PCR/sequence through bad region

Herve Crespeau crespeau at genoscope.cns.fr
Fri Mar 24 04:13:07 EST 2000


Great Paul, You just find the two major problems (I think) in sequencing.

>     Maybe someone can offer some advice.  I am attempting to sequence 2
> 100kb BACs containing Arabidopsis DNA.  However, I am having trouble
> closing one gap in each of them.  One BAC has a homopolymer stretch of Gs. 
> The sequence just DIES once this region is reached from either side.  I
> get about 10 Gs through it and that's it.  We've had success sequencing
> through 20 Gs before but this is proving especially difficult.  PCR
> primers flanking this region were successful in amplifying a 700bp chunk. 
> All the numbers add up but we just can't sequence through it.

OK what you have is a LARGE number of G, if you try to make a PCR product, you will have a 
slippage (?correct in english?) of your enzyme during the "copy" processus, as result in fact 
your PCR product is between, say, 699 and 701 bases long so you try to sequence 3 differents 
templates (in my example) with a common start, but with a difference after 699 bases, and after 
this point 3 differents sequences.
If you try to sequence directly your template you lose more or less rapidly your sequence, if 
you take care of your signals intensities in this sequencable region you will observe importants 
values  for this kind of templates. It's look like that your template "suck" (?) all the dGTP 
(or analog) present in your mix.

So if you want to pass throught this region, simply add more dGTP in your mix 


> The other
> BAC has an even more mysterious gap.  I have tried sequencing directly off
> a Qiagen miniprep of the BAC but the sequence just STOPS when it gets to
> the gap--on both sides.  I haven't been able to determine the size of 
> the gap because I am getting multiple bands from my primers. 
>     I have tried DMSO and increasing the extension temperature during 
> cycle sequencing.  I've even gotten desperate and tried Thermofidelase.  
> It's a DNA binding enzyme that's supposed to help "unlink" DNA allowing 
> polymerase to read through difficult regions.  I feel the 2nd BAC has 
> some kind of secondary structure that just stops the polymerase dead.   

Right we ahve also observed that. when we have this kind of secondary structure, we make longuer 
primer with higher Tm, and make a two step sequencing reaction. With the help of DMSO in some 
case, it's sufficent to destabilize the secondary structure.

Sincerely
RV

Herve CRESPEAU
Genoscope
Centre National de Séquençage
France
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