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

Results

Optimization

The important variables for the ACB-PCR assay to detect the V617F mutation were the annealing temperature, dNTP concentration, and primer concentration. The majority of the conditions were modeled from a previous ACB-PCR assay.[18] Annealing temperatures of 60°C, 62°C, 64°C, and 68°C were initially used with a low concentration of 50 µmol/L of dNTPs to test the specificity of the primers. A low concentration of dNTPs encourages the correct base to be incorporated during the PCR reaction, increasing fidelity and accuracy at the critical 3' end of the primers. In addition, it was previously found that the ideal primer concentration was when the mutant primer and normal primer (with the 3' phosphate blocking group) were used at a 2:1 ratio. The conditions were deemed optimal when repeated negative control DNA samples did not amplify while the positive patient samples were robustly amplified. These conditions are those described in the "Materials and Methods" section. If the ACB-PCR assay is performed under nonoptimal conditions, mispriming may occur from the normal primer and amplification would be observed in the negative control samples.

Detection Sensitivity

The sensitivity of the ACB-PCR assay was assessed using serial dilutions of the HEL cell line DNA (which carries the V617F mutation) with DNA of normal control peripheral blood samples Image 1A. Amplification products were visible to the level of 1%. This level of sensitivity is higher than that of sequencing (10%-40%),[2,3,4,5] pyrosequencing (5%-10%),[7,8] and melt curve analysis (1%-10%).[14,15,16] Furthermore, peripheral whole blood extraction using a commercial kit further simplified the assay because extra steps to isolate granulocytes for low-level disease detection were unnecessary owing to the high sensitivity of the ACB-PCR assay.

Image 1.

Patient Samples

Samples from 26 patients with myeloproliferative disease were analyzed using the ACB-PCR assay Image 1B, and the results were correlated with the sequencing results Table 1 . All ACB-PCR results were concordant with the direct sequencing results. However, analysis of the sequencing chromatograms of 3 patients with ET revealed that in 2 patients, the mutation could not be distinguished from the background and in 1 patient, the mutation was observed but at the limit of detection. No amplification occurred in the ACB-PCR assays of samples from the 16 healthy subjects and 4 patients with lymphoproliferative diseases.

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