Showing papers by "Andres Metspalu published in 1999"

Universal and flexible DNA microarray approach: arrayed primer extension

Abstract: We describe an integrated genetic testing system consisting of DNA oligonucleotide array production, template preparation, multiplex primer extension on the array, fluorescence imaging equipment and data analysis. DNA oligonucleotide (ON) arrays are produced by spotting presynthesized 25-mer ON with a 5' amino group on the glass microscope slide. This approach involves a number of potential problems, such as silanization of the slides, ON selection and immobilization. As a matter of fact, these steps should be more efficient and reproducible. Currently, the spacer separating ON from the glass surface is 2−18 carbons, but longer linker arms seem to be better. Two DNA strands have been analysed simultaneously according to our protocol. The double-stranded DNA template preparation can be described as an one-tube reaction with minimal number of pipetting steps without centrifugation or ethanol precipitation. Arrayed primer extension (APEX) is carried out as a single-step reaction simultaneously with all four dye terminator nucleotides (fluoresceine, Cy3, Texas Red and Cy5) in the presence of the DNA polymerase (Thermo Sequenase). For this purpose, a special four-colour instrument, FD-003, was developed at Asper Ltd. The latter uses a total internal reflection fluorescence (TIRF)-based excitation mechanism combined with four lasers, filter wheel and scientific grade CCD camera. Image acquisition is fast (0.5−2 min for 4-colour) and automatic, followed by software analysis to convert the fluorescence information into DNA sequence data. Signal-to-noise ratio is at least 30:1, allowing comfortable analysis of heterozygous positions in DNA. This system is used for mutation detection in -globin, BRCA1 and BRCA2 genes, resequencing of p53 gene fragments, SNP testing and gene expression using mtComplex I genes.