Plant & Animal Genome IX Conference

Poster: Sequencing & EST
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NEW TOOLS TO STREAMLINE WORKFLOW OF GENOME STRUCTURE ANALYSIS

¹ Fidelity Systems, Inc. Gaithersburg, MD 20879, USA

² Biological Institute of St. Petersburg University, Stary Peterhof, St. Petersburg 198904, Russia

Combinatorial Fimer chemistry is a robust method to generate modified oligonucleotides with diverse properties and select the best fitter to the application. Modifiers are used to change oligonucleotide annealing selectivity, temperature, kinetics, electrophoretic mobility, mass spectra, disrupt formation of dimers or hairpins, modulate interactions with polymerase, attach fluorescent dyes or other labels. We demonstrated feasibility of suppressing non-specific PCR and primer-dimer formation, synergy of chemical and enzymatic tools to read strong stops and long repeats and sequencing directly off crude DNA samples and sub-microgram quantities of microbial genomic templates. We show that finishing of low coverage rough draft BAC sequences can be accomplished without extra shotgun reactions and inventory of thousands of clones. A new ThermoFidelase 2 kit with fimers has been developed to sequence directly from microbial genomic DNA. Achieved high level of sensitivity of DNA sequencing allowed applying this technology to study genome structure of higher organisms. We have successfully sequenced directly from genomic DNA human Y-chromosome 2.47 kb tandemly repeated unit without prior PCR amplification. Similar approach was used to sequence centromeric repeats in avian genomic DNA. A new family of avian centromeric satellites has been isolated from chaffinch genome. FCP (Fringilla coelebs PstI element) was cloned from 500-bp fraction of PstI derived fragments of the chaffinch genomic DNA. Both FISH and PRINS assays on metaphase chromosomes from primary fibroblast culture showed that FCP is clustered in centromeric regions of all chaffinch chromosomes. Sequencing directly from genomic DNA through FCP repeats detected five single nucleotide polymorphism (SNP) sites. Furthermore, direct genomic DNA sequencing has confirmed the tandem organization of FCP repeats in the chaffinch genome. Supported in part by DOE and NIH, DE-FG02-98ER82557 and 2R44GM55485-02