Phosphorescent PCR dye

[Gabriele Borelli]

Hello,
I'm studying real-time PCR and I have a question regarding the dyes. I found on the Internet different types of this molecules, but all of them use the fluorescence. In my opinion the fluorescence is very fast and, from an electronic point of view, is difficult to measure. If a dye based on phosphorescence existed, it would be simpler. Do you know a phosphorescencent dye?
Best regards,
Gabriele

[Jeswin]

Why is fluorescence difficult to measure? qPCR machines use lasers or LEDs to excite fluoropores and sensors to detect the fluorescence. I don't think phosphorescence is suitable for real time PCR. The advantage of fluoresence is its speed in excitation and emission. With each cycle, you can detect the amount of the target gene.

Since phosphorescence emission lasts longer, the emission from the previous excitation will interfere with later detection. Hence, it's unsuitable for continuous detection.

Take a look at this document:
http://www.genelink.com/literature/ps/pg-mb-ver5.3.pdf

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[Gabriele]

Thank you for your answer and for the documents.
I think that phosphorescent could be useful because it is slow. It could be possible to excite the solution, turn off the led/laser and then measure the emission. This will avoid the use of optical filters, which are quite expensive. It's possible to find high speed photodiode which they can measure Fluorescence too, but thery are expensive and not so accurate. So I think phosphorescence cuold be useful in this application. How longer does the phosphorescence take from the excitation to the emission? And for how longer does it emit?

Thank you again.

[John Griessen]

On 05/06/2015 09:30 AM, Gabriele wrote:
> It could be possible to excite the solution, turn off the led/laser and then measure the emission.

That could make a nice low cost detection. Phosphorescence like fireflies have seems to last for a minute or more driven by a
reaction. Glow in the dark plastic seems to last several minutes from just a quick bright exposure.
A system that integrates the dim output light and uses a test standard illuminated just like your samples
would be good. The light source should be controlled so it always puts out the same level, and turned
on and off precisely.

[Jeswin]

On Wed, May 6, 2015 at 10:30 AM, Gabriele <blues...@gmail.com> wrote:
> Thank you for your answer and for the documents.
> I think that phosphorescent could be useful because it is slow. It could be
> possible to excite the solution, turn off the led/laser and then measure the
> emission. This will avoid the use of optical filters, which are quite
> expensive. It's possible to find high speed photodiode which they can
> measure Fluorescence too, but thery are expensive and not so accurate. So I

Are optical filters really expensive? I know that in previous
generations of qPCR machines, they used lasers which needed 220V. The
newer LED machines can use regular 110V so I think that makes it less
expensive. I think a replacement laser for the Applied Biosystems
7900HT costs between $10000 to $15000.

> think phosphorescence cuold be useful in this application. How longer does
> the phosphorescence take from the excitation to the emission? And for how
> longer does it emit?
>

Since the amplification part of qPCR (i.e. PCR) doesn't take too long,
this might where you want to look. Amplification has denaturation,
annealing, and extension. In most cases, you detect fluorescence at
the end of extension. The per cycle time is probably around a minute.
The Applied Bio Quantstudio already has the predefined optimized
cycling program that works with their dyes and that goes under 30 sec.
I worked with Roche Lightcycler before where you had to optimize the
PCR conditions. My notes for those experiments say they cycle for
under 30 sec.

The Applied Bio QuantStudio actually detects the signal thru all 5
filters it has (even though you may only have one type of probe on the
whole plate). I was told this whole process (all 5 filters) occurs in
nanosecond (or pico?). It's very fast.

So if you want to use phosphorescence, you might have to figure out
how to quench the emission signal before the next collection point.

[John Griessen]

On 05/06/2015 11:16 AM, Jeswin wrote:
> you might have to figure out
> how to quench the emission signal before the next collection point.

Not if you go slower than. He's thinking up another way, not how to
"be like" the current industry standard.

Slow, cheap and automated running a queue of samples could be very useful.

By slow, I am thinking of phosphors as used in analog oscilloscopes That put out light for
many milliseconds after a microseconds pulse of input light. Not minutes, not nanoseconds either.

Phosphors that put out for 10 seconds would be quite useful in an automated queue...
and if doing PCR, under the cycle time. Faster than the cycle time is not more useful.

Finding a phosphor that is compatible with and sticks to DNA is where to do some thinking and researching
if you want to figure out a "Phosphorescent PCR dye" method.

[Gabriele Borelli]

By slow, I am thinking of phosphors as used in analog oscilloscopes That put out light for
many milliseconds after a microseconds pulse of input light.  Not minutes, not nanoseconds either.

Hello,
this is exactly what I was thinking at. Yesterday I was reasoning about the bonding mechanism of SYBR Green with a friend of mine who is studying chemical. We reached no relevant results. Could be possible to make a molecole which can bind to any DNA and demonstrate a phosphorescent behaviour? How does SYBR green binding work?

 

Do you confim me that what I wrote are, more or less, SYBR Green timing?

Thank you,

Gabriele

[John Griessen]

On 05/11/2015 06:32 AM, Gabriele Borelli wrote:
> Do you confim me that what I wrote are, more or less, SYBR Green timing?

> How does SYBR green binding work?
>

I'd start a new thread for that question. No I cannot concur. Don't know.
Can't remember what you wrote earlier.