Do you need supercoiled pDNA for ds-seq.?

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Nov 2 16:41:43 EST 1994


MURIANAP at FOODSCI.PURDUE.EDU (Pete Muriana) wrote:
> 
> ... the DNA sequencing facility representative indicated that 
> we need to have **supercoiled** plasmid DNA to sequence from.  Is this true? 
> and why wouldn't a linear double-stranded fragment work? Am I missing 
> something here?
> 
And Roger Weigand responded

: This is nonsense. Both cycle sequencing and Sequenase work fine from
: linear DNA such as PCR products. We have a number of examples where
: nicking or linearizing (either mechanically or using enzymes) plasmids
: results in a 5-10X *increase* in signal strength for cycle sequencing.

First there was  a long background of linear ds sequencing giving
really ugly results.  Then 'supercoil' seq. came along and it was
really pretty.  The lore developed that the supercoil energy
helped the primer compete to get into the duplex, and that by
precipitating out of alkali, you  froze the DNA in some kind of ccc
denatured form that kept the other strand out of the way.

I have always found this hard to swallow, and I wonder if what really
happens is this: What hurts ds seq. is interference by
reannealing of the other strand.  I think that in supercoil seq. you
only extend the stuff that got nicked and strand separated while the
rest snaps back and never primes.  This would artificially reduce your
template conc. and inhibit template/template reannealing while
increasing your primer/template ratio.  

Notice that the same time this all happened, the labelling procedure
got switched from uniform incorporation to a preextension.  I think
the preextension may be an essential part of this story because it
naturally rescues the signal strength by extending further when you
prime fewer templates.  [because you extend until you run out of
'labelling mix'.  The lore that 'you can extend too long' has nothing
to do with polymerization.  It's because you're letting the other
strand bump the labelled oligo off its template thus terminating it
before it gets to see any ddNTPs.  This makes bands in all 4 lanes at
the bottom of the ladder.]

My evidence for all this is only the following.  If you measure the
length of the blanked out region at the bottom of the ladder, you can
estimate the average length of preextension.  If you compare this to
the template and 'labelling mix' concentrations, you can conclude that
most of your template never primes.   However, there is a competing
explanation for this based on the processivity of the enzyme.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San
Antonio
Hardies at thorin.uthscsa.edu




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