Diagnostic Biochip Array for Fast and Sensitive Detection of K-ras Mutations in Stool

摘要: Background: Tumor cells that shed into stool are attractive targets for molecular screening and early detection of colon or pancreatic malignancies. We developed a diagnostic test to screen 10 the most common mutations codons 12 13 K- ras gene by hybridization new biochip array. Methods: DNA was isolated from 26 samples column-based extraction 9 cell lines. Peptide nucleic acid (PNA)-mediated PCR clamping used mutant-specific amplification. biochip, consisting small plastic support with covalently immobilized 13mer oligonucleotides. The read out done confocal time-resolved laser scanning. Hybridization, scanning, data evaluation could be performed in <2 h. Results: Approximately 80 ng obtained 200-mg samples. No inhibition remaining impurities observed. Mutation possible 1000-fold excess wild-type sequence. Discrimination ratios between were >19 as demonstrated tumor line DNA. Stool (n = 26) analyzed parallel PNA-PCR, restriction assay codon mutations, sequencing, biochip. Nine found hybridization, all confirmed sequencing. PNA-PCR alone leads an overestimation because suppression wild type is not effective enough high concentrations only four mutations. Conclusions: well suited fast mutation colorectal cancer screening.