IUBio

Using ET terminators on 3700

Phillip San Miguel pmiguel at purdue.edu
Fri Aug 3 08:08:07 EST 2001


Yes. We did a beta-test for Amersham Pharmacia.  Results were good, but
not as many phred high quality bases as Big Dye produced. But I think
this was because ABI had just released a new mobility file and
basecaller for Big Dye terminators in POP5 that alleviated some of the
shifts that cause ambiguities early in the sequence. (Near the primer).
The trial ET-dye mobility file I was given didn't have this advantage.

Not sure that ET could be diluted the way that Big Dye can, though. We
did 1/2 volume reactions in 384-well plates for the ET dyes. With Big
Dye we typically do 1/8th reactions (pretty much using Bruce Roe's
protocol.) However we generally do 99 cycles with Big Dye. The TaqFS in
Big Dye may be more thermostable than Thermosequenase II in ET. So 99
cycles probably won't help with ET.

ET beats Big Dye in one capacity, hands-down. That is cycle speed. ET
sequencing reactions take about 1/4th the time to run that Big Dye
reactions take. The long (4 min) extension step required for ABI
chemistry isn't required for ET.

The main use we put the ET terminator chemistry to is finishing. Having
an alternate chemistry to finish with helps a great deal. The ET
terminator chemistry seems to have a different set of peculiarities as
to where it fails to produce good sequence than Big Dyes. Hence the two
chemistries are complementary. Where Big Dye (or even dRhodamine)
chemistry fails, ET chemistry often sails through. I hasten to add "and
vice versa."

Phillip SanMiguel
Purdue Genomics Core Facility

Arie Vana wrote:

> Has anyone tried DYEnamic ET terminators (amersham Pharmacia)
> on a 3700 ?
>
> ****************************************
> Arie Vana
> Head, Sequencing Unit
> QBI Enterprises Ltd.,
> PO Box 4071, Nes Ziona 70400, Israel
> Tel: +972-8-9408147  Fax: +972-8-9406476
>
> ---

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