Fig. 3.

MSI genotyping by MALDI-TOF-MS. After PCR amplification of the DNA fragment harboring the microsatellite of interest, primer extension is carried out. Depending on the length of the microsatellite, a defined number of nucleobases is added to the extension primer. Note that due to inherent slippage of the polymerase, a small percentage of heterogeneity can be observed. The signal with the largest area under the peak represents the most abundant microsatellite length. Comparing the peak areas of samples of known genotypes as a reference to unknown samples allows the determination of the microsatellite instability. For internal calibration the mass of remaining unextended primer (P) can be used.