Recent Advances in Probe Amplification Technologies

Abstract

Oligonucleotide probes provide a useful tool for the detection of target nucleic acids by the formation of a double helical structure between complementary sequences. The stringent requirements of Watson–Crick base pairing make hybridization extremely specific. However, the detection of target sequence by hybridization is often insensitive due to the limited number of signal molecules that can be labeled on the probe. In general, the analytical sensitivity of probe hybridization is of the order 106 molecules. Therefore, it cannot meet the needs of most clinical diagnostic applications. Many technologies have been developed to improve the detection sensitivity by amplifying the probe sequence bound to the target. All probe amplification technologies are developed based on the recent advancement in molecular biology and the understanding of in vivo nucleic acid synthesis (i.e., ligation, polymerization, transcription, digestion/cleavage, etc.). A fundamental advantage of probe amplification technologies ascribes to their isothermal nature, (i.e., accomplishing amplification at a constant temperature with the exception of LCR, which requires temperature cycling). Isothermal amplification allows the test to be done using a simple instrument and makes quality control of the instrument easier. In order for the probe to be amplified, the probes have to be specially designed or synthesized. For example, in rolling circle amplification (RCA), a circularized probe is used, whereas the Invader assay employs an overlapping structure within the probes. Finally, maximum amplification is achieved by generating new DNA products (RCA, RAM, SMART, Q-beta replicase, etc.), although some of the technologies (i.e., LCR and CPT) use existing DNA primers without a net increase of DNA products. In addition to amplification of probe sequence to achieve a desired sensitivity, each technology has its unique features, thus unique clinical applications. For example, Invader technology is very useful for single nucleotide polymorphism (SNP) scoring due to specific recognition by the enzyme cleavase to the overlapping structure of two probes. On the other hand, RCA is probably the only technology that can be used for on-chip amplification due to the attachment of product to the primer sequence linked on the chip surface. Therefore, in order to select a