Fabrication and evaluation of a sequence-specific oligonucleotide miniarray for molecular genotyping.

ABSTRACT

PURPOSE: Molecular genotyping relies on the identification of specific microbial DNA sequences. Accurate genotyping not only requires discrimination between low- and high-risk pathogens for effective diagnosis or disease management but also requires the identity of the specific strain or type of the microbe involved in pathogenesis. The majority of these assays require DNA amplification followed by genome identification either through sequencing or hybridization to specific oligonucleotide probes. We evaluated the use of a DNA microchip assay as a simple and easy-to-use procedure for genotyping. METHODS: Various methodological parameters were optimized for single-base mismatch discrimination on a DNA microarray. The fabrication procedures involved substrate chemistry for immobilization. The effect of various buffers and features associated with oligonucleotide sequences were standardized. The assay was evaluated on a low-density genotyping chip containing the sequences of various (Human Papilloma Virus) HPV subtypes. RESULTS: The specific subtype was identified with high specificity by hybridization in miniaturized condition. CONCLUSIONS: The DNA microchip provides a rapid and cost-effective genotyping procedure for microbial organisms and can be implemented easily in any laboratory.