BRAF primer design and representative pyrograms. (A) The modified pyrosequencing assay design from Packham et al.16 The assay detects all mutations in codon 600, including the common V600E (c.1799T > A) mutation as well as the less common variant mutations involving codons 599 through 601. (B-C) Representative pyrograms from an HCLc-expressing IGHV4-34 (case BH18; 92.6% HCL cells) revealing a wild-type result at codon 600 (B), and from an HCLc (case BH21; 77.4% HCL cells) expressing IGHV5-51 showing the c.1799T > A (V600E) mutation (C). The nucleotides on the x-axis (beginning with G) represent their dispensation order during the pyrosequencing reactions, whereas the y-axis displays arbitrary units related to peak height. Peak heights are proportional to the number of nucleotides incorporated into the growing DNA strand. The shaded boxes represent the percentage of nucleotides calculated at a particular interrogated location (AQ score). The arrow in panel C indicates the c.1799T > A mutation. The shaded box represents an AQ score of 41% A and 59% T at that position.
