Team:Imperial College London/Wetlab/Results/RestrictionDigest

=Restriction Enzyme Digestion=

The aim of this experiment was to investigate the effects of methylation on digestion of genomic DNA with different restriction enzyme concentrations. We performed a genome prep assay on 2 different strains of E.Coli - a Dam positive strain (TOP10) and a Dam negative strain (GM2163). The DNA of the Dam positive strain is naturally methylated, whilst the DNA of the Dam negative strain was unmethylated. This allowed comparisons to be drawn between the amount of cleavage by restriction enzymes. A restriction digest was performed using our 2 restriction enzymes (DpnII and TaqI) each at varying concentrations. The results can be seen below:

DpnII Restriction Digest
Here we can see clearly the differences between the Dam positive and negative strains. The Dam-ve strains are fully cleaved at all different concentrations of restriction enzyme. This produces the 'smear' that can be seen, as the DNA fragments are all of varying, but short lengths. This smear is not present in the control without the restriction enzymes. Good contrast can be seen against the Dam+ve genome, of which increasing amounts of cleavage can be seen with increasing restriction enzyme concentration. At 0.2ul restriction enzyme, the cleavage is minimal, and the gel profile is similar to the negative control. At 1.0ul restriction enzyme, there is significantly more cleavage, and the fragments are beginning to form the 'smear' effect seen in the Dam -ve strain.

TaqI Restriction Digest
Again, there are clear differences between the Dam positive and negative strains. The Dam-ve strains again show the same 'smear' pattern as above, meaning they are fully cleaved at all different concentrations of restriction enzyme. This smear is not present in the control without the restriction enzymes. The results of the Dam+ve strain show clear differences against the Dam-ve, although the differences at different restriction enzyme concentrations are not so clear. This is perhaps because of the different recognition sequence of TaqI to the DpnII. As DpnII cuts at the same recognition site (GATC) as the site methylated by the Dam, every sequencee encountered by the enzyme will be methylated. However, with the TaqI, the recognition sequence is different, and therefore the activity will only be blocked when there is overlap with the Dam's recognition sequence. This could perhaps explain the similarity between the TaqI sequences, as these could be the fragments corresponding to cuts where there was no overlap in methylation.