Amplification of circularizable probes for the detection of target nucleic acids and proteins.
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 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.
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.
- SourceAvailable from: Andrée F Maheux07/2011, Degree: PhD, Supervisor: Michel G Bergeron
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ABSTRACT: Microbead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references.Microchimica Acta 08/2014; 181(11-12):1-18. DOI:10.1007/s00604-014-1243-4 · 3.72 Impact Factor
- Chemical Reviews 02/2014; 114(5). DOI:10.1021/cr400354z · 45.66 Impact Factor