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

Department of Pathology, Mount Sinai School of Medicine, New York University, NY 10029, USA.
Clinica Chimica Acta (Impact Factor: 2.82). 02/2006; 363(1-2):61-70. DOI: 10.1016/j.cccn.2005.05.039
Source: PubMed


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.

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    • "However, the addition of the next step, the assembly and ligation of the circular padlock probe, makes this entire assay extremely sequence specific. The stringent strand-matching requirement for the padlock probe ligation has been previously reported (Nilsson, 2006; Zhang et al., 2006). "
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    ABSTRACT: We present an approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single-copy sensitivity and single-base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this technique by successfully detecting six single-copy target sites on human mitochondrial and autosomal DNA. We also demonstrate the high selectivity of this method by detecting X- and Y-specific sequences on human sex chromosomes and by simultaneously detecting three sequence-specific target sites. Finally, we discriminate two target sites that differ by 2 nt. The PNA-RCA-FISH approach is a distinctive in situ hybridization method capable of multitarget visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.
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    • "One of the more promising approaches is that based on proximity probes or circularizable ligation probes. Initially developed as a highly multiplex method for screening for the presence of genes or single nucleotide polymorphisms (Nilsson et al., 1994), they can also be integrated with microarrays to resolve the products of amplification (Zhang and Liu, 2003). The term 'padlock probes' has been coined for these reagents and their use in multiplex diagnostics has been reviewed recently (Landegren et al., 2004). "
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    • "Rolling-circle ampliWcation (RCA), 2 which is isothermal DNA polymerase-driven cyclic replication of small circular DNA probes, is often used as an eYcient method to detect various analytes [14] [15] [16]. It was assumed that the RCA mechanism is diVerent from that of primer-extension reactions using linear DNA tem- plates. "
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