At the recent American Society for Human Genetics (ASHG) meeting in Houston, Behavioral Diagnostics CEO Rob Philibert had a chance to meet with Qiagen’s commercial development team and talk about quantitative PCR and digital PCR.
Here’s an in-depth explanation of digital PCR and what it may do for precision medicine ...
An important feature of Behavioral Diagnostic’s approach for measuring substance consumption is a new digital form of older technology: polymerase chain reaction (PCR). 

PCR was first discovered in the 1970s by the colorful Kary Mullis, who later won a Nobel Prize for his work. Since then, this inexpensive method has been repeatedly refined to become a tool at the heart of many important diagnostic technologies , such as those that detect HIV infections and forms of genotyping. 

But one inescapable fact has limited the use of PCR technologies: As for all chain reactions, it’s difficult to reproducibly control and measure the speed of a PCR reaction at the fastest, most important portions of the amplification curve. As a result, methods like quantitative PCR (qPCR) that rely on measuring the speed of the PCR reaction – as indicated by the production of fluorescent signal – frequently have large error rates .
Digital PCR: A Better Option?
The introduction of digital PCR (dPCR) may solve many of these limitations and spur the development of point of care for precision medicine.

From 50,000 feet, the dPCR concept is simple . Dilute the PCR reaction mixture, which normally contains thousands of amplicons (potential targets for the PCR reaction), to the point that either 0 or 1 copies of the amplicon are present in a reaction mixture. Then conduct PCR as normal. If you see a fluorescent signal in a well, an amplicon is present. If not, no amplicon is present. This reduces the “analog” paradigm of comparing relative rates to a “0 or 1” digital paradigm. 

Like most obvious solutions, however, one simple problem prevented an earlier adaption of this approach: The process of diluting the PCR reaction. Accurate use of the technique was not only logistically challenging, but made a cheap procedure very expensive. 
 
Fortunately, a number of leading instrument companies, such as Bio-Rad and Qiagen , were up for addressing this barrier . Bio-Rad was the first to successfully move forward. Its version of dPCR solves this problem by partitioning a normally 22-microliter reaction volume that contains about 10,000 potential PCR amplicons into 15,000 individual oil encapsulated droplets – and then conducting PCR on the droplets. 

After 40 cycles of PCR, droplets are siphoned through a narrow-gauge needle and serially interrogated with a laser to determine whether the fluorescent signal generated from the repeated copying of an amplicon was produced. 

In addition, because up to four different types of fluors (a molecule that emits light at a particular wavelength or color) can be used together, up to four DNA or RNA targets can be determined for each of the 15,000 droplets from each sample every two minutes.  
The Future of dPCR
Given Bio-Rad’s long track record in flow cytometry, which uses a similar assay procedure to count cells, this approach is a robust solution – and one we’ve adapted at Behavioral Diagnostics.
 
Qiagen introduced a different approach. Building off its recent acquisition of Formulatrix technology, it utilizes an advancement in microfluidics and a “chip” that splits the PCR reaction into a 100x100 grid of 1-microliter wells. After PCR amplification, the presence or absence of fluorescence in each mini-well, as quantified by a digital camera, gives a yes/no signal as to whether a DNA target is present. 
 
Each approach has advantages. It’s likely that the Qiagen and Bio-Rad platforms will find market niches in the rapidly increasing dPCR market. Each of these companies is developing companion products for these applications, such as Qiagen’s Epitect™ bisulfite conversion technologies, which we also use.  









Rob Philibert MD PhD
CEO
Behavioral Diagnostics
Behavioral Diagnostics | Website