Simple two-color array-based approach for mutation detection

The ability to analyze multiple polymorphic/mutation sites rapidly and accu rately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynuc leoside triphosphate terminators (ddNTPs). The oligonucleotide primer endin g one nucleotide short of the mutation site being probed is bound to the sl ide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded compleme ntary templates generated by PCR from genomic DNA. The composite fluorescen ce produced contains peaks of distinct wave lengths corresponding to each C y dye-tagged terminator incorporated, resulting in a fluorescent 'fingerpri nt' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-t agged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template , on-slide temperature cycling and washing conditions. Results from analysi s of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in m any areas of genetic analysis.