Comparison of digital PCR methods
What is digital droplet PCR?
A droplet digital PCR system uses an immiscible fluid in oil to generate tens of thousands of submicroliter droplets. Nucleic acids are encapsulated randomly inside the droplets, which serve as mini reaction chambers.
In a typical ddPCR workflow, the digital droplet PCR reaction is prepared in a tube. The ddPCR mix is then partitioned into individual droplets by a droplet generator. The emulsion is collected in a vial and endpoint PCR is performed. The sample is processed by a flow cytometer where droplets are fluorescently read one by one as they pass in front of a laser excitation source. Just like with any other dPCR protocol, the number of copies of DNA template in the initial reaction can be determined using Poisson statistics.
When deciding over dPCR vs ddPCR, consider the advantages and limitations of ddPCR and if a droplet digital PCR system is a right fit for your requirements.
Benefits and limitations of ddPCR
The advantages of ddPCR include:
- Generates the largest number of partitions
- Established method
Limitations of ddPCR include:
- Droplet variability in size and shape adversely affect robustness and reproducibility of the method
- A droplet digital PCR system consists of multiple instruments (droplet generator, thermocycler, droplet reader) that take up valuable lap space and require trained personnel for operation
- The ddPCR workflow is time-consuming and cumbersome
- Data quality can be affected by coalescence or shearing of droplets caused by thermal oscillation
- A typical ddPCR workflow requires multiple pipetting and transfer steps, which expose the ddPCR reactions to risk of cross-contamination and other errors
- Appearance of ddPCR rain droplets, which result from damaged droplets, non-specific amplification or irregular droplet size, makes setting of threshold that separates positive from negative droplets difficult
dPCR vs ddPCR
In comparison of nanoplate dPCR and ddPCR, the main difference is the partitioning approach. In ddPCR, the partitioning is achieved using a droplet generator, in nanoplate dPCR, the partitioning is achieved using a digital PCR plate. In ddPCR vs dPCR, the use of a nanoplate reduces run times, risk of contamination and eliminates the variability in size and coalescence associated with droplets. Nanoplates also offer a qPCR-like workflow and as less instruments are required, lab space is saved.
For more information, check out a webinar on plate-based dPCR vs ddPCR.
How does plate-based dPCR work?
The advantages of a nanoplate-based workflow is that dPCR method takes only about 2 hours to complete and all reactions occur in one plate. The digital PCR plate workflow is very similar to the qPCR workflow, eliminating the need for extensive training of lab personnel to operate the dPCR system. Nanoplate digital PCR also offers high multiplexing capabilities (up to 5plex) Conveniently, at completion of the dPCR assay, the plate-based dPCR system provides an actual image of the plate, rather than just a table.
To learn more about plate-based digital PCR, watch a video on the dPCR technology, discover the nanoplate digital PCR workflow, watch a webinar on the nanoplate digital PCR technology or find more information on nanoplate dPCR systems.
Other digital PCR methods
Chip-based digital PCR (cdPCR)
Chip-based digital PCR (cdPCR) involves the partitioning of a reaction into nanoliter reaction chambers by a microfluidic device. The dPCR reaction mix is divided into 10,000 to 45,000 partitions on a chip. The chip is subjected to amplification with an endpoint PCR thermocycler. Fluorescence is detected for all partitions on each chip using a high-powered camera reader equipped with a fluorescence filter. The copy number of the target sequence is calculated with accompanying software.
Microfluidic chamber-based dPCR
The microfluidic chamber array consists of a network of fluid lines, valves and chambers. The valves are made of an elastomeric material that deflects under pressure to create a tight seal. The valves are used to regulate the flow of liquids in the array. Single DNA molecules are randomly distributed into nanoliter volume reaction chambers, prior to PCR amplification in the presence of a fluorophore-containing probe.
Microwell chip-based dPCR
This dPCR technology encompasses an “open-array plate” consisting of 20,000 wells/partitions, etched through a microscope slide. The inner surfaces are coated with a hydrophilic surface, so samples can be loaded via capillary action. The plate is immersed in immiscible fluid (oil) to seal the sample on both sides and prevent evaporation or cross-contamination. The chip undergoes endpoint PCR on a conventional thermal cycler and is analysed on a separate instrument.
Crystal digital PCR
This hybrid dPCR approach combines the 2D array format of cdPCR and the use of droplet partitions in as in ddPCR. The dPCR reaction mix is loaded onto a microfluidic chip, and placed in a dPCR system that partitions the sample into 2D monolayer arrays of monodisperse droplets. These droplets are called droplet crystals, because of the spontaneous periodic arrangement of the droplets within the monolayer, similarly to the that of atoms within crystals. The droplet crystals undergo endpoint PCR, then they are transferred onto a fluorescent microscope and imaged to determine the number of amplified partitions.
Chip-in-a-tube digital PCR
This type of dPCR technology also combines a chip-based and droplet digital PCR approach. The dPCR system uses a strip of eight standard PCR tubes. A miniature trapezoidal-shaped chip containing 10,000 partitions is found inside each tube. As dPCR mixture is added into each tube, the liquid is rapidly drawn into the partitions by capillary action. The partitions are sealed with a sealing fluid and the tubes are subjected to PCR in a conventional thermocycler. The tube-strips are then read on a dedicated reader, which concurrently detects fluorescent signals from every partition of the digital PCR reactions.
Semiconductor chip-based dPCR
The lab on an array (LOAA) dPCR approach separates the partitions on a two-dimensional semiconductor chip in a cartridge. The semiconductor plate is based on Micro Electro Mechanical System (MEMS) technology and can be injected by dividing the target molecules into as few as 56 wells and as much as about 20,000 wells. As the number of wells increases, the size of the wells is gradually partitioned into smaller volumes. The amplification of the target nucleic acid and the fluorescence analysis of each well are sequentially performed in one device.
Common dPCR platforms
|Microfluidic digital PCR plate
|Relies on elasticity of the material
|Slipping for partitioning
|Both active (device reconfiguration
or mechanical actuation) and passive
(driven by fluidic properties)
|Pinning of the oil interface
to isolate chambers
|Plug splitting within a network
of chambers pre-wet
with immiscible oil
|Control of the pinned interfaces
for controlled filling
|104 – 106
|10 – 100 pL
|Droplets used as partitions
Benefits and limitations of digital PCR methods
The various dPCR approaches come with their own inherent benefits and limitations. For example, if considering chip-based dPCR vs ddPCR, cdPCR has several advantages over ddPCR including:
- Fast partitioning
- Use of small amounts of samples and reagents
- Many reactions can be integrated into a single device
Despite the limitations of ddPCR, chip-based digital PCR systems also have several disadvantages, including:
- Need to use complex fluidics schemes for partitioning, making the process more complicated and expensive
- Higher cost/sample for high throughput applications
A summary of the digital PCR methods (nanoplate-based dPCR vs ddPCR vs cdPCR) and their performance can be found in the table below:
|12 and 40
|8 hours for
|21 hours for
|2.5 hours for
|2-3 hours for
|25 - 50
|8 hours for
The nanoplate-based digital PCR system
- Screen – for digital PCR system setup
- Tray – for loading one or more nanoplates depending on the configuration
- Barcode scanner – to ensure the correct plate is being read and to give you more control over the process
- Thermocycler – to perform amplification reactions by pre-programmed temperatures and time variations
- Optical module – to capture fluorescence images of the plate using multi-colored detection optics or multi-channel filters
- Software Suite – optimally for both dPCR run setup and analysis software; preferably accessible remotely from other devices
For more information on the QIAGEN digital PCR system, visit the QIAcuity product page to discover features and technical specifications of the digital PCR instrument.