ABSTRACT
Real-time polymerase chain reaction (PCR) is the method of choice for many biomedical applications. Ever since its emergence more than a decade ago, there have been signicant developments such as improvements to the assay design software, instruments, and detection chemistries. Still, the design of an efcient, sensitive and accurate quantitative PCR assay using standard DNA detection chemistry requires adherence to certain fundamental design rules that are dictated by the DNA composition of primers and probes. Melting temperature is determined by the primer and probe sequence and length, design of individual PCR primers and uorescent probes have certain structural requirements, just as these designs must t together under certain relative measures that are again restricted by the composition and sequence context of the desired target. This may, in many cases, complicate or even limit the practical applications of the technology, if, for example, the desired target sequences do not support a design according to guidelines or if the assay complexity prevents simultaneous detection of relevant biomarkers. This tends to be a particular challenge in diagnostic assay development where assay sensitivity and specicity are the key requirements but target sequences may be very limited due to similarity with nontarget sequences. The standard approaches for overcoming such limitations include moving the assay to another sequence, testing multiple suboptimal assays in search of functional assays, or changing cycling or assay chemistry conditions.1 Overall, detection requirements outside the standard designs may require cumbersome optimization cycles and specialized settings that may work against the general preference of being able to run multiple assays under very similar conditions to facilitate high-throughput applications. In the following chapter, we will describe how the use of the RNA analog called locked nucleic acids (LNAs) can dramatically improve the design of qPCR assays through its ability to modify the thermal stability of any oligo while maintaining sequence recognition.
