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This models the relationship between repertoire size and repertoire completeness, and shows that at least 108 1010 different antibodies are needed in the repertoire if a threshold Kd of 10 8 10 10 is required, depending of the probability selected (Fig. 8.3).3 Therefore, for one initial or starting sequence, the rst step in the directed evolution cycle must be capable of generating 108 1010 nucleic acid sequences that code for different proteins. This library or repertoire size is needed for directed evolution according to this model. However, good results have been obtained experimentally (mainly with enzymes) using smaller libraries, of $104 members.6,7 Although early work in this area used physical and chemical mutagenesis, there are two main methods that are now used to generate diversity: error prone PCR and DNA shuf ing. Both techniques require the initial selection and isolation of the gene or genes of interest. Restriction endonuclease enzymes can be used to cleave the selected gene per second from chromosomal genetic material; this is followed by puri cation and eventually ampli cation. Often the starting gene is isolated from the corresponding messenger RNA (mRNA), and the enzyme reverse transcriptase is used to obtain the corresponding complementary DNA (cDNA).8 8.3.1. Polymerase Chain Reaction and Error-Prone PCR PCR is normally used to increase the concentration of a given DNA sequence for analysis. For this primary use, the reagent concentrations and other conditions are carefully controlled to avoid the introduction of mistakes (mutations) during the copying process. In this reaction, a DNA sequence (template) up to 10,000 bases is used. DNA polymerase from a thermophilic bacterium is used. For example Taq-polymerase

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(from Thermus aquaticus) is used to catalyze the extension of two primers by the sequential incorporation of complementary nucleotides, as shown in Figure 109 Taq-polymerase has high thermal stability, retaining activity even after many heating cycles at 95 C Primers are single-stranded, short DNA sequences, frequently 15 25 bases long, complementary to the two sequences at the 50 ends of the region of interest in the double-stranded template DNA In this region, the Taq-polymerase begins to copy the template by extending the primers in the 30 direction, until the template is totally copied or the process is stopped by a temperature increase that denatures the DNA The number of copies obtained is equal to 2n 1, where n is the number of cycles Substrate exhaustion normally occurs after 20 30 cycles Error-prone PCR uses altered reaction conditions in order to increase the rate of production of copying errors.

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Normally, when an application writes to a disk, the disk tells the operating system that the write has completed, so that the application can move on to its next task. When a disk-write cache is employed, the disk reports successful completion of the data write when it is written to the cache, not when it makes it to the disk. Since a cache is nothing more than memory, it is volatile; if power is lost to the cache, its contents are lost with it, regardless of whether or not they made it to the disk. Write caches can live in the disk array, or on the host s disk controller. Caches in the disk array are fine, as long as they have battery backups. Caches without battery backups will lose their cached data if power to the disk array is lost. A write cache on the system controller board is not acceptable in a failover environment. When a failover takes place, the cached data is locked inside the cache, and the other system has no way to access it, or even to be aware of its existence. Prestoserve and Sun s Storedge Fast Write Cache are two examples of system-based disk-write cache boards. Although they can do an excellent job of improving I/O performance on a standalone system, those boards are not acceptable or supportable in shared disk configurations.

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One of the more interesting questions that comes up as engineers configure HA systems is where to place the executables for critical applications. There are, of course, two choices: You can place them on the private disks, along with the boot and system information, or you can place them on the shared disks along with their associated data. If you install the applications on the shared disks, the good news is that you only need to maintain one copy of the executables and of the associated configuration files. If you need to make a change in the application configuration, you only need to make it in one place. If you install the applications on the private disks, then you must maintain two copies of the executables and of their configuration files. Changes must be made twice; otherwise, a failover will not guarantee an identical environment on the other side. However, with just one copy of the applications, it is almost impossible to install an upgrade to the application safely, and with the ability to roll back. With two copies of the application, and system mason active, you can install the upgrade on system dixon and failover to dixon. If the upgrade was successful, then dixon should take over, running the upgraded application correctly; you can then upgrade mason and move on. If the upgrade fails, however, then you need only fail back to system mason, and try the upgrade again on dixon. This is one of those cases where there really is no single right answer. What we can tell you, though, is that more often than not, we have seen a single copy

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