considered sample dependent be cause some genes thought to be single copy can be for und as multiples. Lewin(1993) provides an example of recently poly ploid plants whose genomes are completely repetitive. The RNA situation is more straightforward; 80% of RNa transcripts are present at low abundance, raising the sensitivity requirements for most Northerns or nuclease protection assays(Anderson 1999) If you're uncertain about target abundance, test a series of different target concentrations(van Gijlswijk, Raap, and Tanke 1992; De Luca et aL., 1995). Manufacturers of detection systems often present performance data in the form of target dilution series. Known amounts of target are hybridized with a probe to show the lowest detection limit of a kit or a methe limic this experimental approach to determine your sensitivity requirements and the usefulness of a system. This strategy requires knowing the exact amount of target spotted onto the What Do You Know about Your Probe or Probe template? The more sequence and structural information you know about your probe and target, the more likely your hybridization will deliver the desired result(Bloom et aL., 1993). For example, the size and composition of the material from which you will gener ate your probe affects your choice of labeling strategy and hybridization conditions, as discussed in the question, Which Labeling Strategy Is Most Appropriate for Your Situation? GC content, secondary structure, and degree of homology to the target should be taken into account, but the details are beyond the scope of this chapter. (See Anderson, 1999; Shabarova 1994; Darby, 1999; Niemeyer, Ceyhan, and Blohm, 1999; and http://www2.cbm.uam.es/ilcastrillo/lab/protocols/hybridn.htmfor 402 Herzer and EnglertWhat Do You Know About Your Target? The sensitivity needs of your system are primarily determined by the abundance of your target, which can be approximated according to its origin. Plasmids, cosmids, phagemids as colony lifts or dot blots, and PCR products are usually intermediate to high-abundance targets. Genomic DNA is considered an intermediate to low-abundance target. Most prokaryotic genes are present as single copies, while genes from higher eukaryotes can be highly repetitive, of intermediate abundance, or single copy (Anderson, 1999). However, sensitivity requirements for single-copy genes should be considered sample dependent be￾cause some genes thought to be single copy can be found as multiples. Lewin (1993) provides an example of recently poly￾ploid plants whose genomes are completely repetitive. The RNA situation is more straightforward; 80% of RNA transcripts are present at low abundance, raising the sensitivity requirements for most Northerns or nuclease protection assays (Anderson, 1999). If you’re uncertain about target abundance, test a series of different target concentrations (van Gijlswijk, Raap, and Tanke, 1992; De Luca et al., 1995). Manufacturers of detection systems often present performance data in the form of target dilution series. Known amounts of target are hybridized with a probe to show the lowest detection limit of a kit or a method. Mimic this experimental approach to determine your sensitivity requirements and the usefulness of a system. This strategy requires knowing the exact amount of target spotted onto the membrane. What Do You Know about Your Probe or Probe Template? The more sequence and structural information you know about your probe and target, the more likely your hybridization will deliver the desired result (Bloom et al., 1993). For example, the size and composition of the material from which you will gener￾ate your probe affects your choice of labeling strategy and hybridization conditions, as discussed in the question, Which Labeling Strategy Is Most Appropriate for Your Situation? GC content, secondary structure, and degree of homology to the target should be taken into account, but the details are beyond the scope of this chapter. (See Anderson, 1999; Shabarova, 1994; Darby, 1999; Niemeyer, Ceyhan, and Blohm, 1999; and http://www2.cbm.uam.es/jlcastrillo/lab/protocols/hybridn.htm for in-depth discussions.) 402 Herzer and Englert