Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification

Trimethylaluminum-Triflimide Complexes for the Catalysis of Highly Hindered Diels-Alder Reactions and Their Use Towards the Enantiospecific Total Synthesis of Rhodexin A

Rhodexin A is a cardiac glycoside first isolated from the leaves and roots of Rhodea Japonica in 1951, which shown potent activity against leukemia K562 cells (IC50 19 nM). A previous synthesis of rhodexin A has been accomplished by the Jung group. A novel synthetic approach, utilizing a convergent Diels Alder reaction with two optically active fragments deriving from Wieland-Miescher ketone and carvone, is reported. Additionally a novel Lewis acid system was developed and utilized during the synthesis and its further applications are described in Chapter 2