Myeloid Leukemia

FLT3 Mutations in AML 191
3.1.1. DNA ZOL Reagent
This reagent can be stored at 15°C to 30°C for at least 1 yr. It contains
guanidine isothiocyanate, and may be harmful if contact with skin or eyes occurs.
It is highly recommended that the manufacturer’s instructions be read
carefully before use. Ethanol is also required for the procedure. The extraction
procedure can be performed quickly and easily according to the manufacturer’s
instructions. In brief, cell pellets are lysed in this reagent by gently pipetting.
The genomic DNA is precipitated from the lysate with ethanol.
3.1.2. QIAamp DNA Mini Kit
This kit provides fast and easy methods for purification of genomic DNA
from whole blood and buffy coat. Columns and all reagents, except for ethanol
and phosphate-buffered saline, are included in the kit. Some reagents are harmful
and irritant. It is highly recommended that the manufacturer’s instructions
be read carefully before use. DNA extraction can be performed by the simple
QIAamp spin and vacuum procedures within 20 min according to the
manufacturer’s instructions.
3.2. PCR
3.2.1. Primers
To date, several primer pairs for the detection of FLT3 mutations have been
described. The following primer sequences have been reported by us (5,12):
For the detection of ITD mutations in the juxtamembrane domain:
Forward primer: 5�-CAATTTAGGTATGAAAGCCAGC-3�
Reverse primer: 5�-CTTTCAGCATTTTGACGGCAACC-3�
For the detection of D835 mutations in the kinase domain:
Forward primer: 5�-CCGCCAGGAACGTGCTTG-3�
Reverse primer: 5�-GCAGCCTCACATTGCCCC-3�
3.2.2. Reaction Mixture and PCR Condition
Many thermostable DNA polymerases and their reaction buffers are available.
Neither special polymerase nor buffer conditions are required for the
amplification of the FLT3 gene. We usually use AmpliTaq Gold and the
accompanying reagents (Perkin Elmer, Foster City, CA). AmpliTaq Gold and
reagents must be stored at –20°C in a constant-temperature freezer.
1. Thaw and gently mix each of the reagents.
2. Spin the polymerase solution down in a microcentrifuge before use.
3. Prepare reaction mixture according to Table 1.
4. It is recommended that a master mix of sufficient volume for the number of
samples being analyzed be prepared. This should contain all the reagents except