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Clinical Chemistry 48: 428-435, 2002;
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(Clinical Chemistry. 2002;48:428-435.)
© 2002 American Association for Clinical Chemistry, Inc.

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

Lothar Prix1, Peter Uciechowski1, Beatrix Böckmann1, Michael Giesing1 and Andreas J. Schuetz1a

1 Institut für Molekulare NanoTechnologie, Berghäuser Strasse 295, 45659 Recklinghausen, Germany.

aAuthor for correspondence. Fax 49-0-2361-3000-142; e-mail a.schuetz{at}imnt.de.

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.




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