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54 | Droplet Digital
™
PCR Applications Guide
Rare Mutation and Sequence Detection
RSD Experimental Strategies
Some RSD applications require reliable quantification of rare sequences while others require
detection of a rare sequence. This difference dictates the lower bounds of sensitivity for
a given assay and application. For RSD, the LoD can be defined either in terms of the
total volume of material analyzed or in terms of the number of copies of some type of
background DNA.
In RSD, absolute quantification of the target sequence is often required. In general, ddPCR
can provide an accurate quantification of a rare target sequence at a low concentration.
This eliminates the need for absolute standards and standard curves and improves
reproducibility across experiments and laboratories. In addition, in ddPCR, small variations
in PCR efficiency between wells have no effect on the measured concentration.
When absolute quantification is applied to an RSD application, an important consideration
is the limit of quantification, or LoQ, which is the lowest concentration you can reliably
measure within a predetermined variance, or coefficient of variation (CV). For example,
if you want to quantify within 20%, the LoQ is the lowest concentration with which you can
reliably measure to within ±20% of the real value. For RSD, to reliably detect 1 in 100,000
cells, at least 300,000 background cells must be screened. Similarly, to achieve an LoD of
1 in 1 ml of sample, 3 ml of sample must be screened.
Two different experimental setups are recommended for RSD applications, depending on
whether detection is with respect to a starting volume of sample or with respect to a second
background target that requires quantification.
Case 1: Quantification with Respect to Total Starting Volume
To detect foreign DNA in 5 ml of lake water, 15 ml of lake water must be screened.
The experiment is shown in Figure 5.8 for the case of a single ddPCR well. Depending
on how much total DNA is extracted from the 15 ml sample, more than one well may be
needed to analyze the sample because up to 2.5 µg of total DNA can be loaded into one
reaction well.
Fig. 5.8. Strategy for detecting rare foreign DNA in a sample of defined volume.
DNA from 15 ml
starting sample
Quantify rare sequence
Target
Starting amount