Development of a non-radioactive diagnostic test for the detection of microsatellite instability in colorectal cancer

Abstract

Stepwise accumulation o f mutations in the human genome is the initial step in carcinogenesis. Microsatellites are the regions which are first hit by the mutations resulting from mismatch repair deficiency. Until today microsatellite alterations have been shown mainly in hereditary non-polyposis colon cancer (HNPCC) and several other cancer types. Recent advances indicate that microsatellite alterations can also be detected in DNA samples (such as blood, urine, etc.), which are shed fiOm tumors. This is an important finding for the early diagnosis o f cancer since malignant cells can be detected in tissues other than the primary tumor. Therefore microsatellite analysis, when coupled with an easy, powerful screening technique could have a high diagnostic value for cancer types other than colorectal cancer. Despite its drawbacks the most common microsatellite screening method is based on the use of radioisotopes and autoradiography. However in the clinical setting non-radioactive detection methods are preferred. The aim o f this thesis is development o f a non-radioactive diagnostic test for the detection o f microsatellite instability in genomic DNA which can be used for the early detection o f some forms o f cancer. Therefore we have optimized the PCR conditions for eight microsatellite markers which are: i. mononucleotide repeats BAT25 and intragenic repeat region o f BAX gene, ii. dinucleotide repeats D5S105, D6S291, D11S904, D13S175, D17S855, and in. tetranucleotide repeat FGA. In addition, we have analyzed the mononucleotide repeat markers in blood, paraffin embedded and fi"esh tumor samples o f six colorectal cancer patients with polyacrylamide gel electrophoresis and silver staining