enzymatic cleavage of DNA to small pieces (20-40bp)?
Stewart P Johnson
Johns053 at mc.duke.edu
Tue Apr 28 11:57:00 EST 1998
You can use DNase I to do this. Just do the reaction in the presence of Mn
instead of Mg, which will generate double-stranded breaks in the DNA. You
will need to titrate the enzyme so that you generate fragments 20-40 bp.
After DNase digestion, run the digest on a gel and purify the DNA fragments
of that size. I haven't done this for 20-40 bp fragments, but it worked
well for 100-200 bp fragments.
dspinella at chugaibio.com (Dom Spinella) on 04/28/98 10:53:01 AM
Please respond to dspinella at chugaibio.com
To: methods at net.bio.net
Subject: Re: enzymatic cleavage of DNA to small pieces (20-40bp)?
"Frank O. Fackelmayer" <fof1 at chclu.chemie.uni-konstanz.de> wrote:
> Hi all,
> I=B4m currently thinking about enzymatic cleavage of DNA to small fragm=
> preferentially between 20 and 50bp. Ideally, cleavage should create blu=
> that work for cloning. So, DNase I or micrococcal nuclease are not the
> enzymes of choice, also because they don=B4t stop digestion until the D=
> broken down to TOO short fragments (1-4nucleotides each).
> What I would like to use is a kind of restriction enzyme with a 2bp (or=
> recognition site. I remember that I saw a paper some time back where th=
> authors used something like that, but I don=B4t remember the details (n=
> if I saw the paper published or it was one I had for review...)
> Does anyone know of a method to break down DNA in the way I described?
> Any hints appreciated,
Hi Frank. Sorry I don't know of any 2 or 3 base-recognizing restriction
enzymes, but what about just cutting your DNA with a series of 4-base
blunt cutters? I bet a combination digest with Alu I (AGCT), DpnI
(GATC), HaeIII (GGCC) and RsaI (GTAC) -- all of which leave blunt ends
-- would generate average fragment sizes in the range you are looking
for. It might even be over-kill and you could get away with just 3. In
fact, I remember a protocol in the Red Book a few years ago about
subtracted cDNA libraries that involved generating driver DNA using a
combination of Alu I and Rsa I to yield small fragments like this.
Anyway, just a thought... Good luck. =
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