International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

D27 Advanced Strategies for Direct Sequencing of BACs and Repeated Regions of Mammalian Genomes

A. Slesarev, A. Malykh, O. Malykh N. Pavlov, N. Polushin, S. Kozyavkin. Fidelity Systems, Inc., 7961 Cessna Avenue, Gaithersburg, MD 20879

We have been developing the ThermoFidelase series of thermostable proteins and advanced sequencing protocols aiming to solve problems associated with sequencing of difficult DNA templates and to reduce the cost of genome projects.

Our first application was the use of unlinking activity ThermoFidelase I (TF I) to prepare DNA templates for robust primer annealing and extension during sequencing reactions. Enzymatic unlinking of circular BAC, long bacterial genomic templates and eucaryotic DNA samples with GC- and CT-rich islands with newly developed TF II allows to reduce the duration of the denaturation step during cycle sequencing and results in high yield and quality of Sanger fragments. Furthermore, topological selection of DNA template treated by TF II, which results in incomplete unlinking of the target template while keeping the non-target DNA in a double stranded form, allows sequencing of BAC templates in the presence of excess amount of bacterial DNA.

The current strategy of human and mouse genome sequencing includes mapping BAC clones along chromosomal loci and finding the minimal tiling path for selection of BACs for shotgun sequencing. We have developed an improved procedure for BAC end sequencing which allows for very low amounts of templates and decreasing of cost by reducing of the reagent consumption. The highlights of the protocol include the incorporation of TF II, use of modified primers and reduced reaction volume. This approach happened to be extremely efficient for primer walking on BACs as a method to convert rough draft contigs to ordered and oriented contigs. This strategy will be very useful for closing of dozens of thousands of gaps, which are going to be generated during the "working draft" sequencing phases of human and mouse genome projects.


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