Microsatellite Instability Analysis
The Promega MSI system utilizes fluorescently labeled primers for co-amplification of seven markers including five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and two pentanucleotide repeat markers (Penta C and Penta D) (Table 1). The mononucleotide repeat markers are used for MSI determination since they are highly sensitive to alterations in samples with defective mismatch repair. These mononucleotide repeat markers have few variants, so that most individuals are homozygous for the same allele for each marker, which simplifies data interpretation. The pentanucleotide repeat markers are highly polymorphic and have low MSI potential that serves to identify matching samples.
Adapted from Promega MSI protocol- Note that the MDL uses POP-7 polymer for capillary electrophoresis which produces slightly different allele sizes.
The microsatellite PCR products are subjected to DNA fragment analysis that is performed on an ABI 3130xl 16-capillary genetic analyzer. A 16-capillary instrument delivers superior electropherograms with less signal bleeding between capillaries which is prevalent in higher capillary instruments.