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Detection of MIS 301
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Detection of Microsatellite Instability and Loss
of Heterozygosity Using DNA Extracted from
Formalin-Fixed Paraffin-Embedded Tumor Material
by Fluorescence-Based Multiplex Microsatellite PCR
Joanne Edwards and John M. S. Bartlett
1. Introduction
Microsatellites are widely distributed highly repetitive DNA sequences composed of
di-, tri-, or tetranucleotide repeats (1). They are spread over the whole human genome
and are located between and within genes. Physiologically, they exhibit high levels of
polymorphism, relative to different chromosomal loci, and within different individuals,
different microsatellite lengths can even be noted between two alleles of the same
gene (1). Although the role of microsatellites in the genome and their evolutionary
mechanisms are still incompletely understood, they are widely used tools in genetic
mapping studies and as markers for prediction of disease (2,3).
The length of microsatellite repeats may be used to predict the presence of disease,
for example, the length of the CAG repeat in the AR receptor gene (40–65 repeats)
is a marker of Kennedy’s disease (3). Microsatellites also exhibit a form of genetic
instability characterized by the gain or loss of repeat units at multiple independent loci.
Such alterations have been observed to accumulate in cells with defective DNA repair
mechanisms and are commonly known as microsatellite instability (MIS) (1,2). The
DNA mismatch repair system plays an important role in controlling the accumulation of
somatic mutations and therefore there is an association between DNA repair defects and
carcinogenesis, and the presence of MIS may be used as a marker of certain cancers (4).
MIS has been associated with familial cancer syndrome hereditary nonpolyposis colorectal
cancer, sporadic ovarian, prostate. and pancreatic cancer (1,4,5). Cancer research
also uses microsatellites when investigating inactivation of tumor suppressor genes.
Loss of heterozygosity (LOH) of microsatellites located in or close to a tumor suppressor
gene is an indirect way of testing for inactivation of tumor suppressor genes in
accordance to the classical two-hit model, that is, a recessive mutation is uncovered by
loss of the second copy of the gene, resulting in inactivation of the tumor suppressor
gene (6).
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
301