DNA sequencing - unreadable regions

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Thu Jun 15 11:09:19 EST 1995


David C. Logan writes:

> I'm in the process of sequencing a couple of complete cDNAs and although 
the sequence is mainly of good quality there are a couple of regions which 
cannot be read. Sequencing from dsDNA in pBluescipt.

> The two cDNAs encode related proteins. At each end of each cDNA (I have one 
clone of one cDNA and 5 of the other) there is a region of 20-30 
nucleotides forming a "box" of parallel lines extending across all four 
lanes - it is not compressed. These regions are at the ends of the 5-prime 
and 3-prime UTRs and perhaps this is significant (function?). 

Could it be poly(G) or poly(C) left over from the cloning strategy?
If it's right off the end of the primer, then back the primer up.
In any case, the best bet is to make an internal primer to the
cDNA so you can get the sequence from the other strand.

> Any 
suggestions as to how I can overcome this problem? I am using a 
cycle-sequencing kit. 

        95 degrees * 30 seconds
        55 degrees * 36 seconds
        72 degrees * 84 seconds

> The kit uses 7-deaza dGTP, but as I said I don't think its a compression.

> Is the answer higher annealing a higher annealing temperature?

I'm not optimistic that you can fix this by changing these parameters; but
I suppose a little formamide or DMSO added to the rxns to destabilise
whatever 2ndary str. is doing this may be worth a try.

> 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




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