qPCR
Figure 1
The principle of digital PCR. An extensively diluted sample is partitioned into a large
number of reaction chambers, such that most chambers are empty or contain a single template molecule only. PCR is performed in the chambers amplifying the template
molecules that are present. At the end of the PCR the
number of positive reactions
are scored, which reflects the initial number of template
molecules that were present in the sample
qPCR is extensively used for mutation and SNP analysis. But there is finite possibility an assay with primers targeting an SNP will also amplify wild- type sequence. This gives rise to false positive sig- nal and limits assay specificity. A SNP present in only a fraction of the template molecules may be missed. An alternative approach is to use generic primers and sense sequence variation using probes. The probe, however, binds with finite probability also to wild-type sequence, which limits assay specificity of this design. The specificity of a qPCR assay limits the background of wild-type sequence that is tolerated in a sample. Using regular Taqman assays already, 10-fold background of wild-type sequence is often challenging. qPCR assay speci- ficity can be marginally increased by using modi- fied primers/probes with elevated thermal stability6. Other strategies to enhance qPCR speci- ficity is by sequestering, as used in CastPCR recent- ly made available from Life Technologies7, or by using modified primers such as the myT primers developed by Swift Biosciences8. Final limitation of qPCR is its sensitivity to inhibition. Analysing field sample substances from the sample matrix that have not been removed or carryover of reagents from upstream steps may interfere with the PCR influencing the measured Cqs and thus the estimate of target DNA concentration9. Wild-type Taq as well as several engineered variants are exceedingly sensitive to common inhibitors such as human blood, and major attempts are made to find more resistant variants using either rational design or selection strategies10.
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Already in 1992, even a year before Russ Higuchi described qPCR11, Sykes et al12 had the idea of quantifying target numbers by PCR using limiting dilution. Diluting a sample to such an extent that it contains a very small number of tar- get molecules such that when aliquoted into reac- tion containers most will be empty, while some will contain a single template molecule only (Figure 1). Performing PCR the number of positive reactions will correspond to the number of template mole- cules in the original sample13. In 1999 Bert Vogelstein used the technique to quantify K-ras mutations in stool DNA from colorectal cancer patients and named it digital PCR (dPCR)14. It took time for dPCR to gain popularity as its preci- sion was limited by the rather small number of reaction chambers in conventional 96 and 384- well plate instruments that often had to be filled manually. This changed when the high throughput platforms with integrated loading systems became available. Most convenient is the OpenArray from Life Technologies (Figure 2)2 that we use in our dPCR services in Europe15. The measurement plat- form is a small metal plate the size of a microscope slide with 3072 reaction chambers in the form of small through holes that each hold 33nl of sample. They are arranged in 48 subarrays with 64 (= 8×8) chambers in each. This offers flexibility to tune the loading to the requested resolution and is also con- venient for the serial dilutions initially performed to find optimum loading concentration. Other excellent platforms are the EP1 and the BioMark from Fluidigm2. These use Fluidigm’s ingenious
Drug Discovery World Fall 2011
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