Protein Engineering Protocols - Mycobacteriology research center

NExT DNA Shuffling 1853.9.2. Calibration of the Program and ComparisonWith Experimental ResultsBefore one can compare the measured and calculated values, there is oneimportant point to consider: the incorporation rate of uridine vs thymidine bythe polymerase in the exchange PCR. This value might not only depend on theratio of dUTP:dTTP, but also on the type of polymerase used, the absolute concentrationof nucleotides, and the buffer. Thus, this value needs to be set whenusing the program. The uridine incorporation could be measured directly usingradioactively labeled dUTP and counting the radioactivity of PCR products or,more indirectly, by quantitatively analyzing the fragmentation pattern. Weopted for the latter approach, because it also provided the basis for the comparisonwith our software. However, one should note that this twofold use of thedata is only valid if the fragmentation is complete.For quantitative analyses of fragmentation experiments, we relied on widelyused conditions (standard Taq polymerase [24] and 200 µM dNTPs).Fig. 5. (Opposite page) Comparison of calculated NExT fragmentations with radioactivelylabeled and ethidium bromide-stained fragmentation experiments. (A) Graphicalfront end of NExTProg 1.0 (22). This program reads DNA sequences and calculatesall possible fragments. The program exports lists of fragment length vs normalizedfraction of molecules or mass, as defined by number of nucleotides, respectively.The sequences of all fragments can be generated, whereby identical sequences arecombined, and exported for subsequent assembly calculations. (B) Denaturing polyacrylamideurea gel of radioactively labeled DNA samples. Lanes 1–3: marker oligonucleotideskinased with 32 P-ATP; lanes 4–6: fragments of a gene (CAT_Cd26, 624 bp),based on the indicated amount of uridine and 32 P-CTP in the exchange PCR (each lanecontained 0.3 µCi). The gel was autoradiographed with a phosphor screen (Kodak) andread with a phosphoimager (BioRad Fx). Note the inhomogeneities in the lower thirdof the lanes, which correspond to sequence-specific peaks in the fragmentation. (C)Measured and calculated fragment distributions used to determine the incorporationrate of uridine vs thymidine in the exchange PCR. The gray line represents a linedensityplot of the radioactive 50% U lane in (B), which was converted from relativemigration distance to nucleotide length based on the marker nucleotides, set to integernumbers by averaging the respective values, and normalized. The black line representsthe calculation of NExTProg for the fragment “mass” distribution for the same genewith 50% uridine and an incorporation rate of 0.26, which provided the lowest meanroot square deviation. (D) The gray line is the line density plot of a 20% U reaction(Fig. 1B,C) stained with ethidium bromide, which was converted to fragment lengthand normalized. The black line is the calculation of NExTProg with 20% uridine andan incorporation rate of 0.26. Note that the staining of short single-stranded oligonucleotideswith ethidium bromide is inefficient and, consequently, longer fragments areoverrepresented in the normalized plot. EtBr, ethidium bromide.