DNA sequencing - unreadable regions

bio.tamu.edu bio.tamu.edu
Fri Jun 16 09:20:15 EST 1995


> David C. Logan writes:
stuff deleted
> > Finally, the cDNA for which I have only one clone has a very long polyA 
> tail and the sequence is unreadable after it - any ideas on how to overcome 
> this?
> 
> If the problem is that the ladder fades out, then increase the dATP/ddATP
> ratio 2 to 4 fold by increasing dATP in all rxn tubes.  [or dTTP depending
> on which direction you're sequencing].
> If the problem is too many bands, then either the polymerase is stuttering
> on the A tail, or the template is heterogeneous in the length of the A tail
> due to stuttering during the growth of the BlueScript.  We always see the
> latter with M13, but never with pUC, so I wouldn't have expected it of
> BlueScript.  We haven't had the former problem on pA tails of length
> 30 and DeepVent polymerase; but I don't know if longer tails will do it,
> or if different polymerases have different vulnerabilities to this.  
> I vaguely remember some evidence that lowering the rxn pH might improve
> on this.  As above, the simplest solution is to make an internal primer
> and approach the tail on the other strand.
> 
> Good luck.
> 
> Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
> Hardies at uthscsa.edu

Some other thoughts about sequencing a clone with a very long polyA tail. 
I had a clone with ~100 A tail, which proved impossible to sequence by
changing the ratios of dATP/ddATP.  I used a DNAse I deletion strategy to
try and remove most of the polyA tail (contact me at the E-mail adress
below if you want a reference to this method).  I never found a clone that
still had some of the polyA left, but got one within 15 bp of the tail. 
I'm not sure if this is because the DNAse cuts less frequently in
stretches of polyA, or if I didn't screen enough deleted clones (I
probably looked at a hundred or so).

It really depends how seriously you want the sequence at this end of the
clone and if it needs to be double stranded.  Other possibilities include
sequencing from primer sites located within a transposon that you randomly
insert in your clone.  Again, you need to screen for one inserted at a
more convenient position in the polyA tail.  There is a commercial version
of this system that I don't remember the name of.  Perhaps someone else
out there could provide details? :)

Another possibility is to resynthesize your cDNA clone, using PCR with
oligodT and either a gene specific primer or one homologous to the vector
you cloned into.  Then you can look for clones with shorter polyA tails,
and sequence one (or several if you think PCR errors may be a problem) of
these.  Do you really need double-stranded sequence of this clone??? :)

Hope this helps, and good luck
Kim Snowden
IDMB 
Texas A&M University
ksnowden at bio.tamu.edu



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