Supplement
DNA extraction
Isolation of genomic DNA from one to three 8 μm paraffin wax-embedded sections was
performed with the DNeasy Micro Kit and the QIAcube (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions.
DNA yield, purity and quality were measured photometrically (NanoDrop, Peqlab, Erlangen,
Germany) and validated by capillary gel electrophoresis (QIAxcel, Qiagen, Hilden,
Germany).
Two-step wild-type-blocker allele-specific quantitative PCR (WTB-AS-QPCR)
Step one: WTB-PCR
Primers and probes were designed using open source Primer3-web software
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and were evaluated by Blast
searches at NCBI (http://www.ncbi.nlm.nih.gov/blast/) (Table 1). All primers and probes were
synthesized by Metabion (Planegg-Martinsried, Germany).
WTB-PCR was performed on a Peqlab gradient cycler (Peqlab, Erlangen, Germany), cycling
15 rounds with a reaction volume of 40 µl, containing primers (300 nM), MgCl2 (2 mM),
dNTP (200 nM of each), Taq DNA Polymerase (1 u) (Fermentas, St. Leon-Rot, Germany)
and LNA-WTB-Probe (200 nM) (Primer and LNA-WTB probe listed in Table 1) mixed with
2µl of genomic DNA. The reaction mixture was preheated at 95°C for 2 minutes, followed by
15 cycles at 95°C for 20 seconds, 55°C for 40 seconds and 72°C for 40 seconds, finally
followed by an elongation step at 72°C for 6 minutes.
Step two: AS-QPCR
AS-QPCR was performed on a MX3000p (Stratagene, Amsterdam, Netherlands) and on a
StepOnePlus (Applied Biosystems, Foster City, CA, USA) detection system cycling 40
rounds with typical reactions of a final volume of 25 µl, containing primers (300 nM) and
probes (200 nM) of a p.JAK2 V617F specific SNP assay (Applied Biosystems, Foster City,
CA; Table 1) mixed with the appropriate volume of MasterMix (5Prime, Hamburg, Germany)
and 2 µl of 100fold diluted PCR product of the WTB-PCR. The reaction mixture was
preheated at 95°C for 2 minutes, followed by 40 cycles at 95°C for 20 seconds and 60°C for 1
minute. All experiments were evaluated by performing an identical second run. Briefly, the
results of PCR were validated using sequence specific primers labelled with the IRD 800
fluorescence dye (Metabion, Planegg-Martinsried, Germany). Sequence analysis was carried
out on a LI-COR DNA analyzer (Gene Reader 4200; MWGBiotech). The sequence products
were compared with the published data of the NCBI database by BLAST analyses to exclude
amplification of a false amplicon.