Field of Science

Does local nicking ease uptake?

Posting has become less frequent because proposal writing and teaching are taking my attention away from blogging about research. But yesterday I had a nice idea of another way to probe how USS sequences facilitate uptake.

We're hypothesizing that USSs are sequences that are more easily taken up because they are easily deformed to pass through the narrow secretin pore. If so, a single-stranded nick might take the place of part of the USS, because it should be easy to bend the DNA sharply at the site of a nick.

I remembered old work showing that restriction enzymes, which normally cut both DNA strands at their recognition sites, will cut only one strand in the presence of high concentrations of the DNA-binding dye ethidium bromide. So if we replace part of a USS with a restriction site, we can test whether a nick at that site increases uptake by competent cells. This should be quite easy, as our standard USS for such experiments is cloned into the middle of the restriction sites of plasmid pGEM, giving us lots of built-in sites to work with.

1 comment:

  1. Is there a reason that standard nicking endonucleases would not also work? Sorry if this is obvious: I just happened upon your blog and have used some endonucleases in the past (although almost always standard). But it would seem that if the nicking works, that would be easier than using high conc. of EtBr? From NEB:
    Nicking Endonucleases


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