MSI PCR - Promega MSI System
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 genomic DNA of cells 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.
The mononucleotide repeat microsatellites are prone to polymerase stutter, a PCR artifact, that produces multiple peaks both larger and smaller than the true allele peak. Electropherograms of mononucleotide markers show a number of less intense stutter peaks at 1bp intervals from the most prominent or true allele peak. Higher nucleotide number microsatellites, like the pentanucleotide repeats shown below, are less subject to polymerase stutter, and typically produce a one repeat smaller peak.