Amplification of circularizable probes for the detection of target nucleic acids and proteins

Abstract

Background Circularizable oligonucleotide probe (C-probe) is a unique molecule that offers significant advantages over conventional probes. Methods Closed circular structure can be formed through ligation of the juxtaposed ends of the C-probe after hybridization with a target, and subsequently locked onto its target through the helical turns formed between the complementary sequences of the target and the C-probe (padlock probe). Under isothermal condition, C-probe can be amplified by rolling circle amplification (RCA) to generate multimeric single-stranded DNA (ssDNA). This multimeric ssDNA can be further amplified by a ramification mechanism (RAM) through primer extension and downstream DNA displacement, resulting in an exponential amplification. Usually, an unbiased product is generated by either RCA or ramification amplification method (or RAM) due to the generic primers of C-probe and its localization onto DNA targets. Conclusions These advantages make C-probe amplification very useful for research and molecular diagnosis, especially in areas where other techniques were proved to be inadequate. The development of C-probe-based technologies offers a promising prospect for molecular diagnosis. The applications of C-probe, RCA, RAM, in situ detection, microarray, immunoassay, single nucleotide polymorphism, and whole genome amplification, etc. are discussed in this review.