DOE Human Genome Program Contractor-Grantee
15. Fimer Chemistry for Sequencing off BAC and Genomic DNA Templates
S. Kozyavkin, N. Polouchine, A. Malykh, O. Malykh, and A. Slesarev
Fidelity Systems, Inc., 7961 Cessna Avenue, Gaithersburg, MD 20879-4117http://www.fidelitysystems.com
Robust sequencing off BAC and genomic templates presents a new challenge in technology development. The problems associated with the use of standard oligonucleotides as primers in genomic cycle sequencing protocols include insufficient specificity of primer annealing, non-specific amplification, low sensitivity and premature truncation at secondary structures in template DNA.
To overcome these problems we have developed a new method to generate combinatorial libraries of chemically modified oligonucleotides (fimers). The method is based on the use of our proprietary monomers containing MOX or SUC reactive moieties. We assessed the effects of modifications on DNA melting, electrophoretic mobility and DNA-protein interaction for individual oligonucleotides and their small libraries. We have developed rapid procedure for modification, deblocking and purification of fimers in 96-well plate format. Different design strategies for fimers have been tested with ThermoFidelase-2A, -2B and -2C, deaza-dGTP and dGTP in various thermal cycling protocols. We found that fimer design eliminates many restrictions on choosing primer sequence. Our results demonstrate feasibility of suppressing non-specific PCR amplification and primer-dimer formation after 100-400 cycles, synergy of chemical and enzymatic tools to sequence through strong stop and long simple repeats and sequence directly off sub-microgram quantities of bacterial genomic templates. The implementation of fimers in high-throughput projects will be presented.
We have achieved contiguity and high total and local quality of base calls starting from 2x - 5x shotgun coverage in draft human BAC projects. The major conclusion is that workflow for finishing low-coverage projects differs significantly from that for full shotgun projects and has become manageable due to the increased power of sequencing chemistry. For BAC-end sequencing projects we have developed long fimers and ThermoFIdelase-2E to accelerate kinetics of primer annealing to minute quantities of template DNA and 400x(1 min) sequencing protocol. We have increased detection sensitivity to 10 ng BAC and obtained high quality reads from 30 ng BAC. New protocol is compatible with the yield of BAC DNA from 1-ml cultures in 96 well plate format.
Enhanced reaction chemistry has allowed us to overcome major obstacles in bacterial genomic sequencing associated with high flourescent background and low signal. We obtained high quality reads from as low as 100-300 ng genomic template. Applications of direct genomic sequencing to the discovery of novel genes and characterization of bacterial populations will be presented.
Supported in part by DOE and NIH (DE-FG02-98ER82557 and 2R44GM55485-02).
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