Myeloid Leukemia

192 Kiyoi and Naoe
Table 1
Reaction Mixture for Polymerase Chain Reaction (PCR)
Reaction mixture (for 1 reaction)
Sample DNA 100–200 ng
10X PCR Gold Buffer 5 µL
25 mM MgCl 2 solution 3 µL
2.5 mM dNTP solution 4 µL
Forward primer 50 pmol
Reverse primer 50 pmol
AmpliTaq Gold 2.5 Units (0.5 µL)
Distilled water add up to the total volume 50 µL
for the sample DNA. Importantly, both positive and negative control samples
should be included at every analysis (see Note 2). To avoid cross-contamination,
sample DNA should be added last to the reaction mixture, which has been
aliquotted from the master mix.
3.3. PCR Conditions
The thermal profile for obtaining optimal PCR amplification is dependent in
part upon the characteristics of individual PCR machines and reaction tubes.
The following profiles have been confirmed to be optimal when using
MicroAmp reaction tubes and GeneAmp PCR system 2400, 2700, 9600, or
9700 (Applied Biosystems). For other combinations, the optimal amplification
conditions should first be determined by each investigator.
The thermal profile for ITD-mutations: pre-PCR step, 95°C—9 min; thermal
cycle, 94°C—30 s, 56°C—1 min, 72°C—2 min; 35 cycles; post-PCR step,
72°C—10 min.
The thermal profile for D835-mutations: pre-PCR step, 95°C—9 min; thermal
cycle, 94°C—30 s, 60°C—1 min, 72°C—2 min; 35 cycles; post-PCR step,
72°C—10 min.
Because AmpliTaq Gold is provided in an inactive state, the pre-PCR heat
step is essential. It is recommended by the manufacturer that optimal results
are obtained with a 9- to 12-min heat step at 92–95°C before the PCR cycles.
Amplified products can be stored at –20°C until post-PCR analysis.
3.4. Digestion With Eco RV
To detect D835 mutations, a restriction fragment length polymorphism-mediated
PCR assay is used, because D835 and I836 codons are encoded by the
nucleotide sequence GATATC, which represents an Eco RV restriction site.
Amplified products are digested with Eco RV, and subjected to electrophoresis
in an agarose gel.