Protein Engineering Protocols - Mycobacteriology research center

174 Stebel et al.UDG (13). The result of such a cleavage reaction can be analyzed with high resolutionon denaturing polyacrylamide urea gels (see Subheading 3.4.) fordUTP fractions ranging from 100 to 0% (Fig. 1B) and quantified by imageanalysis (Fig. 1C). For very small fragments, radioactive labeling can be usedfor better visualization (see Note 2).1. For the enzymatic cleavage with UDG supplement, approx 45 µL (typically 7 µgDNA) of the purified PCR product from Subheading 3.2., step 4. with 6 µL ofsupplied 10X UDG buffer and 2 U of E. coli UDG. Adjust the volume to 60 µLwith water, and incubate the digest for 1 h at 37°C (see Note 6).2. To cleave the DNA, add piperidine to a final concentration of 10% (v/v; 6.7 µLpiperidine for 60 µL enzymatic digest) and heat for 30 min at 90°C in a thermocyclerwith a heated lid (see Note 7).As an alternative to piperidine with mild conditions, other endonucleases, suchas endonuclease IV (14), exonuclease III (15), or T4-endonuclease V (16) can beused to cleave the DNA backbone (11). We tested the latter but discontinued itsuse. T4-endonuclease V cleavage after UDG treatment resulted in incompletefragmentations, as seen by the size distribution generated (Fig. 2A). Even moreproblematic was the high error rate inherent to this procedure. Applying a UDGand T4 endonuclease V fragmentation with gel extraction and a reassembly, asdescribed for a CAT wild-type gene, and sequencing six clones with a total of3930 bases gave a mutation rate of 1.75%. We propose two reasons for this finding.First, the DNA backbone is cleaved by the T4 endonuclease V with its lyaseactivity, which catalyses a β-elimination reaction, leaving a 3′ unsaturated aldehyde(4-hydroxy-2-pentanal) attached at the phosphate group (11,17). The furtherchemical reaction leading to the free phosphate group is unlikely to be complete,therefore, such generated fragments are an unfavorable starting point for a polymerase.In addition, as seen by the fragment comparison, not all sites with incorporateduracil in the backbone are cleaved. However, the uracil moiety mightnonetheless be missing, thus base lacking templates might lead to erroneousnucleotide incorporation. Miyazaki (18) proposed the use of E. coli endonucleaseV, however, according to the data provided, cleavage was even less efficient, andshort fragments were not obtained even after prolonged digests (12 h) and highdUTP fractions (75%). Further experiments could be designed to solve theseproblems, but were not performed because the piperidine cleavage worked welland was much more cost-effective.As a chemical alternative to piperidine, we tested NaOH. We replaced thepiperidine solution with a 0.5 M NaOH solution, which was added at 10% (v/v)to the DNA and treated for 30 min at 90°C. The fragmentation result of a piperidineand a NaOH cleavage started from the same uridine-exchange PCR wascompared on a denaturing polyacrylamide gel with subsequent ethidium bromidestaining. The intensity distribution of the two fragmentations was almost