Digital PCR

From Populationgenomics.org

Digital Polymerase Chain Reaction (digital PCR, DigitalPCR, 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. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants and point mutations - and it is routinely used for clonal amplification of samples for "next-generation sequencing."

 

PCR Basics

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase. Conventional PCR is based on the theory that amplification is exponential. Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample. However, many factors complicate this calculation, creating uncertainties and inaccuracies. These factors include the following: initial amplification cycles may not be exponential; PCR amplification eventually plateaus after an uncertain number of cycles; and low initial concentrations of target nucleic acid molecules may not amplify to detectable levels. However, the most significant limitation of PCR is that PCR amplification efficiency in a sample of interest may be different from that of reference samples. Since PCR is an exponential process, only twofold differences in amplification can be observed, greatly impacting the validity and precision of the results.

dPCR Working Principle

Digital PCR overcomes the difficulties of conventional PCR. With dPCR, a sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. (The capture or isolation of individual nucleic acid molecules has been effected in micro well plates, capillaries, the dispersed phase of an emulsion, and arrays of miniaturized chambers, as well as on nucleic acid binding surfaces.) The partitioning of the sample allows one to count the molecules by estimating according to Poisson. As a result, each part will contain "0" or "1" molecules, or a negative or positive reaction, respectively. After PCR amplification, nucleic acids may be quantified by counting the regions that contain PCR end-product, positive reactions. In conventional PCR, starting copy number is proportional to the number of PCR amplification cycles. dPCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and providing absolute quantification.

Development

The digital PCR concept was conceived in 1992 by Sykes et al.[1] using nested PCR. An important development occurred in 1995 with co-inventions by Brown at Cytonix[2] and Silver at the National Institutes of Health[3] of single-step quantitization and sequencing methods employing nano-scale arrays and localized clonal colonies using capillaries, gels, affinity surfaces/particles and immiscible fluid containments, resulting in a 1997 U. S. Patent[4] and subsequent divisional and continuation patents. Vogelstein and Kinzler further developed the concept by quantifying KRAS mutations in stool DNA from colorectal cancer patients[5]. Digital PCR has been shown to be a promising surveillance tool for illnesses such as cancer [6]. Significant additional developments have included using emulsion beads for digital PCR by Dressman and colleagues [7]. 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 (cPCR or clonal PCR) for the identification and sequencing of mixed nucleic acids samples or fragments. It has also proved useful for the analysis of heterogeneous methylation [8].

In 2006 Fluidigm[9] introduced the first commercial system for digital PCR based on integrated fluidic circuits (chips) having integrated chambers and valves for partitioning samples. In November 2010, Life Technologies[10] commercialized a digital PCR product line for the OpenArray system. In March 2010, a patent was published for digital PCR based on emulsions. QuantaLife[11] is developing the technology for release in 2011.

Digital PCR has many potential applications, including the detection and quantification of low-level pathogens, rare genetic sequences, copy number variations, and relative gene expression in single cells. Clonal amplification enabled by single-step digital PCR is a key factor in reducing the time and cost of many of the "next-generation sequencing" methods and hence enabling personal genomics.

References

  1. ^ Sykes, PJ; Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA (1992). "Quantitation of targets for PCR by use of limiting dilution". Biotechniques 13 (3): 444–9. PMID 1389177. 
  2. ^ "Cytonix". http://www.cytonix.com/. 
  3. ^ Kalinina, O; Brown J, Silver J (1997). "Nanoliter scale PCR with TaqMan detection". Nucleic Acids Research 25 (10): 1999–2004. doi:10.1093/nar/25.10.1999. PMID 9115368. 
  4. ^ "U. S. Patent 6,143,496". http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=6,143,496.PN.&OS=PN/6,143,496&RS=PN/6,143,496. 
  5. ^ Vogelstein, B; Kinzler KW (1999). "Digital PCR". Proc Natl Acad Sci U S A. 96 (16): 9236–41. doi:10.1073/pnas.96.16.9236. PMID 10430926. 
  6. ^ Pohl, G; Shih, I-M (2004). "Principle and applications of digital PCR". Expert Rev Mol Diagn 4 (1): 41–7. doi:10.1586/14737159.4.1.41. PMID 14711348. 
  7. ^ Dressman, D; Yan H, Traverso G, Kinzler KW, Vogelstein B (2003). "Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations". Proc Natl Acad Sci USA 100 (15): 8817–22. doi:10.1073/pnas.1133470100. PMID 12857956. 
  8. ^ Mikeska, T; Candiloro IL, Dobrovic A (2010). "The implications of heterogeneous DNA methylation for the accurate quantification of methylation". Epigenomics 2: 561–73. doi:10.2217/epi.10.32. 
  9. ^ "Fluidigm". http://www.Fluidigm.com/. 
  10. ^ "Life Technologies". http://www.lifetechnologies.com/. 
  11. ^ "QuantaLife". http://www.QuantaLife.com/. 

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