Detection of Exon 12 Mutations in the JAK2 Gene

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Detection of Exon 12 Mutations in the JAK2 Gene Todd S. Laughlin, Alison R. Moliterno, Brady L. Stein, Paul G. Rothberg The Journal of Molecular Diagnostics Volume 12, Issue 3, Pages 278-282 (May 2010) DOI: 10.2353/jmoldx.2010.090177 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Fragment analysis showing the effect of the LNA clamp on the relative level of signal from normal DNA and a 6-bp deletion mutant. The genomic DNA was heterozygous for a 6-bp deletion, N542-E543del. As indicated on the left of the figure (% Mutant) serial 10-fold dilutions of mutant DNA in normal DNA (no JAK2 exon 12 mutation) were prepared and used for template. The PCR was done with and without the LNA clamp as described at the top of each column of fragment analysis results. At the bottom of the figure, the positions of the fragments corresponding to the normal (WT) and mutant (Del) are indicated. In each graph, the y axis represents relative fluorescence units and the x axis represents molecular weight. The Journal of Molecular Diagnostics 2010 12, 278-282DOI: (10.2353/jmoldx.2010.090177) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Sequence analysis showing the effect of the LNA clamp on the relative level of signal from normal DNA and a 6-bp deletion mutant. The genomic DNAs were the same serially diluted template DNAs that were used in Figure 1. The genomic DNA mixtures were amplified with the LNA clamp and subjected to dideoxy nucleotide sequencing. The sequence of the mutant DNA is written at the top of the figure, and the normal sequence is written at the bottom with the six nucleotides deleted in the mutant underlined. The percentage of mutant DNA specimen in the template is indicated to the left of the sequence traces. The genomic DNA used for the sequence electropherogram at the bottom was pure normal. The Journal of Molecular Diagnostics 2010 12, 278-282DOI: (10.2353/jmoldx.2010.090177) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Analysis of an E543–D544del mutation present at low allelic load using fragment analysis (A) and sequence analysis (B). The top panels in both parts of the figure show a normal control. The middle and bottom panels used the patient specimen genomic DNA with the PCR done with and without the LNA clamp as indicated. In A the positions of the deletion mutant and normal fragments are indicated at the top. In each graph, the y axis represents relative fluorescence units and the x axis molecular weight. In B the normal sequence is written at the top of the figure with the six nucleotides deleted in the mutant underlined, and the mutant sequence is written between the lower two sequence electropherograms. The Journal of Molecular Diagnostics 2010 12, 278-282DOI: (10.2353/jmoldx.2010.090177) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions