How Absolute Quantification Using Digital PCR Is Revolutionary

With the pressing need to deliver efficient and time-saving services, even the life sciences industry frequently adopts new technologies or improves existing ones. One such advancement is the transition from traditional PCR to digital PCR. Digital PCR (dPCR) comes under the category of quantitative PCR (qPCR). It is a sensitive and reproducible way of measuring the DNA or RNA present in the given sample.

In its principle, dPCR is similar to qPCR in terms of reaction assembly components and amplification process but differs in calculating the sample target. It is a simple and reproducible process independent of the calibration curve for quantifying the given sample target. In this method, no standards or other controls are required.

Droplet Digital PCR (ddPCR), a type of dPCR is actively being employed since 2011. Like quantitative PCR, droplet dPCR technology utilizes the enzyme Taq polymerase in a standard PCR reaction to amplify a target DNA fragment from the given sample. This amplification is done using a pre-validated primer or probe assays. Unlike qPCR, in digital droplet PCR, the initial sample is partitioned into many individual wells before the amplification step. This roughly results in either 1 or 0 targets being present in each well. After the PCR amplification step, the number of positive vs. negative reactions is determined, and the absolute quantification of the target is done using Poisson statistics.

TYPES OF dPCR DEVICES

Based on the methods of reaction mixture separation, dPCR is classified into chip dPCR (cdPCR) and droplet dPCR (ddPCR).

1. cdPCR

Based on microfluidic technology, chip dPCR involves dividing the reaction mixture into nanoliter-sized reaction chambers. After multiple cycles of reaction, fluorescence is detected using an imaging system and an endoscopy. After this, the copy number of a target sequence is measured using imaging software.

BioMark and QuantStudio3D are two commercialized chip dPCR systems. BioMark consists of integrated fluidic circuits with approximately 10000 to 40000 microchambers. QuantStudio3D has an integrated chip with 20000 microchambers. BioMark™ HD System and qdPCR 37K™ IFC are being developed and employed by companies like Fluidigm Corporation.

2. ddPCR

Droplet dPCR makes micro reaction units consisting of water-in-oil and microfluidics. The nucleic acids are subdivided into water-in-oil droplets undergoing PCR independently. After this, a two-color optical system reads the signals in each droplet. The chips have three different shapes, “+,” “T,” and “Y.” The droplets of dPCR are created by a layer that costs lower than chip dPCR. However, the droplets are not even in volume. Therefore, merchandized ddPCR introduced a standardized procedure. The droplets of similar quantities have similar fluorescence. The obtained results are then optimized with Poisson statistics.

QX100/200 and Rain Dro are two main droplet dPCR manufacturers. QX100/200 divides each reaction mixture into approximately 20000 droplets, and Rain dro divided it into 1000000 to 10000000 droplets. Raindance™ Technologies and Bio-rad are pointed as the key players merchandising and using these droplet dPCR systems.

Different companies prefer different types of PCR. According to the conducted research on the trends. It is observed that Multiplex PCR is the most widely used type of digital PCR.

APPLICATIONS OF dPCR

1. High-Precision results

2. Easy quantification of NGS Library

3. Rare Mutation Analysis

4. Differential Gene Expression

5. Viral Load Quantification

6. Low-Level Pathogen Detection

7. Reference and Standard Quantification

8. GMO Detection

ADVANTAGES

Digital PCR is a powerful method, the advantages of which include:

· Independent amplification efficiency

dPCR provides absolute quantification. Standard curves and reference genes positively influence the complete quantification of traditional PCR analyses. Because of this, a 100% absolute quantification cannot be achieved. dPCR expresses the amplification results either as “1” or “0” and quantifies the results with counts (sometimes that is corrected using Poisson statistics), which makes absolute quantification possible.

· Retesting the NGS results

Amplification efficiency is influenced by factors like enzymes, primers, inhibitors, and cycle threshold (Ct); both elements are unstable in traditional PCR. Due to these limitations, the assays with the same design might show poor repeatability. In the dPCR process, primers and probes are reduced, which lowers the mispairing of target sequences. It is with NGS to quantify and analyze the data. On the one hand, dPCR retests the results of NGS. On the other, sequencing data quality is guaranteed, including adaptors, Joint dimer, and long fusion junctions. In all, dPCR suppresses former techniques in all aspects.

· A small volume of sample

dPCR achieves high accuracy with only a small sample volume, especially when detecting hard-to-be-taken samples or the samples containing degraded nucleic acid. For a volume large enough, the clinical specimen is amplified to carry out genomic analyses, like comparative genomic hybridization (CGH) chip, next-generation sequencing (NGS), etc. dPCR is performed based on a minimum sample, reaps reliable results by avoiding the errors generated from preamplification.

· High sensitivity and absolute quantification

Compared to RT fluorogenic qPCR, dPCR is far more sensitive and clinically applied. For dPCR, the standard PCR system is obtained using numerous microunits, which increase the PCR system’s tolerance to inhibitors. This sensitivity of traditional PCR systems can reach up to 1%. Still, dPCR achieves a greater sensitivity up to 0.1%, sometimes 0.001%, making it an ideal tool for trace DNA detection, rare mutation detection, specifically circulating tumor DNA detection. Moreover, dPCR has further been technologically updated into a more compatible device.

INDUSTRY INSIGHTS

Ingenious e-brain conducted an intensive study of the past five years to analyze digital PCR. The key insights were formulated on three subjects. These are:

a) Use of different types of dPCR

Different biotechnology and pharmaceutical companies are employing various types of dPCR types. The conclusions derived after a comprehensive insight into the players’ operations are demonstrated in the chart below.

b) Key active industrial and academic players

The leading companies and academic institutions in this domain are using this technology to optimize their operations.

c) Key emerging players (last two years)

Apart from these leading names, the experts’ team also prepared a list of emerging players. The results demonstrate that the University of Washington has come up with the most number of research initiatives in the domain.

COMMERCIALLY AVAILABLE DPCR SYSTEMS

Some of the commercially recognized systems are:

1. Raindrop™ Digital PCR System by Raindance™ Technologies (Bio-rad).

2. QX200™ Droplet Digital™ PCR System by Bio-Rad.

3. BioMark™ HD System and qdPCR 37K™ IFC by Fluidigm Corporation.

4. QuantStudio™ 3D Digital PCR System by Life Technologies™ (Thermo- fisher).

RECENT INNOVATIONS

It can be concluded from the data presented above that digital PCR is increasingly being applied to replace conventional PCR. As it is a single-molecule-amplification technique with a high signal/noise ratio and increased sensitivity, the droplet type of dPCR is effectively used to detect low levels of DNA. Still, digital PCR is constantly being improved and strategically modified to cater to the biotechnology industry’s demands.

AUTHOR:

Mr. Chetan Marwah is a Patent and Technology Intelligence Expert at Ingenious e-Brain, a broad spectrum of IP intelligence, technology intelligence, and business intelligence services to worldwide companies and legal firms across the globe.