Detection of the JAK2 by sequence analysis, and Pyrosequencing. (A) Sequence traces of 3 individuals found to be wild-type, heterozygous, and homozygous by the ARMS assay. (B) Representative pyrograms for one wild-type, one heterozygous, and 2 homozygous individuals. The sequencing primer is in reverse orientation (immediately abutting the site of the mutation) and the dispensation order GCAGCATAC was used (the 2 Gs are internal controls that should give no peak; E and S indicate enzyme and substrate, respectively). In a wild-type individual (sequence CACATAC) the peak heights of the first C and A are similar. In a heterozygous individual (sequence (C/A)ACATAC) the C peak is reduced in height and the A peak increased above all other peaks as both the mutant A and following A are being read in the same reaction. In a homozygous individual (sequence AACATAC) the first C peak is either much lower than the A peak (third trace) or, occasionally, absent (fourth trace). The ratio of the healthy (C) and mutant (A) alleles is calculated by the Pyrosequencing SNP analysis software. Values are log10 delta-delta CtPRV1/ABL.
