Digital PCR vs. Real-time PCR – Ask TaqMan #30

Digital PCR vs. Real-time PCR – Ask TaqMan #30

As a researcher, you ve been given many choices
in terms of tools and techniques. And for some who are intimately familiar with
real-time PCR, you are probably hearing about digital PCR and its emerging applications. So the question is, when do you use one or
the other or both. Real-time PCR also called quantitative polymerase
chain reaction or qPCR is one of the most powerful and sensitive gene analysis techniques
and is used for a broad range of applications. Digital PCR is the next generation of PCR
technology involving absolute quantitation of nucleic acid target sequences. As digital PCR gains clout among researchers,
many interested scientists ask When should I choose digital PCR over real-time
PCR? The overwhelming advantage of real-time PCR
is that it is a broadly accepted technology with well-established protocols and data analysis
techniques. Other advantages include a wide dynamic range for detection high sample throughputs The major advantages of digital PCR include: no reliance on standard curves and reference
samples; high tolerance to biological and sample prep
inhibitors; and improved performance for applications
requiring higher sensitivity and precision Now, let s take a look at our lab book You can continue using real-time PCR for routine
quantification applications, and add digital PCR for applications requiring enhanced performance. Now, let’s take a look at our lab book Absolute quantitation using real-time PCR
requires standard curves and reference samples, but digital PCR allows you to quantify samples
without using a standard curve. In this example using real-time PCR, notice
how many wells are required to generate the standard curve. This assumes you have appropriate reference
standards for your sample of interest, which may not always be the case. Each standard also must be of known quantity,
such as absolute quantitation of mRNA copy number. Generating such standards is a lot of work
and the standards may deteriorate over time, thus changing the amount of nucleic acid present
in your standards. In the case of digital PCR, your sample is
partitioned into thousands of separate reaction vessels. Taking an end point PCR read for presence
or absence of reactions allows for the absolute measurement of template copies per microliter. Digital PCR is also highly tolerant to PCR
inhibitors by virtue of the massive partitioning we just discussed. These data illustrate how digital PCR is robust
in the face of increasing inhibitor concentrations; whereas, the performance with real-time PCR
dramatically drops off at high inhibitor concentrations. Digital PCR can also provide improved performance
in cancer research, where rare mutations often need to be detected in a background of wild-type
DNA. Digital PCR has the sensitivity to detect
extremely rare target sequences. . So, in the end digital PCR can enhance your
quantitative PCR results, enabling applications that benefit from higher sensitivity, precision,
and absolute quantification. If you ve got more qPCR or digital PCR questions,
remember, just ask Taqman. Submit your question at thermofisher. com/ask
and subscribe to our channel to see more videos like this.

3 thoughts on “Digital PCR vs. Real-time PCR – Ask TaqMan #30

  1. Im still trying to find out where the word digital means in silico or does it actually involve wet lab – molecular hands on work? Digital is misleading if that is the case.

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