CE combined with rolling circle amplification for sensitive DNA detection

Abstract

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.

Tagging the rolling circle products with nanocrystal clusters for cascade signal increase in the detection of miRNA

“…S6 †), and similar phenomena have been reported in the literature. 27,37,38 Moreover, better assay reproducibility and lower detection background were obtained by storing the ZnS NCCs in a solution with a higher BSA content, in order to reduce non-specic adsorption of the NCCs onto the MPs (ESI, Fig. S7 †).…”

“…Recognition of the dye-or nanoparticle-labelled oligonucleotides with the tandem regions permits optical or electrochemical detection of the rolling circle product (RCP). 26 In addition, different sequences, such as digestion site for endonuclease 27,28 and DNAzyme, 16,29,30 can be inserted to the circular probe to facilitate RCP detection without lowering the RCP synthesis rate, owing to the high strand displacement capability of the enzyme phi29 DNA polymerase. Alternatively, hapten-modied dNTPs can be used in RCA to label the RCP.…”

Magnetic beads based rolling circle amplification–electrochemiluminescence assay for highly sensitive detection of point mutation

“…It is believed that the sensitivity of the current assay is comparable with or even exceeded the sensitivity of other reported RCA based methods, such as the reported branched RCA detection of RNA with a sensitivity of 6 amol (Cheng et al, 2009), 2 fmol of CE-RCA assay (Li et al, 2008b), 0.18 fmol of chemiluminescent combined with RCA (Li et al, 2008a), fluorescence detection based on RCA with a sensitivity of 6.4 fmol (Guo et al, 2009), and microsphere-based RCA with a sensitivity of 10 amol. In the proposed assay, the detection limit of 2 amol is achieved.…”

Multiplex ligase‐based genotyping methods combined with CE

“…To visualize the final product of the ligase‐based assay for detection by a CE instrument, a labeling method should be used. A popular method is intercalating dye such as SYBR Green and SYBR Gold . However, because all of the components present in the assay product, including primers, probes, and any nonspecifically formed fragments, should essentially be detected, the assay results can be compromised easily.…”

“…Due to the requirement for postprocessing and low sensitivity, RCA is not a popular option for CE‐based genotyping assays. Nevertheless, RCA has been combined with CE for the analysis of cancer‐related genes and small RNA . In the cited reports, the amplified products were desalted to achieve the desired sensitivity.…”