GliomaDx Nest

The nested approach is optimized for low-input or poor quality samples such as formalin-fixed paraffin embedded (FFPE) DNA, circulating tumor DNA (ctDNA), or biopsy samples. The approach is similar to normal GliomaDx, but involves a high-fidelity multiplex pre-amplification of the mutation regions of TERT promoter and IDH1 and IDH2. Pre-amplification is followed by dilution of the pre-amplified PCR product and allele-specific PCR reaction (nested PCR). A full list of primer sets and cycling conditions is in Supplementary Table 2. Pre-amplification was performed using 50 ng of gDNA input, a mixture of all 6 pre-amp control primers (200 nM final in reaction, without M13 sequences), and the KAPA HiFi HotStart PCR Kit (Final: KAPA HiFi HotStart DNA Polymerase, 0.3U; 0.3 mM each dNTP; 1X KAPA HiFi Buffer) in a 15 μL reaction. The pre-amplified PCR products were then placed at −20 degrees for 1 hour to inactivate the HiFi polymerase (per discussion with KAPA). The pre-amplified PCR products were then diluted to 1:10000 in water. Five microliters of this diluted pre-amplified multiplex PCR product was then used as the input for the nested, allele-specific qPCR reaction of interest, using 15 μL reaction volume total and the KAPA SYBR FAST qPCR Master Mix. All allele-specific reactions were done in triplicate using 400 nM of primer (final). All amplifications were performed using the CFX 96 detection system (Bio-Rad) with a ramp rate of 5⁰C/second.