Achieving primer and probe designs that provide highly sensitive and specific detection of DNA or RNA can be confounded by the sequences of the target region. Target sequences rich in A-T pairs, for example, often have lower melting temperatures, while G rich regions can be effected by guanine-guanine self association. Nanogen has developed modified nucleic acid 'super' bases that can be substituted in primer and probe designs to eliminate many of the problems and improve primer/probe specificity and sensitivity.
The table below summarizes the known sequence contents which pose the greatest trouble to real-time PCR assay developers. In each case, Nanogen's proprietary Superbases can be used to address the problem facing the assay developer.
Super A® & Super T®
Super A and Super T are modified bases that improve the stability of traditionally weaker A-T bonds. By incorporating these modified bases into A-T rich sequences, the melting temperature (Tm) can be raised for hybridization that is more efficient. As a result, Super A and Super T improve the performance of assays in A-T rich regions.
Super G®
Super G is a modified guanosine substitute designed to eliminate guanine-guanine self-association in G rich sequences that interfere with proper hybridization. The modified structure of Super G precludes the hydrogen bonding that occurs between Gs in guanine-guanine self-association and raises the melting temperature for probes and primers specific to G rich regions.
In typical primer/probe designs, sequences containing greater than 50% GC content are avoided. Substitution with Super G allows satisfactory probe performance with greater then 75% GC content in some cases.
Additionally, Super G will not quench adjacent fluorophores attached at the 5' terminus. As a result, Super G allows analysis of G-C rich sequence where other chemistries fail.
Super N™
Super N™ is a universal base designed to bind with near equal efficiency to all bases (regular A, T, G, and C). Super N enables the design of probes and primers to unknown ('N-read') or polymorphic target regions without affecting the analysis of the region of interest. It is particularly useful for analyzing SNP targets with an adjacent polymorphic region.