Myeloid Leukemia

Diagnosis of CBFB-MYH11-Positive AML 167
control. Alternatively, CBFB-MYH11 plasmid constructs may be applied. To
exclude false-positive and false-negative results, the dilution series should be
included in each analysis. The positive control must be diluted in such a manner
that at a certain dilution a conversion from positive to negative signals is
observed. If no conversion from PCR positive to negative signals is observed,
the dilution series may be contaminated. Consequently, false-negative results
may be obtained due to failure to recognize inadequate sensitivity of the assay.
To show that larger transcripts are efficiently amplified in qualitative PCR,
we recommend also including a patient sample positive for CBFB-MYH11 fusion
transcript types D or E (see Fig. 1). To avoid false-positive results, several
control samples negative for the CBFB-MYH11 fusion should be included in
each PCR run.
3.1.1. Qualitative CBFB-MYH11 PCR
1. Prepare the first-round CBFB-MYH11 PCR mix by pipetting in the following
order for one PCR reaction (see Notes 2 and 3): 11.09 µL H 2O, 1.25 µL 50 mM
MgCl 2, 0.4 µL dNTPs, 2.5 µL DMSO, 2.5 µL 10X PCR buffer, 1.0 µL primer cd
(Table 1), 1.0 µL primer mm (Table 1), and 0.26 µL Taq DNA polymerase (total
volume of 20 µL).
2. Mix the first-round CBFB-MYH11 PCR mix and add per reaction 5 µL of cDNA
(50 ng) and a drop of mineral oil (see Note 4).
3. Perform the first-round CBFB-MYH11 PCR using an initial denaturing step of 5
min at 94°C, followed by 27 cycles of 1 min at 94°C (denaturation of cDNA-
RNA hybrids), 1 min at 62°C (primer annealing), 1 min at 72°C (product extension),
followed by a final extension step of 10 min at 72°C (see Note 5).
4. Prepare the second-round CBFB-MYH11 PCR mix by pipetting in the following
order for one PCR reaction: 11.09 µL H 2O, 1.25 µL 50 mM MgCl 2, 0.4
µL dNTPs, 2.5 µL DMSO, 2.5 µL 10X PCR buffer, 1.0 µL primer cmdI
(Table 1), 1.0 µL primer mmd2 (Table 1), and 0.26 µL Taq DNA polymerase
(total volume 20 µL).
5. Mix the second-round CBFB-MYH11 PCR mix and add per reaction 5 µL of a
five times dilution of the first-round PCR product and a drop of mineral oil.
6. Perform the second-round CBFB-MYH11 PCR using an initial denaturing step of
5 min at 94°C (denaturation of first-round PCR product), followed by 25 cycles
of 1 min 94°C (denaturation), 1 min 62°C (primer annealing), 1 min 72°C (product
extension), followed by a final extension step of 10 min at 72°C.
7. Judge CBFB-MYH11 PCR amplification by agarosose gel electrophoresis using
a 1.5% agarose-TBE (0.5X) gel containing 35 ng/mL ethidium bromide. Products
can be visualized after ultraviolet (UV) exposure of the agarose gel. Make
sure to include DNA size markers that allow for proper size determination in the
250- to 1700-bp range. The PCR product lengths obtained for the various CBFB-
MYH11 fusion transcripts after first- and second-round (nested) PCR, respectively,
are indicated in Table 2.