Digital polymerase chain reaction

Digital Polymerase Chain Reaction (digital PCR, dPCR or dePCR) is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA.

Basics

Similar to earlier polymerase chain reaction based methods, digital PCR (dPCR) amplifies nucleic acids by temperature cycling of a nucleic acid molecule with the enzyme DNA polymerase. In digital emulsion PCR (dePCR), the reaction is carried out in the dispersed phase of an emulsion. Theoretically, PCR exponentially amplifies nucleic acids, and the number of amplification cycles and the amount of PCR end-product should allow the computation of starting quantity. However, many factors complicate this calculation, creating uncertainties and inaccuracies. These factors include: initial amplification cycles may not be exponential; PCR amplification eventually plateaus after an uncertain number of cycles; low initial concentrations of target nucleic acid molecules may not amplify to detectable levels; and PCR amplification efficiency in a sample of interest may be different from that of reference samples.

Working Principle

Digital PCR overcomes these difficulties by transforming unreliable exponential data from conventional PCR to digital signals that simply indicate whether or not amplification has occurred. Digital PCR is achieved by capturing or isolating each individual nucleic acid molecule present in a sample within many separate chambers, zones or regions that are able to localize and concentrate the amplification product to detectable levels. After PCR amplification, a count of chambers, zones or regions containing PCR end-product is a direct measure of the absolute nucleic acids quantity. The capture or isolation of individual nucleic acid molecules may be effected in capillaries, microemulsions, arrays of miniaturized chambers, or on nucleic acid binding surfaces.

Development

The digital PCR concept was conceived and developed by the Cytonix Corporation and the National Institutes of Health in 1995, and a first U. S. Patent was issued in 1997. Vogelstein and Kinzler further developed the concept by quantifying KRAS mutations in stool DNA from colorectal cancer patients (Vogelstein and Kinzler, 1999). Digital PCR has been shown to be a promising surveillance tool for illnesses such as cancer (Pohl and Shih, 2004). Digital PCR has many other applications, including detection and quantitization of low-level pathogens, rare genetic sequences, gene expression in single cells, and the clonal amplification of nucleic acids (clonal PCR) for the identification and sequencing of mixed nucleic acids samples or fragments. Clonal amplification enabled by digital PCR is a key factor in reducing the time and cost of sequencing an individual genome.

References

• Kalinina O, Brown J, Silver J, Nanoliter scale PCR with TaqMan detection. Nucleic Acids Research. 25; 1999-2004, 1997.
• Brenan C, Morrison T, High throughput, nanoliter quantitative PCR. Drug Discovery Today: Technologies. 2; 247-253. 2005.
• Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 96; 9236-41, 1999.
• Pohl, G and Shih, I-M Principle and applications of digital PCR. Expert Rev Mol Diagn. 4; 41-7, 2004.
• Blow N., PCR's next frontier, Nature Methods 4, 869 - 875 (2007) doi:10.1038/nmeth1007-869