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K-ras mutation detection by hybridization to a polypyrrole DNA chip
E Lopez-Crapez1, T Livache, J Marchand
1CRLC Val d'Aurelle Paul-Lamarque, Centre de Recherche en Cancérologie, Parc Euromédecine, 34298 Montpellier Cedex 5, France. ecrapez@valdorel.fnclcc.fr
Background:
Detection of mutations in cancer-related genes is of major importance for both basic knowledge and clinical practice. Several strategies have been developed to diagnose these alterations. We describe a method based on polypyrrole DNA chip technology to detect K-ras gene mutations in tumors.
Methods:
An oligodeoxynucleotide array was constructed on a silicon device by copolymerization of 5'-pyrrole-labeled oligodeoxynucleotides and pyrrole. The samples to be analyzed were then amplified by PCR, and the single-stranded biotin-labeled amplified DNA was specifically hybridized to the addressed probes. Perfectly matched duplexes were detected by fluorescence microscopy using R-phycoerythrin as the detection label. The developed methodology was applied to genotype assignment of K-ras in human samples. The genotypes of 75 DNA genomic samples from colorectal cancer patients were analyzed side by side using direct DNA sequencing and a polypyrrole DNA chip.
Results:
The chip method unequivocally defined all of the genotypes. Mutations present at <10% of the wild-type DNA concentration could be distinguished.
Conclusions:
This probe array assay is a rapid and reliable procedure that may be used to detect mutations.