Experimental Design for H3 Mutation Detection. a DNA isolated from patient CSF may contain a small amount of tumor DNA (red). b PCR amplification of H3F3A or HIST1H3B was performed on all extracted DNA. c Specimens with ≥10.5 ng DNA were sequenced for c.83A > T mutation. d Specimens with <10.5 ng isolated DNA were submitted for a second round of PCR with primers designed to selectively amplify the H3F3A c.83A > T mutant allele, yielding a 150 bp product. H3F3A c.83A > T mutation results in lysine 27 codon transversion to methionine (AAG to ATG). The mutation-specific forward primer (red) is designed with the variant base (thymine) at the 3′ end, facilitating anchoring specificity to the mutant allele: this single nucleotide mismatch prevents wild type H3F3A amplification. Reverse primer complementary to the wild type sequence is indicated in blue. Schematic adapted from Zhang et al.