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a All the concentrations are in millimolar (mM). A typical experiment is done with nanograms of template. The [dNTP] is used to calculate the corresponding MgCl2 concentration.9 [Reprinted, with permission, from M. Fromant, S. Blanquet, and P. Plateau, Analytical Biochemistry 224, 1995, 347 353. Direct Random Mutagenesis of Gene-Sized DNA Fragments Using Polymerase Chain Reaction . Copyright # 1995 by Academic Press, Inc.]

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canonical format is carried out by implementing two transformation phases on the platform which hosts the integration solution: proprietary format 1 canonical format proprietary format 2.

9

for error-prone PCR are not trivial: Random DNA mutations produce a bias in amino acid changes, where the probability of incorporation is higher for amino acids coded by several codons, like leucine and arginine. The dNTP concentrations in the starting PCR mixture can be adjusted to partially compensate for this, for example, by reducing the total dGTP dCTP concentration. 8.3.2. DNA Shuf ing In this method of generating genetic diversity, the gene of interest is cleaved with DNAse I into many short double-stranded fragments (10 50 base pairs) that are then puri ed and recombined in a PCR-like process without primers. When the genes are partially recombined, terminal primers are added and full-length sequences are ampli ed. This process is usually used after an error-prone PCR step, where different mutants of the gene are generated. Alternatively, the conditions of DNA shuf ing can be adjusted to introduce mutations, and to avoid regeneration of the original sequence.10 Using a powerful derivative technique called family shuf ing , homologous genes pooled from different organisms are recombined. Figure 8.4 shows a comparison of DNA shuf ing and family shuf ing. Family shuf ing uses genes that have evolved in different microbial species from a common ancestral protein, therefore possessing sequences with a high degree of identity. This is a requirement for the process; > 50% sequence identity is needed. Proteolytic enzymes like subtilisin have been shuf ed, using a large family of 26

Contents Network Service Reliability Network Service Dependencies Hardening Core Services Denial-of-Service Attacks Key Points 10 Data Centers and the Local Environment Data Centers Data Center Racks Advantages and Disadvantages to Data Center Racks The China Syndrome Test Balancing Security and Access Data Center Tours Off-Site Hosting Facilities Electricity UPS Backup Generators Cabling Cooling and Environmental Issues System Naming Conventions Key Points 11 People and Processes System Management and Modifications Maintenance Plans and Processes System Modifications Things to Aim For Software Patches Spare Parts Policies Preventative Maintenance Vendor Management Choosing Key Vendors Working with Your Vendors The Vendor s Role in System Recovery Service and Support Escalation Vendor Integration Vendor Consulting Services Security Data Center Security Viruses and Worms Documentation The Audience for Documentation Documentation and Security Reviewing Documentation System Administrators Internal Escalation Trouble Ticketing Key Points 232 233 236 237 240 241 242 244 244 247 247 248 250 252 253 254 255 257 259 261 263 264 265 266 266 268 269 270 271 271 274 275 275 276 276 277 277 279 280 280 282 283 284 284 287 289 290

Contents 12 Clients and Consumers Hardening Enterprise Clients Client Backup Client Provisioning Thin Clients Tolerating Data Service Failures Fileserver Client Recovery NFS Soft Mounts Automounter Tricks Database Application Recovery Web Client Recovery Key Points 13 Application Design Application Recovery Overview Application Failure Modes Application Recovery Techniques Kinder, Gentler Failures Application Recovery from System Failures Virtual Memory Exhaustion I/O Errors Database Application Reconnection Network Connectivity Restarting Network Services Network Congestion, Retransmission, and Timeouts Internal Application Failures Memory Access Faults Memory Corruption and Recovery Hanging Processes Developer Hygiene Return Value Checks Boundary Condition Checks Value-Based Security Logging Support Process Replication Redundant Service Processes Process State Multicast Checkpointing Assume Nothing, Manage Everything Key Points 14 Data and Web Services Network File System Services Detecting RPC Failures NFS Server Constraints Inside an NFS Failover Optimizing NFS Recovery File Locking Stale File Handles 291 292 292 294 296 296 297 297 298 299 301 302 303 304 305 306 308 309 309 310 311 312 313 314 316 317 318 319 319 320 322 323 324 326 326 327 329 330 331 333 334 334 336 337 337 339 341

Chimeric gene(s) selected Screening (b)