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

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 for 10 of the most common mutations of codons 12 and 13 of the K-ras gene by hybridization to a new biochip array. DNA was...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2002-03, Vol.48 (3), p.428-435
Main Authors: Prix, Lothar, Uciechowski, Peter, Bockmann, Beatrix, Giesing, Michael, Schuetz, Andreas J
Format: Article
Language:English
Subjects:
Biological and medical sciences
Digestive system
Feces - chemistry
Humans
Investigative techniques, diagnostic techniques (general aspects)
Medical sciences
Microscopy, Confocal
Mutation
Oligonucleotide Array Sequence Analysis
Oncogene Protein p21(ras) - analysis
Oncogene Protein p21(ras) - genetics
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Polymerase Chain Reaction
Reproducibility of Results
Sensitivity and Specificity
Tumor Cells, Cultured
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Summary: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 for 10 of the most common mutations of codons 12 and 13 of the K-ras gene by hybridization to a new biochip array. DNA was isolated from 26 stool samples by column-based extraction from 9 cell lines. Peptide nucleic acid (PNA)-mediated PCR clamping was used for mutant-specific amplification. We used a biochip, consisting of a small plastic support with covalently immobilized 13mer oligonucleotides. The read out of the biochip was done by confocal time-resolved laser scanning. Hybridization, scanning, and data evaluation could be performed in 19 as demonstrated by hybridization with tumor cell line DNA. Stool samples (n = 26) were analyzed in parallel with PNA-PCR, restriction assay for K-ras codon 12 mutations, sequencing, and hybridization to the biochip. Nine mutations were found by hybridization, all confirmed by sequencing. PNA-PCR alone leads to an overestimation of mutations because suppression of the wild type is not effective enough with high concentrations of wild-type DNA. The restriction assay found only four mutations. The K-ras biochip is well suited for fast mutation detection from stool in colorectal cancer screening.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/48.3.428