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

Tim Spahlinger txs at po.cwru.edu
Wed Dec 6 16:09:38 EST 2000


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.

Dr. Duncan Clark wrote:

> In article <200012041646.eB4GkZL10932 at 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.
> 
> Duncan
> 
>  






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