Diagnostic biochip array for fast and sensitive detection of K-ras mutations in stool.
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 for 10 of the most common mutations of codons 12 and 13 of the K-ras gene by hybridization to a new biochip array.


METHODS
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 <2 h.


RESULTS
Approximately 80 ng of DNA was obtained from 200-mg stool samples. No inhibition of the PCR by remaining impurities from stool was observed. Mutation detection was possible in 1000-fold excess of wild-type sequence. Discrimination ratios between the mutations were >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.


CONCLUSIONS
The K-ras biochip is well suited for fast mutation detection from stool in colorectal cancer screening.
Prix
Lothar
L
Institut für Molekulare NanoTechnologie, Berghäuser Strasse 295, 45659 Recklinghausen, Germany.
Uciechowski
Peter
P
Böckmann
Beatrix
B
Giesing
Michael
M
Schuetz
Andreas J
AJ
D005243:Feces / Q000737:chemistry*; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D015689:Oncogene Protein p21(ras) / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D015689:Oncogene Protein p21(ras)