Myeloid Leukemia

FLT3 Mutations in AML 193
Table 2
Reaction Mixture for Eco RV Digestion
Reaction Mixture (for one reaction)
Polymerase chain reaction products 10 µL
10X digestion buffer 3 µL
100X bovine serum albumin 0.3 µL
Eco RV 1 µL (20 U)
Distilled water 15.7 µL
Total 30 µL
1. Prepare the reaction mixture according to Table 2.
2. Incubate at 37°C for 2 h.
3. Heat-inactivate the enzyme at 65°C for 10 min.
4. PCR products can be directly used for the digestion procedure without any purification.
However, it is recommended that optimal PCR amplification be confirmed
by gel electrophoresis before the digestion procedure.
3.5. Electrophoresis
In most instances, ITD mutations can be detected by agarose gel electrophoresis
of the amplified products (see Note 3). This procedure usually demonstrates
a wild-type band (329 bp) and a larger-size band that is the ITD
mutation (Fig. 1). In some cases harboring an ITD mutation, loss of the wildtype
allele has been observed (see Note 4). In those cases, only the ITD band is
present (Fig. 1B, lane 5).
D835 mutations are detected by electrophoresis of the amplified products
following digestion with Eco RV. The amplified products of wild-type alleles
are digested to two bands (68 bp and 46 bp) by Eco RV. When amplified products
contain D835 mutations, undigested bands (114 bp) are visualized on agarose
gel electrophoresis (Fig. 2). Inclusion of a negative control is essential to
ensure complete digestion by Eco RV, thereby eliminating the possibility of
false-positive results in patient samples.
3.6. DNA Sequencing
When identifying FLT3 mutations, DNA sequencing is not necessarily
required. However, we recommend the sequencing of mutated FLT3 genes to
confirm the mutation types and to exclude false positives. Mutated bands are
cut out from the agarose gel and purified with gel extraction kits, such as
QIAquick Gel Extraction Kit (Qiagen). Purified samples can usually be
sequenced directly. We use BigDye ® Terminator Cycle Sequencing Kit