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NExT DNA Shuffling 1713. Methods3.1. Cloning StepsThe priming sites of the shuffling primers, containing the restriction sitesused for the NExT-shuffling procedure, should ideally be located shortly beforeand after the gene of interest and have a melting temperature of approx 60°C.We estimate melting temperatures using the 4 plus 2 rule: 4°C for each C andG, and 2°C for each T and A pair.The genes we used in the NExT DNA shuffling procedures were the 657-bpCAT wild-type gene (CATwt; SwissProt: P00483; <strong>Protein</strong> Data Bank: 1NOC:B)and variants coding for an N-terminal 10-amino acid-truncated, C-terminal9-amino acid-truncated, or double-truncated CAT (CAT_Nd10, CAT_Cd9, orCAT_Nd10_Cd9), or a C-terminal 26-amino acid-truncated CAT (CAT_Cd26).These genes and all shuffled genes have been cloned into the vector, pLisc-SAFH11 (7), using the restriction sites XbaI and HindIII, thus, replacing partof the original plasmid. For diversification by error-prone PCR and the uridineexchangePCR (see Subheading 3.2.) the cloned CAT genes were amplifiedusing the primers Pr-N-shuffle (5′-ATTTCTAGATAACGAGGGCAA-3′) andPr-C-shuffle (5′-ACTTCACAGGTCAAGCTTTC-3′) for the wild-type andN-terminal truncated genes; and Pr-N-shuffle and Pr-Cdx-shuffle (5′-CTTCACAGGTCAAGCTTATCA-3′) for the C-terminal truncated and the double-truncatedgenes. Plasmids are best transformed in E. coli using standard methods (8).3.2. Uridine-Exchange PCRUridine was chosen as the exchange nucleotide because dUTP is known to beincorporated into DNA by various polymerases (9). The uridine-exchange PCRamplifies the gene pool of interest and additionally incorporates the exchangenucleotide, uridine. Various ratios of dUTP:dTTP can be used to obtain optimalfragmentation. This can be done either experimentally by analyzing various U:Tratios, as detailed in Subheading 3.4., or using our program that was developedfor this purpose (see Subheading 3.9.). No apparent difference in the amount ofPCR product was observed when using dUTP fractions of up to 50% within thedUTP and dTTP pool. Only the PCR containing uridine alone was found to yieldapproximately a quarter of the product compared with other reactions (Fig. 1A).1. For accurate pipetting, dilute 100 mM nucleotide stock solutions with water to10 mM for dATP, dGTP, and dCTP, and to 1 mM for dUTP and dTTP.2. For the uridine-exchange PCR mixture use 50 ng template (0.017 pmol of a 4340-bpplasmid), 25 pmol of each primer, 0.4 mM of dATP, dGTP, and dCTP each, a 0.4 mMmixture of dUTP:dTTP in various ratios, 5 U Taq DNA Polymerase, and 5 µL of 10XPCR buffer. Adjust the final volume to 50 µL with water (see Notes 1 and 2).3. The thermocycler program is: one cycle of 94°C for 1 min; 25 cycles of 92°C, 30 sdenaturation; 62°C, 20 s annealing; 72°C, 2 min extension; and a final incubation at
172 Stebel et al.Fig. 1. Overview and quantification of NExT DNA fragmentation. (A) 1% agarose gelshowing the uridine-exchange PCR products of CAT_Nd10 clones obtained with differentamounts of uridine in the reactions. Percent U was calculated by {c(dUTP)/[c(dUTP)+ c(dTTP)]} × 100. (B) Polyacrylamide urea gel stained with ethidium bromide showingUDG/piperidine digests of CAT_Nd10 PCR products obtained with various dUTP:dTTPratios (1:0, 0:1, 1:1, 1:2, 1:3, 1:4, and 1:5) to determine an optimal ratio. Lane 1: oligonucleotideswith 58, 48, and 36 bases as size markers; lane 2: 100% dUTP, PCR digested; lane3: 0% dUTP, digested; lane 4: 0% dUTP, undigested; lane 5: 50% dUTP, digested;lane 6: 33.3% dUTP, digested; lane 7: 25% dUTP, digested; lane 8: 20% dUTP, digested;lane 9: 16.7% dUTP, digested; lane 10: 100-bp DNA ladder. Note that residual amountsof piperidine contribute to slightly distorted lanes. (C) Lane density plot of lane 1 and
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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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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 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
Page 202 and 203: NExT DNA Shuffling 1894. Zhao, H.,
Page 204 and 205: 11Degenerate Oligonucleotide Gene S
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222 Fujita et al.The methods that h
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224 Fujita et al.3. Streptavidin an
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226 Fujita et al.Fig. 2. (Continued
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228 Fujita et al.Fig. 3. (Continued
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230 Fujita et al.Fig. 3. (A) Schema
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232 Fujita et al.1. Add 2 µg mRNA
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234 Fujita et al.Innovative Bioscie
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236 Fujita et al.30. Kim, Y., Mlsa,
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238 Ghadessy and Holligeraffinity m
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240 Ghadessy and HolligerFig. 1. (A
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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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