Myeloid Leukemia

FIP1L1-PDGFRA in Hypereosinophilic Syndrome 183
Table 1
Polymerase Chain Reaction Setup
Component Amount needed
Positive control Negative control Water control HES patient
Forward primer 10 pmol 10 pmol 10 pmol 10 pmol
Reverse primer 10 pmol 10 pmol 10 pmol 10 pmol
10X buffer 5 µL 5 µL 5 µL 5 µL
dNTPs (10 mM) 1 µL 1 µL 1 µL 1 µL
Taq enzyme 2.5 U 2.5 U 2.5 U 2.5 U
cDNA from 1 µL* — — —
EOL-1
cDNA from — 1 µL* — —
healthy
individual
Water — — 1 µL —
cDNA from HES — — — 1 µL*
patient
Water to 50 µL to 50 µL to 50 µL to 50 µL
* This is approximately a one-twentieth fraction of the cDNA obtained from 1–5 µg. dNTPs,
deoxynucleotidetriphosphates.
Note: When setting up four reactions with the same primer pair, prepare a fivefold master mix
containing each component (except cDNA).
HES, hypereosinophilic syndrome.
Table 2
Polymerase Chain Reaction (PCR) Program
Step Temperature Time
1 94°C3 min (denaturing of the cDNA)
2 94°C30 s (denaturing of the cDNA/PCR products)
3 58°C30 s (primer annealing)
4 72°C90 s (primer extension by Taq DNA polymerase)
→ repeat steps 2–4 34 times (35 cycles in total)
5 72°C3 min (final extension step)
6 4°Cindefinite (cooling)
This is a basic PCR program. Based on the type of PCR machine, variations on specific times
may be needed.
for FIP1L1 and PDGFRA, the fusion gene cannot be detected so easily at
the DNA level. This is due to the presence of large introns in FIP1L1 and
the variation of the breakpoints over a region of 50 kb (see Note 8). Based
on the fact that the breakpoints in PDGFRA are always located within exon 12