Restriction Enzymes in Droplet Digital PCR

Droplet digital PCR is a method for accurately quantitating copies of DNA or RNA in a sample. Each PCR reaction is separated into thousands or millions of droplets for analysis. Learn more about droplet digital PCR.


What is droplet digital PCR?

Nathan Tanner, Ph.D.:

Droplet digital PCR is a method for accurately quantitating copies of DNA or RNA in a sample of interest.  The droplet name refers to what you do to the reaction.  Where you take a PCR and instead of it being just one big reaction in one tube you break it up into thousands or millions of individual droplets.  So this encapsulates all the copies that are in the reaction, and rather than just measuring everything together, you measure each droplet for a yes or no answer, for presence or absence of the sample.  This is very useful for quantifying very rare targets.  So something that is only present in a very small percentage of the total.  Or for things where you have very limited sample, like a liquid biopsy or cell free DNA.

How can you use restriction enzymes in droplet digital PCR?

Restriction enzymes are useful in droplet digital PCR for a few reasons.  First, with a large genome, like the human genome, it can be very long, and when you put that into the droplet generator, the presence of a lot of DNA can mess up the process of making droplets.  By doing a restriction digest you can break it up into manageable pieces so that the droplet generation works.  Second, for applications like copy number variation, you might have more than one copy on the same molecule of DNA and to get an accurate quantitation you want to make sure to cut that up into individual and discreet pieces for accurate quantitation.    

What is the future of restriction enzymes in Droplet Digital PCR?

Droplet digital PCR is a relatively new method, but it's growing very fast and a lot of new applications come out all the time.  Things like infectious disease, oncology, companion diagnostics.  Restriction enzymes will continue to be helpful for all these methods, especially in applications like copy number variation, where you really want to get a very sensitive detection of copy number fluctuations.  The future of PCR might be digital.  It's helpful that restriction enzymes can really enable these methods.

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