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.