Relative quantitation of the JAK2 exon 14 V617F mutation by real-time polymerase chain reaction (Note: This test does not assess JAK2 exons 12 and 13.)
Performing Laboratory / Facility
UCLA Medical Center Clinical Laboratory (CHS)
Performing Section
Molecular Pathology
Availability
Monday through Friday, 0700-1700
Turnaround Time
14 days from receipt of specimen in performing lab
Methodology
The quantitative allele specific PCR technology is based on the use of specific forward primers for the wild type and the V617F allele, respectively. Only a perfect match between primer and target DNA allows extension and amplification in the PCR reaction. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and hence amplified during PCR. The number of PCR cycles necessary to detect a signal above the threshold is directly proportional to the amount of target present at the beginning of the reaction. Concentrations of V617F and wild type are determined by comparing the V617F and wild type crossing points to their standard curves. The relative concentration (%) of mutant cells in the specimen is calculated by comparing V617F target concentration to total target concentration.
Use
The V617F mutation in exon 14 of the Janus kinase 2 gene (JAK2) was found to be present in several chronic myeloproliferative disorders (MPD), most frequently in polycythemia vera (65% to 97%), essential thrombocythemia (23% to 57%) and chronic idiopathic myelofibrosis (35% to 57%). This point mutation appears to cause constitutive activation of the JAK2 tyrosine kinase. Quantification of the JAK2 V617F mutation is useful both to assist in diagnosis of MPD, and for determining treatment and monitoring, as the aberrant tyrosine kinase appears to be a viable target for pharmacologic approaches.
Relative quantitation of the JAK2 exon 14 V617F mutation by real-time polymerase chain reaction (Note: This test does not assess JAK2 exons 12 and 13.)
Performing Laboratory / Facility
UCLA Medical Center Clinical Laboratory (CHS)
Performing Section
Molecular Pathology
Availability
Monday through Friday, 0700-1700
Turnaround Time
14 days from receipt of specimen in performing lab
Methodology
The quantitative allele specific PCR technology is based on the use of specific forward primers for the wild type and the V617F allele, respectively. Only a perfect match between primer and target DNA allows extension and amplification in the PCR reaction. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and hence amplified during PCR. The number of PCR cycles necessary to detect a signal above the threshold is directly proportional to the amount of target present at the beginning of the reaction. Concentrations of V617F and wild type are determined by comparing the V617F and wild type crossing points to their standard curves. The relative concentration (%) of mutant cells in the specimen is calculated by comparing V617F target concentration to total target concentration.
Use
The V617F mutation in exon 14 of the Janus kinase 2 gene (JAK2) was found to be present in several chronic myeloproliferative disorders (MPD), most frequently in polycythemia vera (65% to 97%), essential thrombocythemia (23% to 57%) and chronic idiopathic myelofibrosis (35% to 57%). This point mutation appears to cause constitutive activation of the JAK2 tyrosine kinase. Quantification of the JAK2 V617F mutation is useful both to assist in diagnosis of MPD, and for determining treatment and monitoring, as the aberrant tyrosine kinase appears to be a viable target for pharmacologic approaches.
