The indicated amounts of DNA extracted from a lymphoma-related cell line (Ramos) were spiked into human leukocyte DNA, as indicated in the figure, and the mutated Ramos target was detected along with 2 internal controls. Using the Type-it Mutation Detect Kit, the mutated 357 bp gene was detected even when only 25 pg of DNA (4 cells) was present. The lymphoma-relevant mutation could not be detected when using preoptimized conditions (Mg2+ and polymerase) with a hot-start DNA polymerase from Supplier I — even when using 25 ng of DNA (4000 cells). Electrophoresis was performed on a 1.3% agarose gel. M: 100 bp ladder.
Two different PCR systems (12-plex and 5-plex), both including the SNP of interest, were amplified using the standard protocol for the Type-it Mutation Detect PCR Kit (Q) or using preoptimized conditions with a hot-start DNA polymerase from Supplier A. With Type-it Mutation Detect Kits, all fragments of interest were amplified without any optimization of PCR parameters and could successfully be used as input for the SNaPshot PCR system. Even after optimization of Mg2+ concentration to 4 mM and varying the polymerase concentrations, amplification using the kit from Supplier A resulted in missing fragments. Electrophoresis was performed on the QIAxcel System. The first and the last bands on each lane are Upper Alignment Marker and Lower Alignment Marker, respectively. M: Alignment marker.