User manual

Droplet Digital
™
PCR Applications Guide | 7
Emerging Applications of Droplet Digital PCR
Sample partitioning allows the sensitive, specific detection of single template molecules as
well as precise quantification. It also mitigates the effects of target competition, making PCR
amplification less sensitive to inhibition and greatly improving the discriminatory capacity
of assays that differ by only a single nucleotide. Digital PCR offers the benefits of absolute
quantification and greatly enhanced sensitivity. Therefore, its application in the following
areas is growing:
■
Absolute quantification — ddPCR provides a concentration of target DNA copies per
input sample without the need for running standard curves, making this technique
ideal for target DNA measurements, viral load analysis, and microbial quantification
■
Genomic alterations such as gene copy number variation (CNV) — CNVs result in too
few or too many dosage-sensitive genes responsible for phenotypic variability, complex
behavioral traits, and disease. ddPCR enables measurement of 1.2x differences in gene
copy number
■
Detection of rare sequences — researchers must amplify single genes in a complex
sample, such as a few tumor cells in a wild-type background. ddPCR is sensitive
enough to detect rare mutations or sequences
■
Gene expression and microRNA analysis — ddPCR provides stand-alone absolute
quantification of expression levels, especially low-abundance microRNAs, with
sensitivity and precision
■
Next-generation sequencing (NGS) — ddPCR quantifies NGS sample library
preparations to increase sequencing accuracy and reduce run repeats. Validate
sequencing results such as single nucleotide polymorphisms or copy number
variations with absolute quantification
■
Single cell analysis — the high degree (10- to 100-fold) of cell-cell variation in gene
expression and genomic content among homogeneous post-mitotic, progenitor, and
stem cell populations drives a need for analysis from single cells. ddPCR enables
low copy number quantification
ddPCR for Absolute Quantification and
Experimental Considerations
In a typical digital PCR experiment, the sample is randomly distributed into discrete
partitions such that some contain no nucleic acid template and others contain one or more
template copies. The partitions are PCR amplified to end point and then read using a droplet
reader to determine the fraction of positive partitions, from which the concentration is
estimated by modeling as a Poisson distribution. The formula used for Poisson modeling is:
Copies per droplet = –ln(1 – p)
where p = fraction of positive droplets.
Droplets are assigned as positive or negative by thresholding based on their fluorescence
amplitude. The number of positive and negative droplets is used to calculate the
concentration of the target and reference DNA sequences and their Poisson-based
95% confidence intervals (Figure 1.12).
Droplet Digital
™
PCR