Efficient extraction of human DNA from stool samples and detection of genetic and epigenetic alterations specific for colorectal neoplasia
Aleksandra Mandic Havelka1,*, Annelie Strålfors1, Lars-Olof Hansson1, Ulrik Lindfors2, Gösta Eggertsen1
1Clinical Chemistry, Karolinska University Laboratory, 2Department of gastroenterology and surgery, Karolinska University Hospital, Stockholm, Sweden
Introduction: Colorectal cancer is a common neoplastic disease with a high mortality rate, mostly because the tumor growth is secretively progressing, why diagnosis often is late. A simple, non-invasive method for detection of tumor-specific mutations is in great demand, to facilitate early diagnosis and initiate accurate and effective therapy.
Objectives: To identify cancer specific genetic aberrations in intestinal DNA extracted from stool samples from patients with colorectal cancer.
Methods: Stool material was collected from patients with diagnosed colorectal cancer in conjunction with open surgery. DNA was extracted using a fully automated system for processing of fecal samples (S2G Scandinavia AB), and the QiaCube extraction system (QIAGENE).
K-Ras and B-Raf mutations were analysed with three different methods. For methylation analysis of the Septin 9 gene in stool and plasma specimens, the Epi proColon Kit (Epigenomics) was used.
Results: A total yield of 50 – 350 ng of DNA/ml was obtained from stool samples. Presence of human genomic DNA was monitored by TaqMan probe based analysis of LCT-13910 C>T using the 7500 Fast Real Time PCR System (Life Technologies).
Three patient samples were investigated for specific mutations in the K-Ras gene. One sample displayed a mutation in codon 12 with all three methods (Gly12Asp ;GGT>GAT). In another sample no mutations were found. Analysis of the third sample was not successful.
Methylation of Septin 9 gene was detected in 5 out of 6 plasma samples and in all 6 stool samples from patients with colorectal cancer. No methylation was observed in plasma or stool samples from healthy individuals.
Conclusion: Efficient extraction and amplification of human DNA could be done from stool samples using an automatic robot system. In purified DNA from patients with colorectal neoplasia genetic and epigenetic aberrations were identified (mutations in the K-Ras gene and methylation of Septin 9 gene).
Keywords: Biomarker, Cancer, Genomics