Applications of Digital PCR

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Overview

Digital PCR is a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. This technique is particularly useful for low abundance targets, targets in complex backgrounds, allelic variants (SNPs) and for monitoring of subtle changes in target levels.

In the Droplet Digital™ PCR (ddPCR™) System, each PCR sample is partitioned into a large number of microscopic droplets prior to amplification. Each droplet is an individual PCR reaction. After end-point amplification, fluorescence is detected in the droplets in which target sequence was amplified. These droplets are scored as positive. Droplets not containing the target sequence show little or no fluorescence and are scored as negative. Using the Poisson distribution law, the fraction of positive droplets is converted to the number of molecules in the starting sample, without the need for standard curves (absolute quantification).

Applications for digital PCR cover different areas of biology. The following describes some of the most popular fields of application.

Copy Number Variation (CNV)

Estimates suggest that over 10% of the human genome is composed of CNVs with sequences larger than 1 kb, and that about 30% of the reference human genome contains CNVs of sequences larger than 100 bp. Some regions can have many copies per cell. A number of CNVs have been linked to diseases. Disease-associated CNVs can be inherited or generated de novo. In addition to diseases, CNVs in dosage-sensitive genes are associated with complex phenotypic and behavior traits. The ddPCR System has been demonstrated to be well suited to high-throughput studies profiling CNV variations in populations of interest (Karlin-Neumann et al. 2011, Boettger et al. 2012).

Digital PCR can provide accurate CNV quantification in single wells. In qPCR assays, many replicates are needed to accurately discriminate CNVs. For example, to ascertain a copy number change from 4 to 5, up to 18 qPCR replicates are required. The precision and sensitivity of ddPCR technology allows the system to distinguish small changes much more readily: the same change from four to five copies can be determined using just one well. Additionally, the accuracy of digital PCR is less sensitive to changes in amplification efficiency, a major cause of inaccuracy in qPCR measurements.

Learn more about Digital PCR for Copy Number Variation Analysis.

Rare Sequence Detection

Increasing detection sensitivity for low-abundance targets will have broad impacts ranging from earlier and more precise detection of cellular changes in research and clinical settings to the development of a wider array of noninvasive tests.

Digital PCR allows for increased performance in the detection and quantitation of rare sequences because target quantification is independent of the number of amplification cycles. Moreover, when detecting related sequences (SNPs, allelic variants, edited RNA), partitioning reduces competition with the more abundant background (wild-type) species. Expanding uses of digital PCR for rare sequence detection include:

  • Detection of cancer below the level detectable by current tests
  • Monitoring for new mutations and duplications in cancer as they arise
  • Detection of viral loads, such as HIV, below those detectable by current testing
  • Noninvasive testing in bodily fluids for infectious diseases and cancer, including circulating cell-free DNA (cfDNA)
  • Noninvasive prenatal testing using cfDNA
  • Detection of transplant rejection in circulating DNA

Learn more about Rare Sequence Detection Using Digital PCR.

Gene Expression

The increased precision of digital PCR can provide higher resolution in many aspects of gene expression measurement. It enables scientists to precisely quantify finer changes in expression levels (less than twofold). Digital PCR also provides a greater sensitivity when quantifying rare targets, or RNA from very limited material.

Digital PCR is used for the analysis of gene expression at both the DNA and RNA levels in the following areas:

  • Detection and measurement of genomic DNA methylation
  • Increased sensitivity in transcriptional analyses with absolute quantification
  • Detection of rare mRNAs and miRNAs with rapid turnover including those in complex matrices such as blood
  • Detection of as few as 2 targets per well using EvaGreen with the QX200™ Droplet Digital PCR System. This allows users to perform gene expression analysis without TaqMan probes.

Learn more about Gene Expression and Digital PCR.

Single-Cell Analysis

Single-cell PCR is challenging. Not only can the isolation of an intact single cell be difficult, but getting accurate results from the low levels of starting material has been technically challenging.

The following characteristics of digital PCR make it a sensitive and robust tool for single-cell analysis:

  • Precise detection and amplification of targets at low template levels
  • No requirement for a standard curve or housekeeping genes
  • Reduced sensitivity to PCR-inhibiting components in crude cell lysates
  • Simultaneous detection of 4 targets through multiplexing
  • Can be combined with other techniques such as next-generation sequencing

Learn more about Single-Cell Analysis Using Digital PCR.

Pathogen Detection and Microbiome Analysis

Perhaps the most extensive use of digital PCR to date has been in microbiology. Both pathogen detection and microbiome analysis often require the detection and quantitation of low-abundance microorganisms in complex backgrounds.

Some areas of pathogen detection in which digital PCR is used are:

  • Detection and monitoring of viral loads that are below the limit of quantification of current tests
  • Pathogen detection both pre- and post-allotransplantation
  • Detection and monitoring of microbial drug resistance and heteroresistance
  • Detection of pathogens in food

The analysis of microbiomes can be difficult and labor intensive due to the large number of species and subspecies in most biomes and their often continually changing nature. The high sensitivity, ability to amplify rare targets in complex backgrounds, and reduced sensitivity to PCR inhibitors such as humic acid are all features of digital PCR that can improve microbiome analysis. Uses of digital PCR include:

  • Tracking changes in population composition
  • Cosegregation analysis including phylogenetic relationships

Learn more about Pathogen Detection and Microbiome Analysis Using Digital PCR.

Next-Generation Sequencing (NGS)

Digital PCR can increase the efficiency and accuracy of NGS, saving both money and time.

Integration of digital PCR into an NGS workflow can occur at several steps:

  • Amplification of target libraries, ensuring better representation of low abundance species
  • Accurate quantification of NGS libraries
  • Validation of sequencing results

Learn more about Digital PCR and Next-Generation Sequencing.

References

Boettger LM et al. (2012). Structural haplotypes and recent evolution of the human 17q21.31 region. Nat Genet 44, 881–885. PMID: 22751096

Karlin-Neumann G et al. (2011). Probing copy number variations using Bio-Rad's QX100™
Droplet Digital™ PCR System. Bio-Rad Bulletin 6277.

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