Digital polymerase chain reaction

Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnology 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, though also more prone to error in the hands of inexperienced users. 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

Main article: Polymerase chain reaction

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

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 estimate the number of different molecules by assuming that the molecule population follows the Poisson distribution. 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, the number of PCR amplification cycles is proportional to the starting copy number. 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 therefore provides absolute quantification.

Development

The original dPCR paper was published by Dr Alec Morley and Pamela Sykes in 1992. The purpose was to quantify PCR targets, rather than the PCR amplified products in an attempt to track and measure the absolute lowest number of leukemic cells in a patient with leukemia. The purpose was to monitor residual disease in leukemia patients, and thereby treat the patients at the earliest possible moment of detection of disease recurrence. Further evolutions of the technology allowed for more practical use of this method with a wider audience, with small partitions created by emulsion droplets and/or microfluidics.

A development occurred in 1995 with co-inventions by Brown at Cytonix and Silver at the National Institutes of Health of single-step quantitization and sequencing methods employing nano-scale physical containment arrays (Brown, Silver), and open chambers (Brown) using localized clonal colonies in 1D and 2D capillaries, macro volumes, gels, free chambers, and affinity surfaces/particles. resulting in a 1997 U. S. Patent, and subsequent divisional and continuation patents. The concepts of electrowetting and digital microfluidics were further introduced (Brown) as one means of manipulating nano fluid volumes.

Digital PCR has been shown to be a promising surveillance tool for illnesses such as cancer, and as a vital front end to determining genomic content, including sequencing the human genome.

Based on the concept Vogelstein and Kinzler developed a technology called BEAMing (Beads, Emulsion, Amplification, Magnetics) and quantified KRAS mutations in stool DNA from colorectal cancer patients.

Significant additional developments have included using emulsion beads for digital PCR by Dressman and colleagues.

It has also proved useful for the analysis of heterogeneous methylation.

In 2006 Fluidigm introduced the first commercial system for digital PCR based on integrated fluidic circuits (chips) having integrated chambers and valves for partitioning samples.

In 2008, Inostics started to provide BEAMing digital PCR services for the detection of mutations in plasma/serum and tissue.

QuantaLife developed a fundamentally distinct method of partitioning, the Droplet Digital PCR (ddPCR) technology, which partitions a sample into 20,000 droplets and provides digital counting of nucleic acid targets. In 2011, Quantalife was acquired by Bio-Rad Laboratories.

In 2013, RainDance Technologies launched a digital PCR platform based on its picoliter-scale droplet technology, which generates up to 10 million picoliter-sized droplets per lane. The technology was first demonstrated in a paper published in Lab on a Chip by scientists from Université de Strasbourg and Université Paris Descartes. Later that year, RainDance Technologies announced a partnership with Integrated DNA Technologies to develop reagents for the digital PCR platform.

Digital PCR has many applications, including the detection and quantification of low-level pathogens, rare genetic sequences, copy number variations, and relative gene expression in single cells. This method provides the information with accuracy and precision. 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.

MIQE

The "Minimum Information for Publication of Quantitative Digital PCR Experiments" or "digital MIQE" guidelines are a comprehensive set of best practices which aim to increase the validity and comparability of digital PCR experiments reported in published literature. The guide was published in 2013 and followed publication in 2009 of "MIQE", a comparable guide for quantitative real-time PCR. In the two years following publication of the "digital MIQE", less than 20% of published digital PCR papers have cited the guideline.

External links

• Digital PCR Protocol