Myeloid Leukemia

FIP1L1-PDGFRA in Hypereosinophilic Syndrome 185
Fig. 3. Overview of the long-distance inverse polymerase chain reaction (PCR)
(LDI-PCR) technique. Genomic DNA is cut with AseI, the digested DNA is purified
and diluted, and subjected to a ligation reaction under conditions that favor self ligation
(circularization). Subsequent PCR reaction using the primers a (Ase-Fa) and c
(PDGFRA-Ra) in a first-round PCR, and the primers b (Ase-Fb) and d (PDGFRA-Rb)
in the second (nested) PCR reaction, yields a PCR product containing the region at the
fusion of FIP1L1 and PDGFRA.
13. Load a fraction of the isolated PCR product again on a 1% agarose gel to check
its concentration and purity.
14. Sequence the PCR product using primer PDGFRA-Rb (see Note 9).
3.4. Detection of the del(4)(q12q12) at the Chromosome Level
3.4.1. Fluorescence In Situ Hybridization (FISH)
The FISH technique is described in detail in a previous chapter in this volume.
The deletion is present in most peripheral blood cells, particularly eosinophils
(up to 70% of white blood cells in the blood of HES/CEL patients), and
neutrophils. FISH can be performed on peripheral blood or on bone marrow.
The probes described here have been successfully used for both interphase and
metaphase FISH (6) (see Note 10).