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ABI 3100/3700 genetic analyzer: substitute NED dye with TAMRA?

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Marcia Macdonald

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Dec 1, 2000, 3:00:00 AM12/1/00
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Has anyone else tried to avoid buying NED-labeled oligos from ABI by substituting TAMRA-labeled oligos?  We just did a quick experiment on our 3100 and found that the TAMRA was detected as yellow peaks just like NED!  Like the other dyes, there was a small amount of leakage into the other colours when the PCR product was over-loaded, but nothing that will interfere significantly with pooling multiple markers  We still have to compare the same marker amplified with both TAMRA and NED dyes to see how their mobilities compare (i.e. if the alleles are labeled as the same size), but that shouldn't prevent us from ordering new markers in the TAMRA dye rather than the NED.  Just thought this might interest anyone genotyping or synthesizing labeled oligos.
 
Marcia
 
Marcia MacDonald
Xenon Genetics, Inc.
100-2386 East Mall
Vancouver, BC 
Canada  V6T 1Z3
604-221-8423 fax

Deanne Bell

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Dec 1, 2000, 3:00:00 AM12/1/00
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Hi
I don't know if the filters are the same on your machines but I am using
the ABI 310. And on my machine, TAMRA is detected as red if you are using
the Virtual filter set 'C'. TAMRA is detected as yellow using virtual
filter set 'A'. When we started using our ABI 310 for AFLPs, Applied
Biosystems put out a kit of primers using TAMRA as the yellow dye. They
then switched over to using NED as the yellow dye in the primer kit. I
think I remember them saying that the TAMRA had more bleeding over into
other colors than the NED. Either which way, I never got the yellow
labeled primers to come off with any height on my electropherograms - be it
NED or TAMRA. I am working with plants and it may be that these primers
just don't amplify much in plants (although ApBiosystems claims to get
amplification in plants with these primers). I have a feeling it is more
likely that the dye just doesn't emit as well as the other dyes such as
JOE, FAM & ROX. I would avoid using a system relying on NED or TAMRA as my
markers / standards. See if you can and IMHO, switch over to a set-up
where you use ROX for your markers / standards.

Deanne Bell
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: db...@qnis.net
phone: 559 453-3170
fax: 559 453-3088

----------
From: "Marcia Macdonald" <mmacd...@xenongenetics.com>
To: met...@hgmp.mrc.ac.uk
Subject: ABI 3100/3700 genetic analyzer: substitute NED dye with TAMRA?
Date: Friday, December 01, 2000 11:05 AM

Marcia


---

Marcia Macdonald

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Dec 1, 2000, 3:00:00 AM12/1/00
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Hi Deanne,
 
Thanks for the info about the C filter set.  I guess I wasn't clear enough.  We're using the filter set D with a ROX size standard and the FAM, HEX and NED dyes for our microsattelite markers.  ROX is detected as red and both NED and TAMRA are detected as yellow.  The NED markers are usually relatively weak compared to the other colours, but we're able to make them work well enough by testing the markers then adding more cycles or using relatively more of the NED samples in the pooled products.  We want to use primers labeled with all 3 available colours to maximize throughput, so replacing NED with TAMRA would allow us to use all 3 colours and avoid ordering ABI custom primers (which are very unreliable).
 
Marcia
 
 
Marcia MacDonald
Xenon Genetics, Inc.
100-2386 East Mall
Vancouver, BC 
Canada  V6T 1Z3
604-221-8423 fax

>>> "Deanne Bell" <db...@qnis.net> 12/01/00 02:08PM >>>

Dr. Duncan Clark

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Dec 4, 2000, 3:00:00 AM12/4/00
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In article <200012012214...@ns.qnis.net>, the eminent Deanne
Bell at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote

> I
>think I remember them saying that the TAMRA had more bleeding over into
>other colors than the NED. Either which way, I never got the yellow
>labeled primers to come off with any height on my electropherograms - be it
>NED or TAMRA. I am working with plants and it may be that these primers
>just don't amplify much in plants (although ApBiosystems claims to get
>amplification in plants with these primers). I have a feeling it is more
>likely that the dye just doesn't emit as well as the other dyes such as
>JOE, FAM & ROX.

NED (and the equally new VIC) is supposed to be a brighter dye. Of
course being cynical one could take the view that the change to NED and
VIC is because they are the only two that are patented and therefore
proprietary. I don't know of any company other than PE/Applied
Biosystems (or whatever they are called this month) that will synthesise
oligos with those labels.

The alternative is to think about FRET oligos to boost the TAMRA or say
a TET signal.

Duncan
--
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk

Deanne Bell

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Dec 4, 2000, 3:00:00 AM12/4/00
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Hi Dr Clark
I have never heard of FRET, can you give a brief description and some
reference I can follow up on?
The TET emission wavelength is 538 and JOE's is 554, I think these are too
close and would not be differentiated by the ABI 310 if I were to multiplex
the dyes. Also, the ABI software does not have a virtual filter that
includes TET and JOE together, they seem to be mutually exclusive.
Thanks

Deanne Bell
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: db...@qnis.net
phone: 559 453-3170
fax: 559 453-3088
>
> In article <200012012214...@ns.qnis.net>, the eminent Deanne
> Bell at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote
> > I
> >think I remember them saying that the TAMRA had more bleeding over into
> >other colors than the NED. Either which way, I never got the yellow
> >labeled primers to come off with any height on my electropherograms - be
it
> >NED or TAMRA. I am working with plants and it may be that these primers
> >just don't amplify much in plants (although ApBiosystems claims to get
> >amplification in plants with these primers). I have a feeling it is
more
> >likely that the dye just doesn't emit as well as the other dyes such as
> >JOE, FAM & ROX.
>
> NED (and the equally new VIC) is supposed to be a brighter dye. Of
> course being cynical one could take the view that the change to NED and
> VIC is because they are the only two that are patented and therefore
> proprietary. I don't know of any company other than PE/Applied
> Biosystems (or whatever they are called this month) that will synthesise
> oligos with those labels.
>
> The alternative is to think about FRET oligos to boost the TAMRA or say
> a TET signal.
>
> Duncan


---

Dr. Duncan Clark

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Dec 6, 2000, 3:00:00 AM12/6/00
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In article <200012041646...@ns.qnis.net>, the eminent Deanne

Bell at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote
>I have never heard of FRET, can you give a brief description and some
>reference I can follow up on?

FRET stands for I think Fourier resonance energy transfer.

Basically you put two fluorophores close enough together on an oligo or
chemically link them together i.e. FAM-NNNNNN-JOE or FAM-X-JOE. 7bp
separation is optimal for best FRET.

If you excite at 488 the emission from the FAM will be transferred to
JOE and you will see emission at the JOE emission wavelength. You will
also see a minor peak of emission at 520 from the FAM. You can link all
sorts of fluorophores even FAM/Cy5.

Look for papers by Glaser et al in Analytical Biochem a few years ago.

This is the principal behind APB's ET primers, Big dye terminators etc.
etc.

Tim Spahlinger

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Dec 6, 2000, 3:00:00 AM12/6/00
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In addition to Dr. Clark's reply, here is an excerpt from an
ABI document: "The probe is an oligonucleotide with both a
reporter fluorescent dye and a quencher dye attached. While
the probe is intact, the proximity of the quencher greatly
reduces the fluorescence emitted by the reporter dye by
Förster resonance energy transfer (FRET) through space.
Probe design and synthesis has been simplified by the
finding that adequate quenching is observed for probes with
the reporter at the 5' end and the quencher at the 3' end."
The TaqMan technology can utilize probes up to 30 to 40
bases long and offers several combinations of reporters and
quenchers. TaqMan technology, however, utilizes rather
narrow PCR reaction conditions.
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