most appropriate probe for your research situation(Anderson 1999: Nath and Johnson, 1998; Temsamani and Agrawal, 1996: Trayhurn, 1996: Mansfield et aL., 1995; Tijssen, 2000). The questions raised in the ensuing discussions demonstrate why only the actual experiment, validated by positive and negative controls, deter mines the best choice The purpose of the following example is to discuss some of the complexities involved in selecting a labeling strategy. Suppose that you have the option of screening a target with a probe generated from the following templates: a 30 base oligo(30mer), a double stranded 800bp DNA fragment, or a double-stranded 2kb fragment 30-mer The 30-mer could be radioactively labeled at the 5 end via T4 polynucleotide kinase(PNK) or at the 3 end via Terminal deoxynucleotidyl transferase(TdT). pNK attaches a single mole cule of radioactive phosphate whereas TdT reactions are usually designed to add 10 or less nucleotides pnK does not produce the hottest probe, since only one radioactive label is attached, but the replacement of unlabeled phosphorous by P will not alter probe structure or specificity. TdT can produce a probe containing more radioactive label, but this gain in signal strength could be offset by altered specificity caused by the addition of multiple nucleotides. A 30mer containing multiple nonradioactive labels ould also be manufactured on a DNA synthesizer, but the pres ence of too many modified bases may alter the probe's hybridiza tion characteristics(Kolocheva et al., 1996) 800bp fragment The double-stranded 800 bp fragment could also be end labeled but labeling efficiency will vary depending on the presence of blunt, recessed, or overhanging termini. Since the complementary strands of the 800 bp fragment can reanneal after labeling,a reduced amount of probe might be available to bind to the target. Unlabeled template will also compete with labeled probes for target binding reducing signal output further. However, probe syn thesis from templates covalently attached to solid supports might overcome this drawback(Andreadis and Chrisey, 2000) Random hexamer- or nanomer- primed and nick translation labeling of the 800bp fragment will generate hotter probes than end labeling. However, they will be heterogeneous in size and specificity, since they originate from random location in the tem Herzer and Englertmost appropriate probe for your research situation (Anderson, 1999; Nath and Johnson, 1998; Temsamani and Agrawal, 1996; Trayhurn, 1996; Mansfield et al., 1995;Tijssen, 2000).The questions raised in the ensuing discussions demonstrate why only the actual experiment, validated by positive and negative controls, deter￾mines the best choice. The purpose of the following example is to discuss some of the complexities involved in selecting a labeling strategy. Suppose that you have the option of screening a target with a probe generated from the following templates: a 30 base oligo (30mer), a double￾stranded 800bp DNA fragment, or a double-stranded 2kb fragment. 30-mer The 30-mer could be radioactively labeled at the 5¢ end via T4 polynucleotide kinase (PNK) or at the 3¢ end via Terminal deoxynucleotidyl transferase (TdT). PNK attaches a single mole￾cule of radioactive phosphate whereas TdT reactions are usually designed to add 10 or less nucleotides. PNK does not produce the hottest probe, since only one radioactive label is attached, but the replacement of unlabeled phosphorous by 32P will not alter probe structure or specificity. TdT can produce a probe containing more radioactive label, but this gain in signal strength could be offset by altered specificity caused by the addition of multiple nucleotides. A 30mer containing multiple nonradioactive labels could also be manufactured on a DNA synthesizer, but the pres￾ence of too many modified bases may alter the probe’s hybridiza￾tion characteristics (Kolocheva et al., 1996). 800bp fragment The double-stranded 800bp fragment could also be end labeled, but labeling efficiency will vary depending on the presence of blunt, recessed, or overhanging termini. Since the complementary strands of the 800bp fragment can reanneal after labeling, a reduced amount of probe might be available to bind to the target. Unlabeled template will also compete with labeled probes for target binding reducing signal output further. However, probe syn￾thesis from templates covalently attached to solid supports might overcome this drawback (Andreadis and Chrisey, 2000). Random hexamer- or nanomer-primed and nick translation labeling of the 800bp fragment will generate hotter probes than end labeling. However, they will be heterogeneous in size and specificity, since they originate from random location in the tem- 404 Herzer and Englert