Path: utzoo!attcan!uunet!husc6!uwvax!speedy!noordewi
From: noordewi@speedy.cs.wisc.edu (Mick Noordewier)
Newsgroups: sci.bio
Subject: Re: Obtaining single-stranded linear DNA
Summary: single-strand restriction endonucleases are no problem
Message-ID: <5729@spool.cs.wisc.edu>
Date: 12 May 88 04:29:47 GMT
References: <1792@aecom.YU.EDU>
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In article <1792@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes:
> Here's this week's biochemical puzzler (you all did well on the
> antisense RNA trick question):
                ^^^^^
Really?  What was the trick?  I thought it was an honest question!

> I am interested in obtaining tritiated (or otherwise labeled)
> single-stranded linear DNA of uniform length (has to be at least 1knt
> long) for exonuclease assays.
> 
> M13 seems ideal since the cells will spit out labeled ssDNA (circular)
> if we cook them with label.  How do we make the DNA linear?  Do we shear
> it, or cut it with a restriction endonuclease with ss activity
> (expensive!) or what? The DNA does not have to be linearized in the same
> place, but it must be linear, and cut once, or at most a few times.

Restriction endonucleases with ss activity are not expensive.  Enzymes
known to cut ssDNA are:

	HhaI, HinPI and MnlI (~50% the rate of dsDNA)

	HaeIII (~10% the rate of dsDNA)

	BstNI, DdeI, HgaI, HinfI and TaqI (~100 fold lower activity
				than on dsDNA)

	AluI, HpaII, MspI, FnuDII, MboI, Sau3AI, DpnI, BbvI, FokI, HphI,
	MboII and SfaNI show no activity on dsDNA.


You should be able to get 
enough with one of these enzymes at a reasonable cost.  Of the above,
BstNI is relatively inexpensive, and cuts M13mp18 7 times.  It 
probably leaves at least some chunks that are a kilobase, but I 
don't have a map available at the moment.  Since you are looking 
for a uniform length, you are presumably going to examine activity
by looking at a gel.  If a >1kb band is well enough resolved then
you could simply ignore lower MW bands.

				Mick Noordewier
				noordewi@cs.wisc.edu