Sequencing through poly A region

Vahe Vahe
Fri Jan 31 11:34:14 EST 1997


>Dear Friends,
>
>Would someone like to share with me his/her experience of sequencing
>through a region of ~22 As in Arabidopsis.  I have tried different
>chemistries from ABI and Amersham but both fail to produce any good
>sequence.  It produces very nice and clean sequence till the end of the A
>stretch but fails right after that. I have not tried using anything
>different in the reaction yet.  Recently I heard about Dynamic ET poly T
>primer kit of Amersham but haven't tried it either.  Any advice of any
>sort
>will be appreciated.
>Cheers!
>

This is a problem that has puzzled me a lot. On some samples we get severe
"slippage" of Taq after even a short stretch of T's (10-15), and the
remainder of the sequence is not readable because of overlapping peaks. On
other samples we can go through 30-40 T's and continue reading without a
problem. It is not clear what makes the difference, but I suspect it is
template purity.

When we do have problems with reading through T's we can sometimes resolve
the problem by using a polyT primer (around 25-30T's) ending with 1-2
additional bases from the sequences after the T's (if we can guess them).
This frequently anchors the primer well enough to continue the read past
the T's.

Vahe Bedian





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