Myeloid Leukemia

258 Campbell et al.
2. Oligonucleotide primers. For these primers, we make a stock solution at 100 µM
and a working solution at 10 µM, diluted in nuclease-free distilled water, and
store at –20°C. Sequences (see Note 2):
a. Common reverse primer (R1):
5�-CTGAATAGTCCTACAGTGTTTTCAGTTTCA-3�
b. Mutation-specific forward primer (F2):
5�-AGCATTTGGTTTTAAATTATGGAGTATATT-3�
c. Control forward primer (F1):
5�-ATCTATAGTCATGCTGAAAGTAGGAGAAAG-3�
3. 10X PCR buffer (Applied Biosystems) supplied with Taq polymerase. Store at –20°C.
4. Magnesium chloride (MgCl 2), 25 mM. Store at –20°C.
5. Deoxynucleoside triphosphates (dNTPs). Stock solution 25 mM each dNTP, and
working solution 2.5 mM each dNTP. Store working solution in small aliquots at
–20°C, as repeated freeze-thawing damages the dNTPs.
6. Ultra-pure (deionized) water.
7. AmpliTaq-Gold (Applied Biosystems) Taq polymerase, 5 U/µL.
8. Thin-walled PCR tubes or PCR plate.
9. MJ Research PTC-200 (or other model or brand) PCR thermal cycler.
2.2. BsaXI Restriction Analysis
1. Template DNA, 40 ng/µL, stable at 4°C.
2. Oligonucleotide primers. Stock solution of each primer is 50 µM, stored at
–20°C.
a. JAK2:
5�-ATCTATAGTCATGCTGAAAGTAGGAGAAAG-3� (forward; F3)
5�-ACCTTCTACTTTTAACTTCATTGCTTTCCT-3� (reverse; R3)
b. SCL:
5�-TCCTGGGGTCTTCTGTCTTG-3� (forward)
5�-CCTGAGAGGCAATGGGAGTA-3� (reverse)
3. 10X PCR buffer (Applied Biosystems), stored at –20°C.
4. Magnesium chloride (MgCl 2), 25 mM. Store at –20°C.
5. Deoxynucleoside triphosphates (dNTPs). Stock solution 100 mM each dNTP,
and working solution 10 mM each dNTP, stored at –20°C.
6. Ultra-pure (deionized) water.
7. AmpliTaq-Gold (Applied Biosystems) Taq polymerase, 5 U/µL.
8. Thin-walled PCR tubes or PCR plate.
9. BsaXI restriction endonuclease (New England Biolabs, cat. no. R0609, 2 U/µL).
10. MJ Research PTC-200 (or other model or brand) PCR thermal cycler.
3. Methods
3.1. Allele-Specific PCR
1. Aliquot 2 µL template DNA (80 ng/reaction) into PCR tubes or a 96-well plate.
Always include a V617F-positive DNA sample, a V617F-negative DNA sample,
and water as controls (see Note 3).