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Automated and simultaneous identification of microsatellite instability by fluorescence-based polymerase chain
M Kinoshita1, J Nakamura, H Kusaka
1Gene-Diagnostic Center, Otsuka Assay Laboratories, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan. kinoshitamo@assay.otsuka.co.jp
Abstract:
Genomic instability is sometimes due to impairment of DNA repair systems, which results in a change in the number of microsatellite repeats in tumor cells, produced by slippage during DNA replication. Such abnormal repeats are manifested as microsatellite instability (MSI). We have devised a simple assay using four-color fluorescence for the detection of MSI by an automatic sequencer. Using this method, MSI and loss of heterozygosity (LOH) at four microsatellite loci can be identified simultaneously. We have also developed an algorithm and software for automated analysis of MSI and LOH with this method. Using our method for the detection of MSI in four microsatellite loci and the algorithm and software that we developed, 18 (94.7%) of 19 patients with hereditary nonpolyposis colorectal cancer (HNPCC), meeting the Amsterdam Minimum Criteria, were found to exhibit MSI.
Insights
This study introduces a new four-color fluorescence assay for detecting microsatellite instability (MSI) and loss of heterozygosity (LOH) simultaneously. The method accurately identified MSI in 94.7% of hereditary nonpolyposis colorectal cancer patients.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Genomic instability, often caused by impaired DNA repair, leads to altered microsatellite repeat numbers in tumors.
- This alteration, known as microsatellite instability (MSI), is a hallmark of certain cancers, including hereditary nonpolyposis colorectal cancer (HNPCC).
Purpose of the Study:
- To develop a simple, automated method for detecting MSI and loss of heterozygosity (LOH).
- To evaluate the efficacy of this new method in identifying MSI in patients with HNPCC.
Main Methods:
- A novel four-color fluorescence assay was designed for MSI detection using an automated sequencer.
- The assay allows simultaneous identification of MSI and LOH at four microsatellite loci.
- A dedicated algorithm and software were developed for automated analysis of MSI and LOH data.
Main Results:
- The developed method successfully detected MSI and LOH simultaneously at four microsatellite loci.
- Out of 19 HNPCC patients meeting the Amsterdam Minimum Criteria, 18 (94.7%) exhibited MSI using this assay.
- The automated analysis software provided efficient processing of the generated data.
Conclusions:
- The four-color fluorescence assay coupled with automated analysis offers a simple and effective tool for MSI detection.
- This method demonstrates high sensitivity in identifying MSI in HNPCC patients.
- The assay has significant potential for the clinical diagnosis and genetic screening of hereditary cancer syndromes.