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Degenerate Oligonucleotide Gene Shuffling 1939. Destaining solution: 1 M NaCl, 5 mM NaOH.10. <strong>Protein</strong> extraction reagent BPER II (Pierce Chemical Company, Rockford, IL).11. Birchwood xylan substrate solution: Add 0.5 g Birchwood xylan to 100 mL of 120mM Universal buffer, pH 6.5. Stir 5 min until homogeneous, then autoclave tocompletely solubilize the xylan.12. PAHBAH stock solution: Add 5 mL of each of 0.5 M Na 3citrate, 1.0 M Na 2SO 3,0.2 M CaCl 2, and 5.0 M NaOH to 25 mL water, mixing after each addition. Add0.76 g p-hydroxybenzoic acid hydrazide (Sigma-Aldrich), mix, and bring volumeup to 50 mL in water.3. MethodsAs an example of the DOGS procedure, we give examples from the shufflingof the D. thermophilum Rt46B.1 xylanase gene, xynB, with five related xylanasegenes, reported by us previously (7). Figure 1 shows schematically the stepsinvolved in the design of primers and the subsequent primer extension steps tocreate the shuffled segments that are incorporated into the final amplified andcloned chimeric genes (see Note 1).3.1. Complementary Degenerate-End Primer Pairs for Efficient PCRAmplification of Gene Segments and Overlap-Extensionof Adjacent SegmentsThe most commonly used strategy to isolate distantly related sequences byPCR has been to design degenerate primers that bind to highly conservedregions of DNA sequences. The difficulty with this method is that, as the primerdegeneracy increases to accommodate more divergent genes, the number ofprimer molecules in a PCR that can correctly prime synthesis drops, and theseprimers may be used up in the first few cycles of the reaction. Nonspecificamplification may then occur because of the abundance of primers that do notparticipate in amplification of the targeted gene, and, therefore, are available toprime nonspecific synthesis, especially because low stringency annealing conditionsare usually needed to detect mismatched templates.Rose et al. (13) described a strategy that overcomes problems of degeneratemethods for primer design, called consensus–degenerate hybrid oligonucleotideprimers (CODEHOP). CODEHOP primers consist of a relatively short3′ degenerate end and a 5′ nondegenerate consensus clamp. Reducing the lengthof the 3′-end to a minimum decreases the total number of individual primers inthe degenerate primer pool. Hybridization of the 3′ degenerate end with the targettemplate is stabilized by the 5′ nondegenerate consensus clamp, whichallows higher annealing temperatures without increasing the degeneracy of thepool. Although potential mismatches may occur between the 5′ consensusclamp of the primer and the target sequence during the initial PCR cycles, they
194 Bergquist and GibbsFig. 1. Schematic diagram of DOGS procedure.are situated away from the 3′ hydroxyl extension site, and, thus, mismatchesbetween the primer and the target are less disruptive to priming of polymeraseextension. Further amplification of primed PCR products during subsequent
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METHODS IN MOLECULAR BIOLOGY 352Pr
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M E T H O D S I N M O L E C U L A R
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PrefaceProtein engineering is a fas
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ContentsPreface ...................
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ContributorsKATJA M. ARNDT • Inst
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Contributors xiKAZUNARI TAIRA • D
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1Combinatorial Protein Design Strat
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Combinatorial Protein Design Strate
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2Global Incorporation of Unnatural
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Incorporation of Unnatural Amino Ac
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3Considerations in the Design and O
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Design of Coiled Coil Structures 37
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4Calcium Indicators Based on Calmod
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Protein-Based Ca 2+ Indicators 73Fi
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Protein-Based Ca 2+ Indicators 7512
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Protein-Based Ca 2+ Indicators 8145
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5Design and Synthesis of Artificial
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Design of Zinc Finger Proteins 853.
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Design of Zinc Finger Proteins 89pr
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Design of Zinc Finger Proteins 91Fi
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96 Koide and Koidewhile retaining t
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98 Koide and Koide5. M9-tryptone: M
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100 Koide and Koidetarget-binding s
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102 Koide and Koide4. Discard the s
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104 Koide and Koideup to 1 mM for h
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106 Koide and Koideplate. Incubate
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108 Koide and Koide1. Perform steps
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7Engineering Site-Specific Endonucl
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Engineering Site-Specific Endonucle
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115Fig. 1. Mapping group-specific r
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8Protein Library Design and Screeni
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Protein Library Design and Screenin
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135Fig. 2. Excel worksheet describi
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137Fig. 4. Excel worksheet describi
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Page 156 and 157: Protein Library Design and Screenin
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Page 170 and 171: Protein Design by Binary Patterning
Page 180 and 181: 10Versatile DNA Fragmentation and D
Page 182 and 183: NExT DNA Shuffling 169This chapter
Page 184 and 185: NExT DNA Shuffling 1713. Methods3.1
Page 186 and 187: NExT DNA Shuffling 17372°C, 4 min.
Page 188 and 189: NExT DNA Shuffling 175Fig. 2. Varia
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Page 192 and 193: NExT DNA Shuffling 179Fig. 3. Reass
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Page 196 and 197: NExT DNA Shuffling 183likelihood of
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Page 200 and 201: NExT DNA Shuffling 187staining. Bec
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Page 204 and 205: 11Degenerate Oligonucleotide Gene S
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Page 235 and 236: 222 Fujita et al.The methods that h
Page 237 and 238: 224 Fujita et al.3. Streptavidin an
Page 239 and 240: 226 Fujita et al.Fig. 2. (Continued
Page 241 and 242: 228 Fujita et al.Fig. 3. (Continued
Page 243 and 244: 230 Fujita et al.Fig. 3. (A) Schema
Page 245 and 246: 232 Fujita et al.1. Add 2 µg mRNA
Page 247 and 248: 234 Fujita et al.Innovative Bioscie
Page 249 and 250: 236 Fujita et al.30. Kim, Y., Mlsa,
Page 251 and 252: 238 Ghadessy and Holligeraffinity m
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Page 255 and 256: 242 Ghadessy and HolligerFinally, t
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244 Ghadessy and Holliger2. Prepare
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246 Ghadessy and Holliger2. After 6
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248 Ghadessy and Holliger21. Oberho
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250 Campbell-Valois and Michnickscr
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252 Campbell-Valois and Michnickfol
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254 Campbell-Valois and Michnick7.
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256 Campbell-Valois and MichnickFig
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258 Campbell-Valois and Michnickres
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260Fig. 3. BL21 pREP4 cells were co
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262 Campbell-Valois and MichnickFig
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264 Campbell-Valois and Michnickvar
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276 Hecky et al.improved half-life
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278 Hecky et al.Fig. 1. (A) Scheme
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280 Hecky et al.11. Reverse primer
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282 Hecky et al.high variability in
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284 Hecky et al.coding sequence for
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286 Hecky et al.4. Analyze the resu
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Table 1Amino Acid Substitutions Sel
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290 Hecky et al.Fig. 4. Structure o
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292 Hecky et al.Fig. 5. Expression
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294 Hecky et al.Table 2Kinetic Para
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296 Hecky et al.The thermoactivity
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298 Hecky et al.Fig. 8. Unfolding o
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300 Hecky et al.for the first trans
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302 Hecky et al.7. Thompson, M. J.
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304 Hecky et al.40. Wang, X., Minas
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306 IndexCCalcium indicators, see C
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308 Indexchemiocompetent cellprepar
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310 Indexmaterials, 169, 170mutatio
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312 IndexTerminal truncation, see a
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