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Unit Code 83872:
JAK2 V617F Mutation Detection

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Method Description

Genomic DNA is extracted from whole blood or bone marrow 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 =        mutated/wild-type (sample)

                                      mutated/wild-type (calibrator)

 

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 >1x10(2) are considered positive.

 

(Instruction Manual:  Roche Applied Science Technical Note No. LC

13/2001. Relative Quantification; LightCycler 480, 2006)

Performing Laboratory Location

Rochester

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