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
cc:
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=
ents,
> preferentially between 20 and 50bp. Ideally, cleavage should create blu=
nt ends
> 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=
NA is
> 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=
 3bp)
> recognition site. I remember that I saw a paper some time back where th=
e
> authors used something like that, but I don=B4t remember the details (n=
ot even
> 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,
> Frank
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.  =
--Dom Spinella










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