Digital polymerase chain reaction

Digital Polymerase Chain Reaction (digital PCR or dPCR) 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.

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Basics

Similar to earlier polymerase chain reaction based methods, dPCR amplifies nucleic acids by temperature cycling of a nucleic acid molecule with the enzyme DNA polymerase. 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; 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 each individual nucleic acid molecule present in a sample within many separate chambers or regions prior to PCR amplification. A count of chambers containing detectable levels of PCR end-product is a direct measure of the absolute nucleic acids quantity.

Development

The digital PCR concept was developed cooperatively by the Cytonix Corporation and the National Institutes of Health and was published by Kalinina and colleagues (Kalinina et al., 1997, Brenan and Morrison, 2005). 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 quantitization of nucleic acids for pathogens detection and the clonal amplification of nucleic acids (clonal PCR) for the identification and sequencing of mixed nucleic acids samples or fragments.

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.