BIOC0005: Tutorial 2 exercise Below are five experimental scenarios. Think about the questions raised in each case, and if necessary use the web links given and other background reading to come up with explanations that you can discuss in your tutorial on Monday 16th October. 1. The mystery of the fickle restriction enzyme I amplified a 1.0 kb region of a human gene using the polymerase chain reaction. To check that I had amplified the expected region I decided to do a restriction enzyme digest of the PCR product. Based on the Genbank sequence for the gene there was a single site for SexAI and for NcoI within the amplified region, so a digest of the linear PCR product with either enzyme should give two fragments in each case (see figure). Sure enough, digests of the PCR product with SexAI or NcoI gave two fragments of the expected sizes. I then cloned the fragment into a 3.0 kb plasmid vector and isolated the recombinant plasmid from an E. coli transformant. Again, I decided to check that all was fine by cutting the plasmid with each enzyme. Since I knew that the vector lacked any sites for either SexAI or NcoI, then I expected to see a single 4.0 kb in each case. Being a good scientist, I also did a negative control where I set up mock digest but didn't add any enzyme. When I ran the digests on a gel, the NcoI one gave a single band of 4.0 kb as expected, but the SexAI digest appeared to have failed since the DNA pattern I got was identical to the uncut control. I repeated the digests of both the PCR product and the plasmid, and got the same result each time - NcoI would cut both DNAs, but SexAI would only cut the PCR product. SexAI NcoI SexAI NcoI PCR product (1.0 kb) Plasmid vector (3.0 kb) Recombinant plasmid (4.0 kb) What is going on with SexAI ? Useful links https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231299/pdf/nihms573347.pdf https://international.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation 1
Answer: DNA propagated in E. coli is methylated by the dam and dcm methylases at GATC and CCWGG sites, respectively. For some restriction enzymes whose target sites match (or overlap) these sequences, then the methylation can prevent the enzyme from cutting. Effectively, the methylation pattern matches that of the RE's corresponding modification enzyme. SexAl is sensitive to dcm methylation so won't cut DNA propagated in E. coli. However, DNA synthesised by PCR is unmethylated, so SexAI will cut this. NcoI is not affected by either dam or dcm methylation so will cut both types of DNA. Note: A work-around to this problem is to propagate DNA in a dam- or dcm-minus strain of E. coli. (This could also lead into a discussion about the importance of knowing the genotype of the E. coli strain you are using in an experiment by checking sites such as: https://openwetware.org/wiki/E ._ coli_genotypes). 2
2. End-labelling DNA with T4 polynucleotide kinase - some like it hot I wanted to make a