Myeloid Leukemia

Detection of BCR-ABL Mutations 99
of the tube.
13. Pipet 23 µL of the master mix into 0.2-mL tubes.
14. In a laboratory that is separate from the PCR setup and cDNA addition laboratories,
add 2 µL of the purified PCR products that require the second-step PCR and
2 µL of the first-step no-template control to the 0.2-mL tubes. Mix thoroughly
and centrifuge briefly to collect the mix at the bottom of the tube.
15. Transfer the reactions to the thermal cycler and amplify with the following conditions:
1 cycle at 94°C for 2 min.
10 cycles at 94°C for 10 s, 60°C for 30 s, 68°C for 2 min.
30 cycles at 94°C for 10 s, 60°C for 30 s, 68°C for 2 min—increase the 68°C step
by 20 s at each cycle.
1 cycle at 68°C for 7 min.
16. After completion of the PCR, assess the size of each amplification product by
electrophoresis of 5 µL of the PCR product mixed with 1 µL gel loading buffer
on a 2.0% agarose gel, using the SPP1 size marker. The no-template control
sample should not contain an amplified product, but if it does, the PCR should be
repeated. If a product of the correct size (863 bp) is obtained from the patient
samples, purify the PCR product using the UltraClean Purification kit according
to the manufacturer’s instructions. Additional bands may be present on the gel
that may or may not interfere with the sequencing reaction (see Note 3).
17. The samples are ready for the sequencing reaction and are stored at 4°C after
completion.
3.2. Sequencing Reaction
Sequencing reactions are performed in the forward and reverse directions.
The primers used are the second-step forward primer and the first-step reverse
primer. The primers will allow sequencing of a region that encompasses the
entire BCR-ABL kinase domain. The amount of purified product added to the
sequencing reaction may be varied if required (see Note 4).
1. Thaw the Big Dye 3 sequencing reagent on ice, while protecting from the light.
Thaw the primers, mix, and centrifuge briefly to collect the material at the bottom
of the tube.
2. Prepare two master mixes on ice for the forward and reverse sequencing reactions,
one containing the forward primer and one containing the reverse primer.
For each mix, pipet the following into a 1.5-mL tube for each sample + 1: 4 µL of
Big Dye 3 sequencing reagent, 0.3 µL primer (final concentration of 0.75 µM),
and sterile water to a total volume of 18 µL. The volume of master mix prepared
may need to be adjusted for samples that produce faint amplified products (see
Note 4).
3. Mix the tubes thoroughly and centrifuge briefly to collect the mix at the bottom
of the tube.
4. Pipet 18 µL (see Note 4) of each master mix into 0.2-mL tubes.
5. In a laboratory that is separate from the PCR setup and cDNA addition laborato-