1
$\begingroup$

Is there a way to linearize plasmids in a sequence-independent manner?

The circular plasmid should be cut ideally only once. It does not matter where. But as soon as it is linearized, it should not be cut any more.

We are talking about plasmids up to about 20kb.

$\endgroup$
5
  • $\begingroup$ Do you have any information on your plasmid? $\endgroup$
    – Chris
    Commented Nov 9, 2022 at 14:27
  • $\begingroup$ The method should not be dependent on the host/origin or on any other characteristics. It should work for bacterial plasmids, constructs, etc. So basically as long it is circular double stranded DNA the approach should work. $\endgroup$
    – Michael
    Commented Nov 9, 2022 at 14:30
  • $\begingroup$ When you write "it does not matter where", does that mean it can cut in the insert of a recombinant, or is it necessary that the cut be in the plasmid vector itself? $\endgroup$
    – David
    Commented Nov 10, 2022 at 23:04
  • $\begingroup$ @David Yes, it can cut the insert as well. It really does not matter where in the plasmid. $\endgroup$
    – Michael
    Commented Nov 11, 2022 at 14:05
  • 1
    $\begingroup$ Ok. Well, I think my answer is ok. If it had to cut the plasmid you might have to engineer a DNAase attached to a protein that specifically binds to the origin of replication or the like — a major undertaking if it doesn’t already exist. $\endgroup$
    – David
    Commented Nov 11, 2022 at 14:12

1 Answer 1

3
$\begingroup$

I was going to write a facetious comment asking if the poster believed in fairies, but I restrained myself. Why? 1. because it would not have been nice, 2. because it occurred to me that there was a hack you might be able to use to do this:

Let Nature do the work for you.

Long, long ago, I used to do DNA cloning myself, and so I frequently ran plasmids on agarose gels. I have lost most of the photos, but I happened to contribute a chapter to a text book in which I presented an example, so I reproduce a photo below:

pBR322 on agarose gels

What this shows is that in practice (at least they way I did it) a plasmid preparation does not consist of just a single band representing supercoils of the double-stranded closed circular DNA. Typically, and to varying degrees, there are two more species produced by shearing (or perhaps slight nuclease contamination) — relaxed nicked circles and doubly-nicked linear species.

The linear species would appear to be what the poster wants. (He doesn’t say what he intends to do with it, so I can't be sure.) If so, the band in the gel could be cut out, placed in a dialysis bag in suitable solution, and subjected to an electric field, when the DNA will be removed from the gel into solution by electrophoresis. (I can’t remember the details of the latter. I used to do it routinely, so it should be well documented on the Internet, and there may well be modern, more efficient ways of retrieving DNA from agarose gels.)

But if you wish to improve on Nature…

The proportion of linear species in the gel above is relatively low, so one might wish to go one better. In fact my original thought was to use low concentrations of DNAase calibrated to produce just the two cuts that would generate linear species, before I realized that there was probably no need to go to the trouble of calibration as plasmids contained nicked linear species already. There are many commercially available non-specific endonucleases such as bovine DNAase I that I imagine one could use. I am not quite sure how one would terminate the reaction and inactivate the DNAase. Heat perhaps; one could check how this is done in other procedures like footprinting.

It is possible that the nicking is caused by physical shearing, in which case one could perhaps vortex the plasmid to increase this instead of using DNAase.

The general idea of calibration to obtain the ‘right’ incomplete reaction is seen in the chain-termination method of DNA sequencing, for example. Any incomplete reaction will always give a range of species, of course.

$\endgroup$

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .