Protein Engineering Protocols - Mycobacteriology research center

186 Stebel et al.1. Label DNA radioactively by adding 32 P-dCTP in the uridine-incorporating PCR(see Subheading 3.2. and Note 2).2. Run a denaturing polyacrylamide gel (see Subheading 3.4.).3. Autoradiograph the gel with an Imager (see Note 23). This avoids signal distortionscaused by inefficient staining of short fragments.4. Create line-density plots for each lane with an image analysis software program(e.g., QuantityOne (BioRad) or NIH Image/Scion Image/ImageJ).5. Calculate the relative migration for the fragment smear and the radioactivelylabeled markers from the line-density plots.6. Fit the equation rel.distance = a × ln(length in basepairs) + b, with the variables aand b being the relative distance and length of the markers.7. Convert the relative distance of the fragment smear to nucleotide length using thereverse of the above equation.8. Convert the intensity signal vs a continuous length distribution to a intensity signal vsinteger numbers (only discrete oligonucleotide lengths can be present) by combiningrounded nucleotide length values and by averaging the respective intensity values.9. Normalize the distribution by dividing each intensity value through the sum of allvalues.An autoradiograph of such a denaturing gel is shown in Fig. 5B. The normalizedintensities taken from the gel are shown as “%mass” in Fig. 5C. This experimentwas compared with program calculations to determine the relative uridinevs thymidine incorporation value. For the calculations, incorporation values from0.2. to 0.3, in 0.01 increments were used. Setting a value of 0.26 resulted in thebest agreement of measurement and prediction based on a root mean squareanalysis of the fragment size range between 10 and 150 bases, where the lengthcalculation is likely reliable and between 4 and 200 bases (Fig. 5C). In the caseof using the range between the markers (20 and 100 bases), a factor of 0.25 scoredmarginally better. It is remarkable how well these plots overlay, and how wellpeaks and dips within the fragment smear (indicated by arrows in Fig. 5B,C) canbe explained. Importantly, the scaling of the y-axis falls into place without furtheradjustment. We are very confident that the uridine incorporation rate has beendetermined and not a factor accounting for incomplete fragmentation, because ofthe nearly perfect agreement of calculation and experiment, and the many experimentalconditions tested for fragmentation. Measurements of the radioactive33.3% uridine fragmentation and calculations agreed equally well. The radioactive25% dUTP fragmentation produced significant amounts of fragments beyondthe 100-nucleotide marker, and showed some deviations caused by scaling problems.However, using the same incorporation rate value of 0.26 and comparingthe results with the ethidium bromide-stained fragmentation results, which weremore accurate for longer fragments because of the available markers, demonstrateda good agreement (Fig. 5D) considering the length dependency of the gel