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Last week I tried to introduce single-strand nicks into a closed circular plasmid (pUSS-R) by doing restriction digestion in the presence of ethidium bromide (background is here). I tested two different dilutions of HindIII, a restriction enzyme that should cut this plasmid once, with three different concentrations of ethidium bromide, to find conditions where a convenient range of incubation times gave a range of partially digested molecules.
I was hoping to see what's shown in the upper gel drawing - appearance of a novel band that migrated slower than the fully digested DNA in the rightmost lane and much slower than the supercoiled DNA in the leftmost lane. This new band would contain plasmid that had undergone a single-strand nick at its single HindIII site. I didn't know whether I might also see eventual appearance of linear DNA. But instead I saw what's shown in the middle gel drawing - gradual appearance of a linear-sized band as the supercoiled band disappeared.
I wondered if the HindIII now being sold no longer causes nicking (maybe New England Biolabs has 'improved' their HindIII clone...). So I did the control shown in the lower gel drawing, incubating pUSS-R with a very low concentration of DNase I, an enzyme widely used to create nicks in double-stranded DNA. Again I expected to see a novel, slow-migrating band, but instead saw only a linear-sized band. Damn!
I also ran samples from a few old plasmid preps. Most of these contained several bands, but I don't know if the slowest one is relaxed circles or dimers, because the plasmids may have been prepared from rec+ cells. I've now also checked the old literature, just in case I was mistaken in expecting nicked/relaxed DNA ("form II") to migrate slowly, but indeed it should (see for example the marker lanes in Fig. 2 on PNAS 86:1309).
Now what? Is the problem that the nicked DNA migrates at the same speed as linear DNA? Is this specific to this particular plasmid? Or do I not have any nicked DNA? Should I try another plasmid?
Rosie -- I don't have my old notebooks handy, but IIRC, the relative gel mobilities of supercoiled (Form I), relaxed/nicked (Form II), and linear (Form III) DNAs for a given plasmid depended hugely on gel concentration, gel buffer, and voltage gradient ... sorry I don't remember the details, but maybe this comment will jog someone else's memory.
ReplyDeleteCould you use another method to produce (unspecific) nicks and run it on the gel as a control? That would tell you how or where nicked DNA runns.
ReplyDeleteFirst in relation to what Guy wrote, if you would run it on a lower density agarose gel (0.7% for example) and with lower voltage gradient you should theoretically be able to get better separation of these molecules.
ReplyDeleteSecond, HindIII R.E. from NEB is a time saver enzyme meaning it digests DNA really fast. I’m guessing that the different time points where achieved by heat inactivation of the reaction, there for with an enzyme that fast I don’t know how effectively you'll be able to see the difference between the time points in the aspect that interests you.
If it bugs you this much I think you have several options, you could:
a) Theoretically, every plasmid extracted from bacteria should give 3 bends upon electrophoresis 1 for each mode (linear relaxed etc.).
Do miniprep of this plasmid along with another plasmid just as control, after that electrophores them to see if 3 bends appear on the gel. If so to every reaction add a non cut control when running it on gel.
b) Increase the concentration of the plasmid in the reaction while lowering that of the RE if you continue using a fast digest enzyme.
c) Conceder changing a plasmid
d) Take the more expensive and longer (but potentially shorter) option I have mentioned at the earlier post :)
Ido.