Sequencing by hybridization with the generic 6-mer oligonucleotide microarray: An advanced scheme for data processing

DNA sequencing by hybridization was carried out with a microarray pf all 4( 6) = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucle otides immobilized in 100 x 100 x 20-mu m polyacrylamide gel pads of the ge neric microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel For all microchip duplexes with a fluorescence microscope equipped with CCD camera. This allowed us to discriminate the perfect duplexes formed by the oligonucleotides, which are complementary to the target DNA. The DNA seque nce was reconstructed by overlapping the complementary oligonucleotides pro bes. We developed a data processing scheme to heighten the discrimination o f perfect duplexes from mismatched ones. The procedure was united with a re construction of the DNA sequence. The scheme includes the proper definition of a discriminant signal, preprocessing, and the variational principle for the sequence indicator function. The effectiveness of the procedure was co nfirmed by sequencing, proofreading, and nucleotide polymer phism (mutation ) analysis of 13 DNA fragments from 31 to 70 nucleotides long.