self-ligation of small dsDNAs

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Jun 25 07:42:00 EST 1992


In article <24JUN199219324913 at wccf.mit.edu>, huh at wccf.mit.edu (GENE SIMON HUH) writes...
>Dear Fellow Netters,
> 
>     I have a question which seems appropriate for this group.  Does anybody 
>know the smallest length of double-stranded DNA which will recircularize 
>in a standard ligation reaction?  That is, what is the length of the smallest 
>circular DNA that can be formed without introducing undue contraint upon DNA 
>bendability?
> 
>Thanks in advance,
> 
>Gene Huh   
>huh at wccf.mit.edu  or  huh at mitwccf
> 
Gene,
	There really are two questions here, and I added a third
1. What's the smallest piece of dsDNA that can be ligated and
2. What's the chance of having 2 ends of the same oligo find each other.
3. What's the product of a ligation reaction.

	And the answers are:
1. DNA ligase requires at least a 5-mer (efficiency is greater with longer
   fragments, but 5-mers seems to be the smallest that will work)
2. I have no idea because the only way most of us do this expt is to use
   some means of biological selection (i.e. circularize a linearized plasmid)
   and after transformation, grow in selection media (i.e. antibiotic) and
   only those cells expressing the phenotype (i.e. antibiotic resistance)
   will grow because the harbor the plasmid.
3. The vast majority of the products are linear joins of several pieces
   to form very large oligos and the probability of joining two ends of
   the same piece to form a circle is very small.  However, in most instances
   we never see these because we then transform and let the cells purify
   the circles away from the linear for us.  Even if we only have >1%
   closed circles, the bugs with the smallest circular plasmids (with ori's
   and antibiotic resistance) will predominate and in the end you'll never
   see the linear stuff.

Cheers,
	Bruce Roe
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