User manual

Droplet Digital
™
PCR Applications Guide | 47
Rare Mutation and Sequence Detection
Figure 5.2 depicts a bulk 20 μl solution containing 40 mutants and 40,000 wild-type DNA
molecules with a mutant abundance of 0.1%, which is beyond the limit of quantification by
real-time PCR. After random partitioning into 20,000 1-nl droplets, there are 40 droplets
containing target molecules at a relative mutant abundance of 33%, which are easily
distinguishable from the 19,960 droplets containing wild-type molecules only.
Rare Mutation Detection
Rare mutation detection occurs when a biomarker exists within a background of a highly
abundant counterpart that differs by only a single nucleotide. Many methods for mutation
analysis have poor selectivity and fail to detect mutant sequences with abundances of less
than one in 100 wild-type sequences (Scott 2011, Benlloch et al. 2006, Whitehall et al. 2009).
If enough DNA sample is available for testing, a limit of detection of 1 mutant in 100,000 wild
type can be detected using a well-designed assay and proper experimental setup.
ddPCR for Rare Allele Detection and Experimental Considerations
The partitioning effect, which is a hallmark of ddPCR technology, has an important impact
on the sensitivity and specificity of a PCR reaction. For applications reliant on measuring a
low-abundance rare mutant allele in a large excess of wild-type DNA, partitioning the sample
into droplets increases the sensitivity by orders of magnitude by effectively diluting away
the background. This means that the mutant target is present in droplets at a much greater
relative abundance than it would be in bulk solution. Considering a rare allele detection
scenario where the desired mutant has an abundance of 0.1% relative to wild-type DNA,
Figure 5.2 shows that randomly partitioning the sample into 20,000 1-nl droplets increases
the relative abundance by 330-fold, resulting in drastic improvement of the achievable
sensitivity compared to real-time PCR.
Fig. 5.2. Effect of partitioning on the relative abundance of a mutant target in an excess
of wild-type DNA.
40 droplets with mutant
33% mutant abundance
19,960 droplets without mutant
40,000 wild-type molecules
40 mutant molecules
0.1% mutant abundance
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
C
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
Bulk sample = 20 µl
Partitioned sample = 20,000 x 1 nl
A
C
G
C
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C
A
C
G
T
A
C
A
C