Detection of target nucleic acids and proteins by amplification of circularizable probes

Abstract

Circularizable oligonucleotide probe (C-probe) is a unique molecule that offers significant advantages over conventional probes. Closed circular structure can be formed through ligation of its ends after hybridizing onto a target and locked on its target due to the helical turns formed between the complementary sequences of the target and the C-probe (padlock probe). Under an isothermal condition, C-probe can be amplified by rolling circle amplification, to generate multimeric single-stranded DNA. This multimeric single-stranded DNA can be further amplified by a ramification mechanism through primer extension and upstream DNA displacement, resulting in an exponential amplification. Usually, an unbiased product is generated by either rolling circle amplification or ramification mechanism due to the generic primers of C-probe and is localized on targets. These advantages make C-probe amplification very useful for research and molecular diagnosis, especially in the areas where other techniques are not adequately helpful. The development of C-probe-based technologies iniates a new future for molecular diagnostics. The applications of C-probe, rolling circle amplification, ramification mechanism, in situ detection, microarray, immunoassay, single nucleotide polymorphism and whole genome amplification are discussed.