Myeloid Leukemia

Detecting JAK2 V617F Mutation in MPD 263
a 1-h incubation at 37°C, although we routinely use 4 U for 4 h to ensure complete
digestion.
12. All SCL PCR products should digest with BsaXI, generating products of 356 bp,
110 bp, and 30 bp. In contrast, the mock-digestion control sample should remain
as a 496-bp fragment. If this 496-bp fragment is ever observed in a PCR sample
incubated with BsaXI, incomplete or ineffectual digestion must have occurred,
preventing accurate assessment of the JAK2 locus. We recommend either repeating
the restriction analysis with a new supply of BsaXI (in case the current stock
has lost its biological activity), or using a commercially available kit to purify the
PCR reaction prior to repeating the restriction analysis (see Note 10).
13. The wild-type (V617F-negative) JAK2 allele digests with BsaXI to produce products
of 206 bp, 174 bp, and 30 bp, whereas the mutant (V617F-positive) JAK2
allele remains as an undigested 410-bp fragment (Fig. 2). As outlined in the Introduction,
unfractionated peripheral blood or purified granulocyte samples are
likely to consist of a mixture of wild-type cells and cells from the malignant
clone, with the ratio varying from patient to patient. This phenomenon prevents
discrimination of V617F-heterozygous and V617F-homozygous genotypes. Total
peripheral blood or granulocyte samples analyzed by the BsaXI restriction
method should therefore be listed as “V617F-positive” if the 410-bp JAK2 PCR
fragment is observed in a reaction in which BsaXI digestion of the SCL PCR
fragment is complete. However, because cells present within a hematopoietic
colony are clonal, the JAK2 genotype of individual colonies can be determined
by the BsaXI restriction pattern. A wild-type colony will have the 206-bp, 174bp,
and 30-bp restriction fragments, a heterozygous V617F colony will have all
four restriction fragments (410 bp, 206 bp, 174 bp, 30 bp), and a homozygous
V617F will have only the 410-bp restriction fragment.
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