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Can the size of a supercoiled plasmid DNA be determined by using standard DNA size fragment electrophoresed in parallel? 2. An unknown DNA molecule was cleaved using several restriction enzymes individually and in various combinations. The DNA fragment sizes were determined by agarose gel electrophoresis and the restriction enzyme recognition sites were mapped. Subsequently, the DNA was sequenced and an extra recognition site was found for one of the enzymes. However, all the other mapping data was consistent, within experimental errors, with sequence data. What are the simplest explanations for this discrepancy? Assume the DNA sequence had no errors.

My answer:

the extra enzyme recognition site was cut-off with the fragment due to partial digest that occurred because of insufficient enzyme was added or the rxn was stopped after a very short time.

Am i wrong? my logic here was such that they only found the extra recognition site after they sequenced the DNA thus it was not available during the rxn and thus it was either cut off unevenly in one of the fragments OR it could have had the complementary sequence to it that was methylated that hid it.

Detailed answers are appreciated!!

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  • $\begingroup$ The scenario in your question can be explained if the "extra" recognition site mapped very close to another recognition site for the same enzyme. $\endgroup$ – mdperry Oct 30 '15 at 4:47
  • $\begingroup$ you mean if say i had ECOR1___HindIII___ECOR1 the extra recognition site would be neglected? $\endgroup$ – Jx1 Oct 30 '15 at 6:40
  • $\begingroup$ Yes, exactly. Although I was envisioning, for example, 2 EcoRI sites separated by between 0-50 bp of plasmid DNA. Depending on the type of gel being used, the resulting tiny DNA fragment generated could be difficult to detect (either because they run off the bottom of the gel, or because they don't resolve). Similarly, depending on the size of the adjacent fragments in the various digests, and the assay's limit on estimating fragment sizes, you might not detect that an "extra" fragment was generated, and "missing". $\endgroup$ – mdperry Oct 30 '15 at 10:33
  • $\begingroup$ thanks perry, could you copy your answer and post it under answers? I would love to list you as a the correct anwer owner $\endgroup$ – Jx1 Oct 31 '15 at 1:10
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The scenario in your question can be explained if the "extra" recognition site mapped very close to another recognition site for the same enzyme.

For example, if there were 2 EcoRI sites separated by between 0-50 bp of plasmid DNA, then depending on the type of gel being used, the resulting tiny DNA fragment generated could be difficult to detect (either because small fragments run off the bottom of the gel, or because they don't resolve well unless the gel contains a higher percentage of agarose or acrylamide).

Similarly, depending on the size of the adjacent fragments in the various digests, and this assay's limitations on estimating fragment sizes, you might not detect that an "extra" fragment was generated, and "missing".

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