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
• a low per-sample experiment cost.
• 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
You can continue using real-time PCR for routine quantification applications, and add digital PCR for applications requiring enhanced performance.
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.
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