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Digital PCR is a next-generation approach for specific detection and quantification of nucleic acid sequences. PCR has the ability to generate detectable quantities of specific DNA sequence from a single molecule through the exponential amplification of sequences. This is done through the use of primers and a thermostabile DNA polymerase.
By monitoring this amplification in real-time, quantities can be measured with great accuracy. Since this is compared to an external reference, measurements are relative quantifications. Digital PCR builds on these same principles of DNA quantification, enabling an absolute value with much higher precision compared to standard real-time PCR, and WITHOUT the need for any reference.
If you're already doing real-time PCR, moving toward digital PCR is straightforward. Your sample preparation and TaqMan assay remain the same. Just swap out your real-time instrument with a standard thermal cycler and digital PCR instrument. Digital PCR starts with the partitioning of your reaction mix into hundreds to thousands of independent PCR replicates. Some partitions receive the nucleic acid sequence of interest and others do not.
Partitions with a target molecule will amplify via PCR to a detectible level. These are categorized as positive reactions. Partitions without the target molecule will read as negative reactions. In the end, the number of positive reactions is directly proportional to the total number of molecules present in the sample enabling an absolute count to be established.
To understand the absolute count, it's necessary to understand that some reaction wells will receive more than one target molecule during partitioning. Uncorrected, this can lead to undercounting, as only positive reactions are counted. This mathematical issue may seem daunting, but fortunately a digital PCR instrument such as the QuantStudio 3D from Life Technologies, figures this out automatically with the use of standard statistics.
Following identification of positive and negative wells, a Poisson model is used to calculate the probability of a given reaction receiving zero, one, two, three or more copies. This correction factor enables all molecules in the starting sample to be accounted for, yielding absolute quantification.
Within absolute quantification are performance attributes that enable applications to go beyond the limits of real-time PCR. Digital PCR enables increased sensitivity and specificity of target detection as the partitioning of samples into individual reactions reduces competition of background DNA.
Volume sampled also affects sensitivity. For example, if an inadequate volume is sampled, there isn't enough sensitivity to detect a rare target. Increasing the volume sampled naturally increases sensitivity.
Precision is another performance attribute of digital PCR. This is driven by the number of replicates (or partitions) that are run. Increasing replicates increases the statistical significance of your answer, thereby giving more confidence that the value determined represents the actual target quantity in the sample. and makes it possible to directly compare results across experiments and labs.
And with these qualities, digital PCR is ideal for applications such rare mutation detection, pathogen detection and quantification, genetically modified organism detection, accurately quantifying NGS libraries and validating sequencing results.
And developing a reference for your real-time experiment when it doesn't exist ...just to name a few.
While real-time PCR still maintains its advantages of enabling higher throughput and a wide dynamic range for your assays, you'll find that digital PCR is a great compliment to real-time PCR.
This is evident in cases of a very late Ct in real-time experiments, where the precision in the measurement is not adequate to make a determination. The added sensitivity and precision of digital PCR can rescue your results, eliminating the need to re-design experiments.
Digital PCR application possibilities are extensive, and it's never been easier to put the latest technologies right at your fingertips. What will you do with digital PCR?
25 апр 2013
