Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 83
9. Transfer the reactions to the thermal cycler and amplify with the following conditions:
a. 1 cycle at 94°C for 2 min.
b. 10 cycles at 94°C for 10 s, 65°C for 30 s, 68°C for 2 min.
c. 20 cycles at 94°C for 10 s, 65°C for 30 s, 68°C for 2 min—increase the 68°C
step by 20 s at each cycle.
d. 1 cycle at 68°C for 7 min.
10. 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.
11. The size of the b2a2 BCR-ABL product is 1886 bp, the b3a2 BCR-ABL product is
1961 bp, and the e1a2 BCR-ABL product is 483 bp. The presence of other sized
bands is indicative of an atypical BCR-ABL transcript. Sequence the atypical band
to determine the transcript type. Sequences of unknown origin that are present
within the BCR-ABL transcript may by derived from intronic sequences. An
inverted intronic sequence has been described that was inserted due to the introduction
of cryptic splice sites (23).
3.7. Qualitative PCR for p210 BCR-ABL
Our laboratory uses the quantitative PCR and atypical BCR-ABL PCR methods
to assess all CML patients. However, a qualitative nested PCR method has
been designed in our laboratory to specifically amplify the p210 BCR-ABL
transcript, and the method is presented here. The primers will amplify only the
b2a2 or b3a2 BCR-ABL transcripts and will not amplify atypical BCR-ABL
transcripts. We use a sample that is known to contain both the b2a2 and b3a2
BCR-ABL transcripts as a quality control.
1. Primers were designed using Primer Express software. The sequence of the primers
are:
First-step forward primer: 5� tgc aga tgc tga cca act cgt
First-step reverse primer: 5� cca ctg gcc aca aaa tca tac ag
Second-step forward primer: 5� tga aac tcc aga ctg tcc aca
Second-step reverse primer: 5� ggt cca gca gga agg ttt t
2. The patient RNA is extracted as outlined under Subheading 3.1., and cDNA is
prepared as outlined under Subheading 3.2.
3. Thaw the PCR reagents and primers and mix thoroughly before use (except for
the enzyme, which is stored at –20°C until added to the master mix). Centrifuge
briefly to collect the material at the bottom of the tube.
4. Prepare a master mix for the first and second-step PCR by adding the following
to a 1.5-mL tube for each patient sample + 1 positive control + 1 no-template
control + 1: 2 µL MgCl 2, 2.5 µL of 10X PCR buffer, 0.625 µL dNTP (10 mM
mix), the forward and reverse primers at a final concentration of 0.5 µM, 0.25 µL
of 5 U/µL of AmpliTaq gold, and sterile water, to a total volume of 22.5 µL for
the first-step mix and 23 µL for the second-step mix. Mix the tubes thoroughly
and centrifuge briefly to collect the mix at the bottom of the tube.