Digital PCR analysis helps researchers to detect target nucleic acids in complex samples while elevating the shortcomings of traditional qPCR assays. In the digital PCR method, the study sample is divided into multiple sub-reactions where each partition has few or no nucleic acid sequences of interest. After the PCR reaction, the fraction of positive partitions is used for quantifying the target sequences. Notably, partitioning a sample efficiently concentrates the nucleic acid sequence of interest within each isolated microreaction. This concentrated micro sample has a reduced template competition and can effectively detect rare mutations. The current article discusses digital PCR services in nucleic acid quantification.
Digital PCR analysis for nucleic acid quantification
Although device miniaturization has been at the core of development in digital PCR analysis, digital PCR systems were developed using microtubes or microplates. Laboratories such as PK CRO and assay service providers suffered a lot from these traditional formats due to limited automation, limited partitions, and associated costs of large amounts of reagents. Here, microfluidics technology has added advantage in the parallel partitioning of samples and advancements in digital PCR platforms. Microfluidics is based on microfabrication systems adopted from microelectronics. From liquid droplets to physical partitions, researchers have used several passive and active microfluidic methods for compartmentalizing study samples. Many of these approaches use limited reagents and facilitate basic automation.
However, there are also several partition-free methods. For example, an early method using a silica capillary assay reaction vessel performed PCR analysis using diluted DNA molecules. This technique is used in line fluorescence detectors to count the number of amplified products after electromigration. Fused silica capillaries-based PCR reactions relied on the diffusion of the generated and amplified products and their migration during electrophoresis. The generated signals correspond to the target sequences present in the study samples.
Similar to qPCR expression analysis, digital PCR uses two primary chemistries: hydrolysis-based probes and DNA intercalating dyes. Both these detection strategies yield a fluorescent signal that is proportional to the DNA in the sample. DNA binding dyes are inserted into double-stranded DNA. Post interaction with this double-stranded DNA, DNA dyes are excited, resulting in a strong fluorescence signal. They are nonspecific and interact with the double-stranded DNA irrespective of their sequences.
On the other hand, hydrolysis-based probes are very specific to the sequence and hence require unique chemistry. The 5’ nuclease method uses the 5’ to 3’ exonuclease activity of DNA polymerases to cleave the fluorescent-labeled oligonucleotide probe once it hybridizes with the target sequence. The fluorescently labeled reporter dye at the 5’ end is released, generating a fluorescent signal.
Multiplex qPCR analysis can detect several targets in a single study volume using different fluorescence types attached to the probes. Hence, the number of target analytes quantified by qPCR is spectrally limited. Consequently, qPCR assay development is challenging to multiplex compared to ddPCR assays. Researchers can code different target sequences with different fluorescent dyes and diverse fluorescence intensities in ddPCR assays. Besides, with options such as the ddPCR method, scientists can now effectively partition samples into thousands of droplets and evaluate each micro reaction for detecting and quantifying nucleic acids.