JAK2 V617F Mutation Detection, Blood
Method Description Describes how the test is performed and provides a method-specific reference
Genomic DNA is extracted and 2 PCR reactions are used for each sample. In each reaction, a short fragment of genomic DNA, including the mutation site, is amplified using quantitative PCR in a real-time PCR instrument (LightCycler 480, Roche). In 1 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 wild-type 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 wild-type 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, LightCycler 480 Relative Quantification Software is used to calculate the mutated:wild-type ratio, which is expressed as a unitless normalized ratio following correction with the calibrator data.
The formula for the normalized ratio is as follows:
Normalized ratio =
Samples with a normalized ratio of 0 are considered negative.
Samples with a normalized ratio of <1x10(2) are considered below the cutoff level for positivity.
Samples with a normalized ration of > or =1x10(2) are considered positive.
(Instruction manual: Roche Applied Science Technical Note No. LC 13/2001. Relative Quantification; LightCycler 480, 2006)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday; 12 p.m.