After the PCR discovery due to Kary Mullis, several improvements have been obtained in order to amplify and quantify nucleic acids. Among them, the Real-Time PCR, also called quantitative polymerase chain reaction (qPCR), is probably one of the most powerful and sensitive techniques for quantitative gene expression analysis and pathogen detection.
As the name suggests, the real-time PCR measures PCR amplification as it occurs, at the contrary of what happens on standard PCR, where results are collected after the reaction has been completed, so making impossible to determine the starting concentration of nucleic acid. On the contrary Real-Time PCR focusses on the exponential phase of the amplification and calculates the Threshold line (that is the level of detection at which a reaction reaches a fluorescent intensity above background) and the PCR cycle at which the sample reaches this level that is called the Cycle Threshold (Ct). The Ct value is used in downstream quantitation or presence/absence detection. By comparing the Ct values of samples of unknown concentration with a series of standards, the unknown amount of template DNA can be accurately determined.
It is quite easy in theory, but not so easy in practice… since the choice of controls, normalization methods and quality control management have serious implications for the reliability, relevance and reproducibility of qPCR experiments so that two researchers can perform the same real-time PCR experiment and get different results (as reported here in BioTechniques). I’m not trying to convince you that the qPCR does not work, simply I do not like this technique at least for my in vivo experiments in aphids.
This is why I’m reading with great interest the lastest news about digital PCR that could represent a relevant improvement for DNA quantitation in respect to qPCR. Digital PCR is a new approach to nucleic acid detection and quantification that differs in respect to qPCR, because it directly counts the number of target molecules rather than relying on reference standards or endogenous controls (qPCR vs digital PCR at a glance from Invitrogen). As you can see in the figure below (from Invitrogen), digital PCR works by partitioning a sample into many individual PCR reactions; some portions of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.
As summarized by Monya Baker in Nature Methods (2012):
“Digital PCR (dPCR) uses the same primers and probes as qPCR but is capable of higher sensitivity and precision. In standard implementations, qPCR cannot distinguish gene expression differences or copy number variants smaller than about twofold. Identifying alleles with frequencies of less than about 1% is difficult because such tests would also detect highly abundant common alleles with similar sequences. In contrast, dPCR can measure a 30% or smaller difference in gene expression, distinguish whether a variant occurs in five versus six copies and identify alleles occurring at a frequency of one in thousands. It can also be used to standardize qPCR assays.”
The more partitions, the greater the resolution. For instance with new techniques for digital PCR your sample will be partitioned in 20.000 droplets (as reported here by Biorad), each working as a single qPCR experiment that means that you could have a quantitative analysis similar for precision of doing 20,000 replicates of a quantitative PCR!
Bio-Rad (using the QuantaLife technology) has machines that offer many sample partitions using a sort of droplet digital PCR, where reaction chambers are separated not by the walls of a well but by carefully titrated emulsions of oil, water and stabilizing chemicals. First, samples are put into a machine where they are mixed with all the necessary reagents and dispersed into tiny droplets. The droplets for each sample are transferred into tubes that can be placed in a thermocycler for PCR. Afterward, the tubes are transferred to a droplet reading machine, which functions like a flow cytometer to analyze each droplet for whether or not a reaction has occurred. Would you like to know something more? Look at this nice Biorad video:
At the moment, digital PCR is a specialist approach that is much more costly than quantitative PCR mainly for the digital PCR apparatus (you have to pay about 100.000 €) rather than for the PCR chemistry, but as the next generation sequencing recently showed, as the technology matures… the costs come down so that there are few users for digital PCR apparatus today, but there will be more tomorrow.
Baker, M. (2012). Digital PCR hits its stride Nature Methods, 9 (6), 541-544 DOI: 10.1038/nmeth.2027