PCR/sequence through bad region

Phillip San Miguel pmiguel at purdue.edu
Fri Mar 24 09:14:30 EST 2000


Paul Shinn wrote:

>     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.[...]

Paul,
    Have you tried the dGTP terminator kit? (The standard ABI kit uses dITP
instead of dGTP. ABI's polymerase has a tough time getting through 'G' heavy
regions.) dRhodamine? These are suggestions from Bruce Roe's web site[1]. Also
mentioned there is the "shatter library" method, where if you can get a PCR
product you shear it down to very small fragments (200-500bp). These are
cloned, sequenced and assembled. Or you could clone the PCR band and use
transposon based methods to sequence. I've sequenced lots of stuff using a
natural transposon using in vivo methods. But PEBio, NEB and Invitrogen all
sell in vitro transposon kits.
    If you have sub-clone of the containing the gap, you could try a direct
dye primer read using a non-cycle method (Sequenase, for example), but you
need lots of template compared to cycle sequencing. Also, if you have access
to a Licor, a CEQ 2000 or an Alf express, you can try their chemistry. Not
that these chemistries are  inherently more robust than ABI's, but they are
different--so they may have different weaknesss. (Actually the Alf express
using thermosequenase is pretty robust, but there is really no phred support
for their chromatograms. So you won't have accurate quality values.)
    For your mysterious gap, it might be worth sub-cloning a restriction
fragment containing it. You could check your sequence for fairly rare cutting
enzymes so you could get the right fragment. This is presuming you don't
already have an M13 clone from your shearing library--this is a clone gap,
right?

Phillip San Miguel
Purdue University Genomics Core Facility

[1]http://www.genome.ou.edu/SeqStrategy.html


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