K-ras mutation detection by hybridization to a polypyrrole DNA chip.
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.
Lopez-Crapez
E
E
CRLC Val d'Aurelle Paul-Lamarque, Centre de Recherche en Cancérologie, Parc Euromédecine, 34298 Montpellier Cedex 5, France. ecrapez@valdorel.fnclcc.fr
Livache
T
T
Marchand
J
J
Grenier
J
J
D015179:Colorectal Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D015689:Oncogene Protein p21(ras) / Q000235:genetics*; D011108:Polymers; D011758:Pyrroles; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D011108:Polymers; D011758:Pyrroles; C067635:polypyrrole; D015689:Oncogene Protein p21(ras)