A Simple, Rapid, and Sensitive Method for the Detection of the JAK2 V617F Mutation

Angela Y.C. Tan, PhD; David A. Westerman, MBBS, FRACP, FRCPA; Alexander Dobrovic, PhD

Disclosures

Am J Clin Pathol. 2007;127(6):977-981. 

In This Article

Materials and Methods

Samples

The peripheral blood samples of 26 patients with myeloproliferative disease (PRV, 8; ET, 15; IMF, 2; postpolycythemic myelofibrosis and myeloid metaplasia, 1) and 4 patients with lymphoproliferative disease were analyzed by the Pathology Department, the Peter MacCallum Cancer Centre, East Melbourne, Australia. The patients were undergoing studies for hematologic disease. Normal peripheral blood samples were obtained from 16 healthy volunteers. All samples were collected into tubes containing EDTA and were obtained in accordance with the Peter MacCallum Cancer Centre Ethics of Human Research guidelines.

DNA Extraction

DNA was isolated from the peripheral blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer's instructions.

ACB Assay

Approximately 100 ng of DNA was amplified in a total volume of 25 µL containing 200 nmol/L of Normal_f primer 5'-GCATTTGGTTTTAAATTATGGAGTATGTG-phosphate (a phosphate group was added to the 3' end to prevent extension from the normal primer, blocking amplification of the normal allele), 400 nmol/L each of Mutant_f primer 5'-GCATTTGGTTTTAAATTATGGAGTATGAT and reverse primer 5'-ACTGACACCTAGCTGTGATCCTG, 2 mmol/L of magnesium chloride, 50 µmol/L of each deoxynucleotide triphosphate (dNTP), 0.5 U of HotStar Taq (Qiagen, Hilden, Germany), and 1 × buffer (Qiagen). All primers were purchased from Sigma Aldrich (Castle Hill, Australia). The cycling conditions were 95°C (15 minutes) and 45 cycles of 94°C (30 seconds), 64°C (30 seconds), 72°C (30 seconds), and a final 72°C extension for 10 minutes. The 139-base-pair products were visualized on a 2% ethidium bromide-stained agarose gel. The presence of a band indicated that the sample was positive for the V617F mutation. All samples were first amplified using the control PCR reaction of JAK2 (see the next section). Each sample was set up in triplicate in the ACB-PCR assay to ensure that no false-negative results were obtained, and at least 3 negative control samples were run alongside the test samples to exclude false-positive results.

Control Reaction PCR of JAK2

A control PCR reaction that amplifies exon 14 of JAK2 was performed for all samples to exclude false-negative results in case the extracted DNA samples were not of adequate quality to amplify. The amplification was performed as described for the ACB-PCR assay except the primers Control_f 5'-TTCCTTAGTCTTTCTTTGAAGCAG and Control_r 5'-ACTGACACCTAGCTGTGATCCTG were used with 200 µmol/L of dNTPs. The 191-base-pair products were visualized on a 2% ethidium bromide-stained agarose gel.

Direct Sequencing

To validate the ACB-PCR assay, each sample was analyzed by direct sequencing of the control reaction PCR products (exon 12, JAK2) using a standard fluorescent dye method. Briefly, products were incubated with EXO-SAP-IT (USB, Cleveland, OH), following the manufacturer's instructions. The enzymatically cleaned PCR products were used as a template for the cycle sequencing reaction. Forward and reverse sequencing were performed using Big Dye Terminator version 3.1 chemistry (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. After cycle sequencing was completed, products were ethanol precipitated and, after addition of Hi-Di (Applied Biosystems), analyzed on an ABI 3100 (Applied Biosystems).

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