PCR/sequence through bad region

Paul Shinn pshinn at mail2.sas.upenn.edu
Wed Mar 22 19:56:46 EST 2000

    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.  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.  
Anyone have any ideas how to get through these regions?


Paul Shinn    
Sequencing Coordinator                                    ,___o
pshinn at neomorph.bio.upenn.edu                            _-\_<,
Arabidopsis thaliana Genome Center                      (*)/'(*)
(215) 573-7256

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