Myeloid Leukemia

190 Kiyoi and Naoe
most frequent genetic alteration identified in AML. Several large-scale studies
in well-documented patients published to date have demonstrated that FLT3
mutations are strongly associated with a poor prognosis and a high leukemia
cell count in patients with AML, suggesting that FLT3 mutations are involved
in disease progression (6,10,11). Both ITD and D835 mutations are detected
by means of a conventional genomic PCR-mediated technique (2). Because
the detection of FLT3 mutations is fast, easy, and inexpensive, mutation analysis
can be performed as a routine test to facilitate assessment of individual
patient prognosis.
2. Materials
1. DNA extraction reagent or kit (e.g., DNAZOL Reagent, Gibco BRL,
Gaithersburg, ND; and QIAamp® DNA Mini Kit, Qiagen, Chatsworth, CA).
2. Ethanol.
3. Oligonucleotide primers.
4. Thermostable DNA polymerase (e.g., AmpliTaq Gold, Perkin Elmer, Foster
City, CA).
5. Polymerase chain reaction (PCR) reaction buffer (usually supplied with polymerase).
6. dNTP solution (usually supplied with polymerase).
7. MgCl2 solution (usually supplied with polymerase).
8. Water (autoclaved deionized, ultrafiltered, or glass-distilled water is recommended).
9. PCR equipment (thermal cycler).
10. Eco RV endonuclease.
11. Reaction buffers for EcoRV (usually supplied with Eco RV endonuclease).
12. Agarose (e.g., NuSieve ® GTG agarose and SeaKem® GTG agarose).
13. Sample loading buffer.
14. TBE buffer: 89 mM Tris-borate, 2 mM ethylenediamine tetraacetic acid (EDTA),
pH 8.0.
15. DNA molecular-weight marker (e.g., 100-bp ladder markers for the detection of
ITD, 10-bp ladder markers for the detection of D835 mutations).
16. Ethidium bromide.
17. Electrophoresis equipment.
18. DNA sequencing kit and equipment.
3. Methods
3.1. DNA Extraction
Genomic DNA for the detection of FLT3 mutations is extracted from bone
marrow and/or peripheral blood cells by any one of a number of standard methods.
Many reagents and/or kits for DNA extraction are commercially available.
We usually use DNAZOL Reagent (Gibco BRL, Gaithersburg, ND) or
QIAamp DNA Mini Kit (Qiagen, Chatsworth, CA).