User manual

72 | Droplet Digital
™
PCR Applications Guide
8 Additional Applications
Linkage Analysis
Physical linkage of two alleles in Droplet Digital
™
PCR (ddPCR
™
) can be thought of in at
least two ways:
■
Tandem repeats of the same sequence
■
Proximity of two sequences (targets) physically linked on the same piece of DNA
We recommend using restriction enzymes to digest the DNA in a copy number variation
(CNV) study and physically separate the two target copies (in order to ensure random
target distribution into droplets). If the copies are not separated, then the target can be
undercounted because the copies will always segregate into the same droplet. This is
especially true for tandem copies near each other. The extreme sensitivity of ddPCR,
however, allows you to study linkage between targets and to infer the haplotype of samples
by comparing digested vs. undigested DNA.
Figure 8.1 represents a study of different human samples using restriction digestion in
comparison with undigested DNA for the same sample for the MRGPRX1 gene, which has
variable copy numbers (CNs) in different populations. In samples 4 and 5, the undigested
(uncut) samples have lower CNs compared to the digested (cut) DNA of the same sample,
which suggests the copies in those samples are linked on the same chromosomal strand.