Agricultural Microbes Genome 2 Conference Agricultural Microbes Genome 2 Conference
Poster: Functional Genomics
P05_01.html
ThermoFidelase 2 has a unique combination of activities that are not found in other proteins. As a thermostable topoisomerase, it unlinks and denatures topologically constrained DNA, as a DNA binding protein, it accelerates primer annealing, helps polymerase to pass through secondary structures and protects DNA from thermal degradation. Fimers are primers with proprietary chemical modifications that inhibit non-specific amplification and premature truncation at secondary structures, increase sensitivity and quality of genomic sequencing with small quantities of templates. Using ThermoFidelase 2 and Fimers we have established a number of records: sequencing through very long hairpin and >1.5 kb simple nucleotide (C,T) repeat region, sequencing unique regions off 5 Mb microbial genomic DNA and repeated regions off 3 Gb human genomic DNA, highly sensitive sequencing off 10 ng BAC and 100-300 ng bacterial genomic DNA. A high throughput procedure for primer walking on bacterial genomic DNA has been developed. It allows rapid acquisition of sequences of bacterial protein genes without a need for multiple steps of library construction, blot hybridization, screening or PCR amplification. To screen transposon knockout libraries thousands of clones have to be analyzed. Unfortunately, the yield of genomic DNA in high throughput DNA purification methods varies dramatically from sample to sample. Our previous sequencing protocol for genomic DNA sequencing required at least 2,000-3,000 ng of DNA per reaction. For some bacteria it is problematic to grow enough cells in culture to obtain such quantities of DNA. We have developed a new protocol for high-throughput direct genomic DNA sequencing of transposon insertion sites.