DIGITAL PCR (dPCR)
Doing digital PCR means to carry out a PCR reaction with only one single amplification step nonetheless resulting in absolute/quantitative values. For dPCR, in contrast to conventional real-time PCR (qPCR), no standard curves, calculating of Ct-values or standard references are required because the single amplification step is performed to obtain a pure yes (positive) or no (negative) answer.
The principle behind dPCR is to divide a given sample into a high number of samples of very small volume (femto litre) of which each small sample contains ideally only one DNA molecule of interest. The higher the number of small samples the more precise will be the result of the evaluation/analysis as the probability increases to have only one single copy or none in a given volume if the volume decreases. Therefore first evaluation of the samples is according to a “Yes” (positive) and “No” (negative) principle. After counting the positive and negative signals the ratio between positive and negative samples and the absolute amount of positive samples is calculated to get the absolute content of target molecules in the original sample. For this procedure no internal or external standards are necessary.
By performing only one single amplification step there is no chemical restriction regarding the consumption of e.g. dNTPs, degradation of Taq Polymerase etc., or no physical restriction, e.g. instrumental specifications or mathematical by calculation of a Ct-value. Additionally the dynamic range and precision of this procedure is much higher due to the low detection limit.
Like in qPCR detection is done by measuring the fluorescence of PCR probes.
To have samples containing more than only one single copy of the target is a possible source of error. To overcome this problem all results will be recalculated and compensated by the mathematical method referred as Poisson Algorithm.
Applications of digital PCR:
- Absolute quantification of samples, e.g. diagnostics of pathogens
- Detection of rare Alleles and Mutations
- Absolute quantification of nucleic acid standards
- Absolute quantification of Next Generation Sequencing libraries
- Absolute quantification of gene expression
- Absolute quantification of reference standards for qPCR
Advantages of digital PCR
- dPCR is very tolerant against inhibitors
- Sample preparation is the same as for qPCR
- Variations in the genome can be detected precisely (e.g. transgenic variations)
- Highly reproducible with high precision
Analysis of miRNA expression of stem cell differentiation has been described by Jiang et al. (Jiang, K., Ren, C., & Nair, V. (2013). MicroRNA-137 represses Klf4 and Tbx3 during differentiation of mouse embryonic stem cells. Stem Cell Research, 11(3) 1299-1313).
dPCR encounters increasing interest in the fields of cancer mutations, circulating RNA and DNA, for example in cancer patients or the case of detecting low abundant events such as mutant sequences that can be detected very specifically against a high background of wild-type sequences. Somatic mutations can be detected specifically due to the high selectivity of dPCR. For patients, this means a significantly higher chance for an earlier, minimally invasive diagnosis. The detection of low abundant alleles or mutations is a further advantage.
qPCR: Suited for high amounts of different samples with only little amount of work. Only relative measurements possible. Quantification only possible against reference standard.
dPCR: absolute measurement of nucleic acids possible. No calculation of Ct-values necessary. Very useful to screen for low abundant alleles, mutations or other differences in genes of interest.