qPCR
integrated fluidic circuits that are able to run 12 samples, each partitioned into 765 reaction cham- bers (12.765) and 48 samples each partitioned into 770 reaction chambers (48.770). Reaction volumes are 6nl and 0.85nl, respectively.
Many of the limitations of qPCR are alleviated in dPCR and the technology has important advan- tages in several applications. Most popular dPCR application is copy number determination. In theo- ry it should be possible to determine absolute copy numbers with dPCR, ie, loading a sample and measuring the number of target DNA copies it con- tains directly without having to use a reference. In practise, this is not so easy because of dead vol- ume, losses during processing and handling of the sample, and ambiguity in separating positive and negative reads16. In the future some of these prob- lems may be solved, perhaps by calibration, which would bring dPCR a step closer to becoming an absolute standard and as such exceedingly impor- tant as a reference method in nucleic acid testing. For now, copy number determinations, even with dPCR are performed as relative measurements. One may measure the relative abundance of one target in two samples or the relative amounts of two targets in a single sample. The latter is a typi-
cal comparison with an endogenous control, such as a conserved sequence present in exactly one copy per haploid genome. One such assay is ValidPrime17, which targets a non-transcribed region in the genome and is therefore not amplify- ing any cDNA. One of the obvious applications is to determine allelic imbalance such as gene dupli- cation/amplification, genetic aberrations, allelic loss and similar variations in gene copy numbers. The precision in the dPCR determination depends on the number of reaction chambers used, and has been detailed mathematically18 and confirmed experimentally19. For example, four from five copies can be distinguished using some 1,200
Figure 2
Digital PCR service in Europe. Left: Dr Anders Ståhlberg loading the OpenArray at the TATAA Biocenter digital PCR service facility in Gothenburg, Sweden. Above: The OpenArray through hole plate with 3072 reaction chambers
Drug Discovery World Fall 2011
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