Myeloid Leukemia

96 Branford and Hughes
kinase domain. The PCR product is directly sequenced in the forward and
reverse directions using dye terminator chemistry and a 3700 DNA Sequencer
(Applied Biosystems, Foster City, CA, USA) (16,28). Mutation Surveyor software
(SoftGenetics, LLC, State College, PA) is used to analyze the sequence.
RNA extracted from peripheral blood or bone marrow is suitable for analysis.
The technique is also applicable for imatinib-treated patients with Philadelphia
chromosome-positive acute lymphoblastic leukemia (ALL). These patients
may have a BCR-ABL transcript that is the same as that present in most patients
with CML (b2a2 or b3a2 BCR-ABL), or the transcript may involve the fusion
of BCR exon 1 to ABL exon 2 (e1a2 BCR-ABL). For patients with the e1a2
transcript, an alternate 5� PCR primer is required.
The ability to detect mutations is very dependent on the quality of the RNA,
which is assessed prior to mutation analysis by measurement of the level of
normal BCR mRNA using real-time quantitative PCR (29). A cutoff level based
on the number of detectable BCR transcripts in the sample was established as
an indicator of degraded RNA. Samples with BCR levels below the cutoff are
not assessed for mutations. This technique permits the detection of single or
multiple mutations in patients with disease status ranging from a complete
cytogenetic response (Philadelphia chromosome negative) to overt relapse, at
a sensitivity of 20%. To date, we have detected 24 different point mutations,
spanning the entire kinase domain and ranging from amino acid 244 to 486
(GenBank accession number M14752). Identification of these mutations is of
clinical significance, because virtually all CML patients with detectable mutations
acquire resistance to imatinib (16,22).
2. Materials
2.1. Semi-Nested PCR Reaction
1. Sterile 0.2-mL capped PCR tubes (Interpath Services).
2. Sterile 1.5-mL capped PCR tubes (Interpath Services).
3. 500-µL single-use lots of autoclaved MiliQ water. Store at room temperature in
1.5-mL tubes.
4. PCR primers. All primers are ordered as a 50 µM solution and stored in 30-µL
lots at 20°C. Primers were designed using Primer Express software (Applied
Biosystems).
5. 15 mM stock of dNTPs (Amersham Biosciences). Store at –20°C in 100-µL lots.
6. 25 mM MgCl 2 (Applied Biosystems). Store at –20°C.
7. Expand Long Template PCR System (Roche). Store at –20°C.
8. PCR cabinet with ultraviolet light.
9. UltraClean PCR Clean-up DNA purification kit (Mo Bio Laboratories). Store at
room temperature.
10. Agarose, molecular-biology grade (Progen). Store at room temperature.
11. SPP1/EcoR1 DNA molecular-weight marker (Geneworks). Store at –20°C.