sequencing AT rich genes

Mark Dowton mdowton at uow.edu.au
Wed Aug 29 18:44:38 EST 2001


Hi Marcio,

The quickest way around this problem is probably to design a second, internal
primer from your sequence data, and use that in a second sequencing reaction
to complete the sequencing of your fragment.  Getting any more than 500 bases
of reliable data from a single sequencing reaction is problematic in any
case, and the AT-content of hymenopteran mitochondrial genes certainly
doesn't help.  I know that symphytans, that have a more balanced AT content,
generally give cleaner, longer sequencing reads compared with anything within
the Apocrita.



Marcio Pie wrote:

> Hi all,
>
> We've been trying to sequence around 1kb of ant mitochondrial DNA.
> Although the PCR works fine, I'm having problems with the sequencing. We
> can't get sequences larger than 500bp, and we suspect that it is because
> of the very high AT content of the ant mtDNA. Has anyone had similar
> problems?
>
> I've been using an Applied Biosystems 377 automated DNA sequencer with XL
> upgrade.
>
> Thanks
>
> Marcio
>
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> Marcio R. Pie
> Department of Biology
> Boston University
> 5 Cummington St.
> Boston, MA 02215
>
> Phone: (617) 353-6974
> FAX:   (617) 353-6340
> http://people.bu.edu/pie/
>
> ---

--
Mark Dowton
Institute for Conservation Biology
Dept Biology
Wollongong University
Wollongong  NSW  2522
AUSTRALIA
Phone: 61-2-42215897
Fax: 61-2-42214135
Email: mdowton at uow.edu.au

And

Centre for Evolutionary Biology & Biodiversity
Dept Applied and Molecular Ecology
Waite Campus, Adelaide University
PMB #1
Glen Osmond  SA  5064
AUSTRALIA






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