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PCR cycler with end product fluorescence detector

cycler PCR fluorescence detector

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#1 Indrek Keskkyla

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Posted 05 January 2017 - 10:40 AM

Hello
I want to check an idea that a molecular biologist gave me. I am an electrical engineer myself who got convinced to build this device. The device should contain a simple PCR cycler (slow, very little flexibility in program, robust) and a fluorescence detector for 6-FAM color(decides positive-negative based on light intensity from the excited sample). The detector is not in the cycler well but separate pocket. So you would need to pick the reaction tubes and place them into the detector one by one. This is for cost reasons.
Now when the prototype is ready i (suddenly) realized that it would be wise to double-check the initial thesis. The thesis, that this type of device would be useful at all. 

Perhaps with specific primers to solve some particular issue somewhere....

But what do you think. You are professionals in the field of genetics. Do you see any possible use cases? Pitfalls? Potential problems?

Thank you in advance
Indrek

 


#2 bob1

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Posted 05 January 2017 - 02:46 PM

While there is a possibility of a market perhaps in remote diagnostic labs and schools. However, there are much simpler and cheaper ways of checking if a PCR has run correctly, such as the standard agarose gel electrophoresis. All this requires is some agarose, buffer, gel tank and trays, and a DC power source. You can also buy things to check the product on a "chip" which is basically a very very small gel enclosed inside a small cartridge (maybe 2 cm each side) that sucks the DNA in and separates it giving you a read out at the end, but it is expensive.

 

The potential pitfalls of yours is that it will almost certainly be expensive (based on the dye alone), and the presence of a signal doesn't mean that your PCR has worked - you could have primer dimers or off-target bands in the reaction that would give positive signal, but no way of telling if the signal is a result of these artifacts. Also if you are setting up PCR, especially a lot of PCR, then many people use a plate, rather than using individual tubes (imagine trying to label 96 tubes individually...) - is your device capable of handling plates or strip tubes? 



#3 Indrek Keskkyla

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Posted 09 January 2017 - 10:55 AM

Thanks for the reply. I got lots of new information.

Could you provide a link for the gel "chip" ? Sounds cool.

About the question you asked: My prototype is a 4 x 4 plate with wells for 0.2 ml tubes. I think strips would be ok if you cut them in the length of 4, since the heater i am using is salvaged from an older device. But plate is a no-go.



#4 mdfenko

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Posted 10 January 2017 - 04:45 AM

Could you provide a link for the gel "chip" ? Sounds cool.
 

i beleive that bob1 was referring to the agilent bioanalyzer.


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#5 bob1

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Posted 10 January 2017 - 06:45 AM

 

Could you provide a link for the gel "chip" ? Sounds cool.
 

i beleive that bob1 was referring to the agilent bioanalyzer.

 

Correct.



#6 Trof

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Posted 10 January 2017 - 10:44 AM

Did you have any specific uses in mind while designing the prototype? 
Because I can't really think of any. There are simple field-work flurescence scanners for on-site positive/negative PCR testing, that only have a simple super-fast cycler (battery operated or attached to some generator) and fluorescence check to see if the product is possitive.
Of course all the problems mentioned above applies. But you can have assay so well optimized, that you can really expect negative to be negative and positive to be positive. But this is for field work, soil bacteria PCR and so, the assays are created and optimized in classic laboratory under rigorous conditions to make sure they will work in the tent or wherever. Of course all the preps are accustomed to this, no isolation just quickly adding to pre-made mixes that contain inhibitor-proof polymerase mix.
(today most likely small precast gel units with intergated fluorescence are a better solution anyway)

That is only use for a end-point single color fluorescence I can think of, you can use end-point for dual color genotyping assays, but mostly, you need to optimize it too and just using a real-time cycler does the same thing, these assays are pretty expensive so if you can't afford cycler, it's out of question.


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