Protein Engineering Protocols - Mycobacteriology research center

NExT DNA Shuffling 17372°C, 4 min. An extended elongation time of 2 min was chosen based on a test seriesshowing that the yield was significantly improved compared with shorter times (datanot shown). Depending on the efficiency of the PCR, it can be necessary do four ormore 50-µL reactions to obtain enough product (optimally ~7 µg after gel extraction;see Note 3).4. Combine the PCR samples and separate product from template using a 1%agarose gel (see Note 4). If there is enough product, the DNA can be seen in theagarose gel as a thin red line even without UV light. Purify the excised gel bandusing one or two spin columns (see Note 5) of a gel extraction kit and elute with50 µL elution buffer.5. Determine the concentration of the PCR product by taking the baseline corrected260 nm value of an absorption spectrum from 220 to 350 nm. We usually used a1:30 dilution and measured in a 140-µL microcuvet. The optimal amount for theNExT DNA shuffling procedure is approx 7 µg of DNA. Lower amounts mightwork as well, but sufficient DNA permits a trouble-free gene reassembly (seeSubheading 3.7.). Ideally, use all of the DNA for the next steps.In this study, we focused solely on uridine as the exchange nucleotide, however,the technique could equally be applied to the incorporation of several otheranalogs. One such example, 8-oxoguanine, can be cleaved out by 8-oxoguanineDNA glycosylase (formamidopyrimidine-DNA glycosylase; ref. 10). This basecould prove useful in AT-rich regions, in which DNA cleavage by UDG is too frequent,or in GC-rich genes, in which thymidines are seldom found. Alternatively,it is feasible that a combination of several exchange nucleotides together couldgenerate fragments of the desired range for reassembly. Additionally, incorporationof exchange nucleotides into the primers of the incorporating PCR shouldensure that these regions of the gene library are also shuffled.3.3. Enzymatic Digest and Chemical CleavageTo fragment the gene, the exchange nucleotide is excised by incubating theproduct of the uridine-exchange PCR with the enzyme UDG (11). This enzymeis capable of attacking double-stranded as well as single-stranded DNA usinga hydrolytic mechanism to remove, with high specificity, the uracil moiety bya nucleophilic attack at the C1′ position of deoxyuridine (12). Piperidine isused to split the backbone positions where the uracil has been removed byFig. 1. (Continued) lanes 5 to 10 of (B), detailing the fragment sizes based on the fractionof dUTP used. For the shuffling of all CAT variants, a uridine-exchange PCR containing33.3% dUTP was used, producing fragments ranging from 30 to 200 bases inlength (thick line). The image was acquired with a FluorS Multiimager, and the plot wasgenerated using the Quantity One software program (Bio-Rad). For clarity of the plot, thesignal of the 100-bp ladder was shifted by –750 counts and the signal of the oligonucleotidesby –500 counts. Dig., digested; undig., undigested; nt, nucleotide.