Sequencing AT rich Templates

David Fritzinger dfritzin at macNoSpam.com
Tue Nov 11 19:36:47 EST 2003


In article <bor5mr$2tc$1 at mercury.hgmp.mrc.ac.uk>,
 Mick Jones <m.d.jones at imperial.ac.uk> wrote:

> Hi
>
> What is the best method to sequence AT-rich templates on a 3700 capillary
> sequencer.
>
> We have been given samples to sequence that contain ~800+ bp inserts in a
> standard pUC based plasmid.  The inserts have been generated by PCR
> amplification of bisulphite treated DNA (Cs gone to Us, 5meC remain as C) so
> they are very AT-rich.
>
> We seem to get good data on some templates, on others we get slippage.  Also
> the signal strength drops rapidly.
>
> Any advice gratefully received.
>
> Mick Jones
>
> Michael D Jones, BSc, PhD, ILTM
> Head Genomics Core Laboratory
> MRC Clinical Sciences Centre
> Hammersmith Hospital Campus
> London, UK
>
IIRC, the PCR amplification can cause problems if you have runs of >10
or so A's or T's, and this is where the slippage occurs. You might want
to sequence these templates from the oroginal plasmids, rather than from
a PCR product. I'm not sure why you are getting the loss of signal
intensity. Could be that you are just running out of terminators?

HTH

David C. Fritzinger, Ph.D.
Cancer Research Center of Hawaii
Honolulu, HI 96825, USA
>
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