User manual

46 | Droplet Digital
™
PCR Applications Guide
Fig. 5.1. Rare mutation detection and rare sequence detection assays.
Rare Mutation and Sequence Detection
Measurement
Units Examples
Assay
Components Assay Schematic
Rare Mutation Detection
Ratio
(% or a/[a+b])

Tumor biopsy: 0.01%
mutant detected,
or 1 in 10,000

Single nucleotide
polymorphisms (SNPs)

Small indels
1 primer pair,
2 competitive probes
Rare Sequence Detection
Case 1 Copies/volume

Invasive species
monitoring
(copies/ml lake water)

Human immuno-
deficiency virus (HIV)
detection
(copies/ml plasma)
1 assay
(1 primer/probe set)
Case 2 Ratio
(% or a/[a+b])
or copies/unit
reference

Gene expression
of rare transcripts

Viral staging

Indels
2 independent assays
(2 primer/probe sets)
FAM
T
HEX
C
Target DNA
A/G
SNP
FAM
T
Target DNA
A
FAM
T
HEX
C
Target DNA #1
A
Target DNA #2
G
For any rare detection assay, standard design rules should be used in the design of TaqMan
probes and primer sets (see Chapter 2). In general, primers should be designed to have
melting temperatures (T
m
) of ~60°C (1 M NaCl, 1 µM concentration) and should be within
2°C of each other. Furthermore, the probe must have a melting temperature 3–10°C higher
than the primer T
m
. A temperature binding enhancer, such as a locked nucleic acid (LNA)
can be used to shorten the number of nucleotides in the probe while maintaining a higher T
m
.
Finally, the mutant site should be positioned near the middle of the probe sequence.
Rare detection assay designs should be validated with a temperature gradient to ensure
the highest specificity between the mutant and wild-type clusters. The optimal annealing
temperature is defined by the condition in which the mutant probe exhibits no false positives
in the wild-type–only sample and the relative distance between the FAM-only (mutant) and
HEX (or VIC)-only (wild-type) clusters is maximal.
To enable the ultrasensitive detection of mutant targets, very high loads of DNA
are required. However, for ddPCR, amounts of intact human DNA exceeding 66 ng
(20,000 genome equivalents)/20 µl reaction negatively affect the accuracy of DNA
quantification. To mitigate this effect, gDNA must be fragmented by restriction digestion
using an enzyme that cuts around the amplicon(s) of interest. Once fragmented, the human
gDNA concentration can exceed 1 µg/20 µl reaction without affecting DNA quantification.