PCR amplification and sequencing of the entireCYP6P5–CYP6P9b intergenic region
To validate the poolseq results, which indicated the presence of a
structural variant in the upstream region of CYP6P9b , the entire
intergenic region between CYP6P5 and CYP6P9b was
amplified. Primers were 6P9dplF CCC CCA CAG GTG GTA ACT ATC TGA A
located at 19bp before the stop codon of CYP6P5 and the 6P9Ra/b
TAC ACT GCC GAC ACT ACG AAG located at 35bp after the start codon ofCYP6P9b and the Phusion high fidelity DNA polymerase (Thermo
Scientific) was used for the amplification. The Phusion Taq PCR mix
consisted of 3µl of 5x HF buffer, 0.12µl of 25mM dNTPs, 10mM forward and
reverse primers, 0.15µl Phusion Taq, 9.71µl of deionised water and 1µl
of DNA for 15µl reaction. Thermocycler conditions were: pre-denaturation
at 98°C for 1 minute; 35 cycles of denaturation at 98°C for 10 seconds,
annealing at 62°C for 30 seconds, extension at 72°C for 4 minutes; a
final extension at 72°C for 10 minutes. PCR amplicons were stained with
Midori Green Advance DNA Stain (Nippon Genetics Europe GmbH) and
visualised and size-scored following (1%) agarose gel electrophoresis,
using an ENDURO GDS (Labnet) UV transilluminator. PCR products were
gel-purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden,
Germany), ligated into PJET1.2 blunt-end vectors and Sanger-sequenced
from each end of the 5.3 kb fragment using the plasmid-specific
sequencing primers pJET1.2F and pJET1.2R. To sequence the complete
fragment, four additional internal sequencing primers were used (Table
S1). Sequence data were analysed with BioEdit software (Hall, 1999).