Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brain tumor 
with a median survival of 1 year after diagnosis. It has been reported recently 
that about 80% of DIPG cases and 70% of midline glioblastomas contain a mutation 
at one allele of the H3F3A gene (encoding histone H3 variant H3.3), replacing 
the lysine 27 with methionine (K27M). In order to facilitate diagnosis of DIPG 
patients, a quick and reliable method to identify the H3F3A K27M mutation is 
needed. Here, we describe a real-time PCR-based procedure involving a 
mutant-specific primer, a blocker oligonucleotide, and a reverse primer that can 
differentiate samples with H3F3A K27M mutation from those that do not. We first 
tested four different mutant-specific primers for their ability to selectively 
amplify H3F3A K27M-mutant allele and found that one primer amplified the mutant 
allele more efficiently than the rest. We then determined the optimal 
concentration of blocker oligo that significantly improved amplification of the 
H3F3A K27M-mutant allele. Using this optimized real-time PCR assay, we analyzed 
eleven samples, two of which containing H3F3A K27M mutation, and found that 
these two samples were differentially amplified from the nine others. In 
addition, we were able to discern the H3F3A K27M mutation in a newly obtained 
pediatric brainstem glioblastoma sample whose H3.3 status was not known 
previously, and in three other DIPG samples as well as paraffin embedded 
samples. These results demonstrate that we have developed a new reliable 
procedure for detecting the H3F3A K27M mutation in pediatric glioblastoma 
patient samples.