|Advances of Fimer Technologies
Fimers are primers, which include nucleotides with proprietary chemical modifications. Incorporation of the modified nucleotides restricts utilization of DNA molecules (such as Fimers themselves or products of Fimer extension by DNA polymerases) as templates for DNA synthesis. When non-modified oligonucleotides are used in cycle sequencing protocols, two major non-specific events interfere with the sequencing reaction. If a primer can form an extendable dimer, then primer dimer extension can occur, typically at low temperatures.
Annealing of non-modified oligonucleotides to the elongated DNA chains produces non - specific PCRs that interfere with linear amplification in sequencing. The non-specific reactions become predominant at extended cycle sequencing protocols. Primer extension reactions are completely inhibited when DNA polymerase encounters the modified nucleotides in template strands. Thus, using Fimers in sequencing reactions can inhibit non-specific primer dimer extensions and PCRs and allow > 100 cycles of DNA amplification.
In addition, Fimer technologies offer unique opportunities to enhance specificity of annealing to distinctive regions of DNA templates, which are especially important for sequencing large genomic DNAs with many potential sites for non-specific primer annealing.
Use of Fimer technologies with ThermoFidelase allows for direct sequencing large DNAs, such as Bacterial Artificial Chromosomes (BAC), entire Bacteriophages and Microbial genome DNAs, and regions of animal DNAs, which require up to 400 cycles of linear amplification and enhanced specificity of annealing to the templates. Fimers also allow carrying out robust sequencing of plasmids directly from bacterial cell culture or isolated colonies, and sequence difficult DNA templates that are not accessible by other means of automatic DNA sequencing.
Covered by U.S. Patents 5,427,928, 5,656,463, 5,902,879, 6,548,251. Patents pending. Fidelase, ThermoFidelase, D-Strap and Fimer are trademarks of Fidelity Systems.