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(> 5), the process is exothermic at room temperatures. The oligonucleotide sequence and length of the DNA probe determines the selectivity of detection of a target species. For example, statistical considerations have shown that a 17base probe may be used to locate a single, unique segment in a randomly ordered, six-billion base DNA sequence, such as the human genome.20 Of course, DNA does not possess a statistically random sequence, and longer probes are generally used. Hybridization on the membrane is used to form a heteroduplex, as shown in Figure 9.16. Heteroduplexes may be dissociated at low ionic strength or at high temperatures. The temperature at which the double-to-single-strand transition occurs is called the melting temperature, Tm . Probes with high G C content have higher Tm values, because a greater number of hydrogen bonds are present in the heteroduplex. The DNA probes may be labeled with single atoms (i.e., radiolabeled), functional groups such as uorophores, or side chains attached to enzymes, to allow the detection of bands in a separation pattern that contain the sequence of interest. The label attached to the DNA probe must not interfere with the hydrogen bonding of the probe to the target sequence. The hybridization step is carried out by immersing the nitrocellulose or nylon membrane in a solution containing excess DNA probe, and incubating at a temperature just below the Tm for the heteroduplex. Excess probe is then removed by rinsing, and the membrane is examined for the location of the label. The DNA probes are often labeled by the nick-translation method.21 This involves the combined activities of three enzymes: DNaseI, 50 ,30 -exonuclease and

If you carry that principle further, you can use the single backup node to protect more hosts, say, 10 (10 percent overhead) or 12 (about 8 percent overhead), in a larger cluster This model, where some number of clustered systems failover to a single dedicated standby node for the whole cluster is called N-to-1 failover The other serious limitation of SCSI-based N-to-1 clusters is that since only the one node (the backup for all the active systems in the cluster) can see all of the disks, when the failed node returns to service, it is necessary to fail its services back to it, freeing up the one node to takeover for another set of service groups As you will see, failing services back isn t necessary for N-plus-1 clusters SANs have greatly simplified the construction and design of N-to-1 clusters, as shown in Figure 17.

3' 5'

8, where we have taken the same 4-to-1 cluster depicted in Figure 177 and migrated it to a properly redundant SAN infrastructure (For more about SANs, please refer to 8, SAN, NAS, and Virtualization ).

Figure 3.26. Example of routing rule definition ( Axway, 2006)

17

50 ,30 -polymerase. The latter two enzymes are present in the E. coli DNA polymerase complex. As shown in Figure 9.17, DNaseI randomly introduces singlestranded breaks, or nicks, by the hydrolysis of double-stranded DNA to create free 30 -hydroxyl groups. The exonuclease then removes one or more of the bases from the 50 -phosphoryl side of this nicked region, while the polymerase catalyzes the incorporation of labeled nucleotides onto the 30 -hydroxyl side, to ll the gap with labeled bases. The labeled nucleotides that are incorporated are complementary, that is, the resulting strand has the same sequence as the original strand. As the reaction proceeds, the nick shifts by one base in the 30 -direction; this process is allowed to continue until 30 60% of the total bases have been replaced by labeled nucleotides. Puri cation of the labeled probe species is unnecessary if the original DNA sample contained only the sequence of interest. By adjusting the DNaseI concentration, it is possible to produce short probes with many labels incorporated, or long probes with few labels. The nick-translation method was originally used to incorporate radiolabeled nucleotides into DNA probes. More recent work has shown that nucleotides labeled with biotin can also be incorporated using this method, to yield probes of equivalent sensitivity. Uracil and adenine nucleotides that have been labeled with biotin are shown in Figure 9.18. By itself, biotin is not a detectable label. It is used as a label because a subsequent irreversible reaction with the proteins avidin or streptavidin (K > 1012 M 1 ) can be used for ultrasensitive detection, if these proteins have been labeled with an enzyme and an activity stain is used. Conjugates of these proteins with alkaline