Detection of microsatellite instability by real time PCR and hybridization probe melting point analysis.

Microsatellite alterations can be found in a number of tumors. There are two types of alterations: loss of heterozygosity (LOH), which can be detected in the majority of colorectal cancers (CRC), and microsatellite instability (MSI). MSI occurs in about 15% of CRC with a mutator phenotype and are the hallmark of hereditary nonpolyposis colorectal cancers (HNPCC). Furthermore, MSI can also be detected in other tumors which are part of the HNPCC tumor spectrum (eg, gastric, ovarian, and endometrial carcinomas). Usually, a set of microsatellite markers is amplified by PCR followed by gel or capillary electrophoresis to separate PCR amplicons and by detection of the markers using autoradiography (Thibodeau et al, 1993), silver staining (Schlegel et al, 1996), or fluorescence techniques (Gyapay et al, 1996; Mansfield et al, 1994). We have established a technique to detect MSI by LightCycler PCR and melting point analysis using sequence-specific hybridization probes (HyProbes) labeled with LightCycler dyes, LCRed640 and LCRed705. Amplification of microsatellites by real-time PCR is followed by melting point analysis to display alterations in the length of repetitive sequences, thereby avoiding any electrophoretical separation of amplified DNA. Two mononucleotide markers (BAT25 and BAT26) were tested in 81 formalin-fixed and paraffin-embedded colorectal cancer samples with matched normal tissues from 21 MSI tumors and 60 tumors with microsatellite stability. Amplification and melting point determination of BAT26 and BAT25 was possible in 129/162 (80%) and 123/162 (76%) formalin-fixed and paraffin-embedded tissue samples, respectively. MSI could be detected specifically with both BAT25 and BAT26 markers only in MSI-high tumors (> or =40% MSI rate, determined with microsatellite reference panel, BAT25, BAT26, D5S346, D2S123, D17S250; Boland et al, 1998; Dietmaier et al, 1997). This new technique allows MSI detection within less than a hour and provides a basis for fast, high-throughput MSI analysis.