Page 299 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Supplementary Figure 1. Illustration of molecular methods
ddPCR: Droplet Digital PCR (ddPCR)3 is a method based on water-oil emulsion droplet technology.
DNA isolation is performed using MagNA Pure Compact Nucleic Acid Isolation Kit I (Hoffmann-La Roche, Basel Switzerland). A DNA sample is fragmented into tens of thousands of droplets (A) and PCR amplification of DNA is present in each droplet (B). PCR is based on standard TaqMan probe-based assays. In this study, we used FAM and HEX fluorescent labels, shown in red and blue (primers and probes used are; F-primer: CTGATCTTCATCTTCATTCC, R-primer: AGGTGGGAGAATTGTTGCTAT, probe: HEX-CCAGTTTCATCTTGTTCTGTCCGAACAA-BHQ , F-primer: TCACTGAACTAAAGGCTGA, R-primer: CAACTTATGCCAATTCTCTTG, probe: FAM-CTTTCCAGACAACATAAGTGTGATTTAAC-BHQ). In patients with two variants on a single allele (in cis) most droplets will emit both fluorescent labels, resulting in a combined color purple, where in patients with two variants on different alleles (in trans) most
Supplement
ddPCR
A create droplets
sample oil oil
droplets
B DNA reads per droplet
in cis in trans
C expression per droplet
D determine phasing
in cis in trans
PacBio
Plasmid
A labeled nucleotides
A ligation
AGCT
plasmid vector
PCR product
B fluorescent pulse
B transformation
cell
C two peaks at once
C sequence one strand
GTTTGCAACC
CT
AGACGGTCAA
D determine phasing
D determine phasing
in cis
in trans
GTTTGCAACC GTCTGCATCC
GTTTGCATCC GTCTGCAACC
AGACGGTCAA in cis
AGACGGTCAA in trans
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