Method Description

Genomic DNA is extracted, and 2 polymerase chain reactions (PCR) are used for each sample. In each reaction, a short fragment of genomic DNA, including the variant site, is amplified using quantitative PCR in a real-time PCR instrument. In the first reaction, the 5' terminal base of the reverse primer matches the mutated sequence, and the PCR conditions are such that it will only bind mutated DNA. In the second reaction, the 5' terminal base of the reverse primer matches the wildtype sequence, and the PCR conditions are such that it will only bind the wild-type sequence. In both reactions, the PCR is monitored using TaqMan probe chemistry. The amount of mutated DNA and the amount of wildtype DNA is measured for each sample. In each run, the amount of mutated and wild-type DNA in a calibrator DNA sample is also measured. The calibrator is a mixture of DNA from a positive cell line (HEL) and a negative cell line (HL60) that is frozen in aliquots and expected to give an identical result in each run. Deviations in the calibrator result are assumed to be due to deviations in the run conditions and the sample results are corrected accordingly. Following each reaction, Relative Quantification Software is used to calculate the normalized mutated:wildtype ratio, which is expressed as a unitless ratio following correction with the calibrator data.

The formula for the normalized ratio is as follows:

The final result is reported as percent JAK2 V617F of total JAK2 (ie, [mutated/mutated + wildtype] x 100%), calculated from the normalized mutated:wildtype ratio.(Unpublished Mayo method)