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Detection of the JAK2 V617F Mutation by LightCycler PCR and Probe Dissociation Analysis  Marla Lay, Rajan Mariappan, Jason Gotlib, Lisa Dietz, Siby Sebastian,

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Presentation on theme: "Detection of the JAK2 V617F Mutation by LightCycler PCR and Probe Dissociation Analysis  Marla Lay, Rajan Mariappan, Jason Gotlib, Lisa Dietz, Siby Sebastian,"— Presentation transcript:

1 Detection of the JAK2 V617F Mutation by LightCycler PCR and Probe Dissociation Analysis 
Marla Lay, Rajan Mariappan, Jason Gotlib, Lisa Dietz, Siby Sebastian, Iris Schrijver, James L. Zehnder  The Journal of Molecular Diagnostics  Volume 8, Issue 3, Pages (July 2006) DOI: /jmoldx Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Principle of PCR-RFLP assay for detection of the V617F mutation in JAK2. Samples are amplified with fluorescently labeled primers, followed by restriction digestion with BsaXI (5′… 9(N)AC(N)5CTCC(N)10… 3′) and subsequent sizing by capillary electrophoresis with ABI fluorescence detection. A wild-type sample will have a green peak at 97 bp and blue peaks at 140 and 170 bp. The JAK2 V617F mutation results in the loss of a restriction site and will appear as a blue/green peak at 267 bp. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Results of a dilution series of homozygous positive cell line TIB180 diluted in wild-type DNA at 20% (1), 10% (2), 5% (3), 3% (4), and 1% (5). A: LightCycler assay in which the JAK2 wild-type allele appears as a melting peak at 61°C and the V617F mutant allele appears as a melting peak at 54°C. B: ARMS assay in which the JAK2 wild-type allele appears as a band at 279 bp and the V617F mutant allele at 229 bp. An amplification control band at 463 bp also amplifies. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Time and motion comparison. White bars indicate hands-on time; gray bars indicate instrument time (not requiring technologist's labor). Inter., interpretation; Set dig., set-up of restriction digestion; Set seq. rxn, set-up of DNA sequencing reaction. The Journal of Molecular Diagnostics 2006 8, DOI: ( /jmoldx ) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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