Myeloid Leukemia

Detecting JAK2 V617F Mutation in MPD 259
2. Make a master mix for the number of reactions set up with:
a. Common reverse primer (R1) (10 µM), 5 µL;
b. Mutation-specific forward primer (F2) (10 µM), 2.5 µL;
c. Control forward primer (F1) (10 µM), 2.5 µL;
d. 10X PCR buffer, 5 µL;
e. Magnesium chloride (25 mM), 3 µL;
f. dNTPs (2.5 mM each), 4 µL;
g. AmpliTaq-Gold polymerase (5 U/µL), 0.5 µL;
h. Ultra-pure (deionized) water, 25.5 µL;
i. Template DNA, (2µL).
Final volume 50 µL. Mix well and aliquot 48 µL into each tube (see Note 4).
3. Place the tubes or plate into a thermal cycler and PCR amplify using the following
program (see Notes 5 and 6):
a. 94°C for 11 min (if using AmpliTaq Gold or other hot-start Taq);
b. 36 cycles of: 94°C for 30 s, 58°C for 30 s, 72°C for 30 s;
c. 72°C for 6 min;
d. 4°C thereafter.
4. Run the PCR products for samples and controls on a 1.5% agarose gel with
ethidium bromide. The control forward primer and common reverse primer amplifies
a 364-bp product from all samples, and the mutation-specific forward
primer and common reverse primer amplify a 203-bp product only from V617Fpositive
DNA (see Note 7 and Fig. 2i).
3.2. BsaXI Restriction Analysis
1. Aliquot 2 µL purified template DNA into two individual PCR tubes or wells of a
96-well plate. Alternately, when analyzing individual hematopoietic colonies,
aliquot 5 µL crude cell lysate into two individual PCR tubes or wells of a 96-well
plate (see Note 8). Also include V617F-positive and V617F-negative DNA
samples as controls (see Note 3).
2. Make two PCR master mixes: one with primers specific to the JAK2 gene (F3
and R3), the other with primers specific to the SCL gene. The volume of each
master mix should be sufficient for the total number of reactions (n) to be performed;
we generally make enough for 1.1n to allow for pipetting inaccuracies.
Each reaction consists of:
a. Forward primer (50 µM), 1.0 µL;
b. Reverse primer (50 µM), 1.0 µL;
c. 10X PCR buffer, 5.0 µL;
d. MgCl 2 (25 mM), 3.0 µL;
e. dNTPs (10 mM each), 1.0 µL;
f. Taq polymerase (5 U/µL), 0.5 µL;
g. Water to 50.0 µL final reaction volume.
Mix well and aliquot 48 µL (45 µL if cell lysate is being used) into each tube or
well.
3. Place the tubes or plate in a thermal cycler, and amplify using the following
program: