All patients had the mutational status for JAK2 V617F determined using the allele specific-PCR assay on DNA purified from granulocytes, as reported [19].
The presence of mutation m.14484T>C in these patients was confirmed at the Kunming Institute of Zoology and at the Zhongshan Opthalmic Center, independently, by using the allele-specific PCR, SSCP (single strand conformation polymorphism), and/or sequencing [22], [23].
PCR reactions were done for 35 cycles consisting of 95°C for 30 sec, 61°C for 30 sec, and 72°C for 1 min. Products were amplified using the allele-specific primers designed by Jennifer Richer (University of Colorado) listed below.
The PIK3CA rs141178472 was genotyped using the allele-specific PCR assay on the S1000 thermal cycler (Bio-Rad).
Diagnostic allele-specific PCR was performed according to a previously described method [ 35] using the allele-specific primers shown in Table 4.
A total of 18 single nucleotide polymorphisms (Barker et al. 2011) were genotyped using the allele-specific primer extension (ASPE) technique, (Luminex, Austin, TX, USA), following the modified protocol outlined in Orantes et al. (2012).
Using the allele-specific primer sets Vrn-P1F/R ~ Vrn-P4F/R, we showed that 161 out of 173 winter wheat cultivars possessed the recessive vrn-A1 allele at the Vrn-A1 locus.
There were 79,533 A/T SNPs with a 'G' or 'T' base in the 3rd nucleotide upstream, or a 'C' or 'A' base in the 3rd nucleotide downstream: these SNPs can be efficiently genotyped using the allele-specific PCR method [ 30].
JAK2V617F mutation was detected in DNA samples using the allele-specific PCR method previously described by Baxter et al. [ 16] The quantification of JAK2V617F allele burden on genomic DNA was performed using the JAK2V617F MutaQuant® Allele kit (Qiagen, Milano, Italy), according to the manufacturer's instructions.
