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Clin Chem. 2010 Nov;56(11):1750-7. doi: 10.1373/clinchem.2010.150680. Epub 2010 Sep 17.

Microsatellite instability detection by high-resolution melting analysis.

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  • 1Department of Molecular and Regenerative Medicine, Hematology, Oncology and Transfusion Medicine Center, Vilnius University Hospital Santariskiu Clinics, Vilnius, Lithuania. ramunas.janavicius@santa.lt

Abstract

BACKGROUND:

Microsatellite instability (MSI) is an important marker for screening for hereditary nonpolyposis colorectal cancer (Lynch syndrome) as well as a prognostic and predictive marker for sporadic colorectal cancer (CRC). The mononucleotide microsatellite marker panel is a well-established and superior alternative to the traditional Bethesda MSI analysis panel, and does not require testing for corresponding normal DNA. The most common MSI detection techniques-fluorescent capillary electrophoresis and denaturing HPLC (DHPLC)-both have advantages and drawbacks. A new high-resolution melting (HRM) analysis method enables rapid identification of heteroduplexes in amplicons by their lower thermal stability, a technique that overcomes the main shortcomings of capillary electrophoresis and DHPLC.

METHODS:

We investigated the straightforward application of HRM for the detection of MSI in 70 archival CRC samples. HRM analysis for 2 MSI markers (BAT25 and BAT26) was evaluated, and 2 different HRM-enabled instruments were compared-the LightCycler® 480 (Roche Diagnostics) and the LightScanner(TM) (Idaho Technology). We also determined the analytical sensitivity and specificity of the HRM assay on both instruments using 11 known MSI-positive and 54 microsatellite-stable CRC samples.

RESULTS:

All MSI-positive samples were detected on both instruments (100% analytical sensitivity). The LightScanner performed better for analytical specificity, giving a combined specificity value of 99.1% compared with 92.3% on the LightCycler 480.

CONCLUSIONS:

We expanded the application of the HRM analysis method as an effective MSI detection technique for clinical samples.

PMID:
20852132
DOI:
10.1373/clinchem.2010.150680
[PubMed - indexed for MEDLINE]
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