Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets

Гарафутдинов, Р.Р.; Сахабутдинова, А. Р.; Gilvanov, Aidar R.; Чемерис, А. В.

doi:10.1134/s1068162021060078

Cited by 18 publications

(3 citation statements)

References 139 publications

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“…A primer complementary to the template strand is then introduced and anneals with the template strand. Moreover, strand displacement polymerase creates a new complementary strand, and this newly synthesized strand displaces the original single-stranded region of the template, thus creating a new single-stranded region for the following primer to bind [ 99 , 100 ]. The RCA process is depicted in Figure 2 on the right.…”

Section: Rca With Paper Microfluidicsmentioning

confidence: 99%

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

Reynolds,

Loeffler,

Leigh

et al. 2023

Biosensors

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Isothermal nucleic acid amplification tests have recently gained popularity over polymerase chain reaction (PCR), as they only require a constant temperature and significantly simplify nucleic acid amplification. Recently, numerous attempts have been made to incorporate paper microfluidics into these isothermal amplification tests. Paper microfluidics (including lateral flow strips) have been used to extract nucleic acids, amplify the target gene, and detect amplified products, all toward automating the process. We investigated the literature from 2020 to the present, i.e., since the onset of the COVID-19 pandemic, during which a significant surge in isothermal amplification tests has been observed. Paper microfluidic detection has been used extensively for recombinase polymerase amplification (RPA) and its related methods, along with loop-mediated isothermal amplification (LAMP) and rolling circle amplification (RCA). Detection was conducted primarily with colorimetric and fluorometric methods, although a few publications demonstrated flow distance- and surface-enhanced Raman spectroscopic (SERS)-based detection. A good number of publications could be found that demonstrated both amplification and detection on paper microfluidic platforms. A small number of publications could be found that showed extraction or all three procedures (i.e., fully integrated systems) on paper microfluidic platforms, necessitating the need for future work.

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Section: Rca With Paper Microfluidicsmentioning

confidence: 99%

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

Reynolds,

Loeffler,

Leigh

et al. 2023

Biosensors

View full text Add to dashboard Cite

show abstract

“…In order to circumvent these and other disadvantages of PCR (e.g. ampli cation time and the mandatory trained staff), various alternative methods are available, which are commonly referred to as "isothermal ampli cation" methods [9][10][11][12] . By using a wide variety of complex molecular reaction mechanisms with speci c enzymes and strand displacing polymerases as well as special designed primer sets, all of these methods allow DNA to be ampli ed at a constant and low temperature without the energetically and technically expensive thermal cycling of the PCR process 13 .…”

Section: Introductionmentioning

confidence: 99%

Labelling of RPA amplicons: an experimental comparison between primer and nucleotide labelling used for multiplex detection of antibiotic resistance genes

Warmt,

Broweleit,

Fenzel

et al. 2023

Preprint

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Direct labelling of amplification products using isothermal amplification is currently done most frequently by incorporating previously labelled primer. Although this method is well proven and widely used, it is not a universal solution due to some weaknesses. Alternatively, labelled nucleotides could be used, whose application and functionality have been already partially demonstrated. It remains to be determined how this method performs in comparison to traditional labelling, in particular combined with isothermal amplification methods. In this work, we show a detailed analysis of the labelling efficiency under different conditions and compare the results with the traditional primer-labelling method in the context of RPA amplification. Impressively, our results showed that using Cy5-labelled dUTPs can achieve much more efficient labelling for fragments above 200 bp, while using them for smaller fragments does not bring any relevant disadvantages, but also no major benefit. Furthermore, this work successfully demonstrate for the first time a quadruplex microarray for the detection of resistance genes using RPA and direct labelling with Cy5-dUTP as a potential application scenario. The sensitivities achieved here extend to SNP discovery for the detection of the proper blaKPC variant.

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“…In another of our prior works, , we utilized an amplification protocol for the detection of miRNAs nonspatially using rolling circle amplification (RCA). RCA is an isothermal amplification scheme. − Unlike RT-qPCR, we utilized RCA to amplify the detection element rather than the biomarker, and RCA can be readily incorporated into our existing nanoliter well array. , …”

mentioning

confidence: 99%

Sensitive Multiplexed MicroRNA Spatial Profiling and Data Classification Framework Applied to Murine Breast Tumors

Mohd,

Heng,

Wang

et al. 2024

Anal. Chem.

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MicroRNAs (miRNAs) are small RNAs that are often dysregulated in many diseases, including cancers. They are highly tissue-specific and stable, thus, making them particularly useful as biomarkers. As the spatial transcriptomics field advances, protocols that enable highly sensitive and spatially resolved detection become necessary to maximize the information gained from samples. This is especially true of miRNAs where the location their expression within tissue can provide prognostic value with regard to patient outcome. Equally as important as detection are ways to assess and visualize the miRNA's spatial information in order to leverage the power of spatial transcriptomics over that of traditional nonspatial bulk assays. We present a highly sensitive methodology that simultaneously quantitates and spatially detects seven miRNAs in situ on formalin-fixed paraffin-embedded tissue sections. This method utilizes rolling circle amplification (RCA) in conjunction with a dual scanning approach in nanoliter well arrays with embedded hydrogel posts. The hydrogel posts are functionalized with DNA probes that enable the detection of miRNAs across a large dynamic range (4 orders of magnitude) and a limit of detection of 0.17 zeptomoles (1.7 × 10 −4 attomoles). We applied our methodology coupled with a data analysis pipeline to K14-Cre Brca1 f/f Tp53 f/f murine breast tumors to showcase the information gained from this approach.

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Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets

Cited by 18 publications

References 139 publications

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

Recent Uses of Paper Microfluidics in Isothermal Nucleic Acid Amplification Tests

Labelling of RPA amplicons: an experimental comparison between primer and nucleotide labelling used for multiplex detection of antibiotic resistance genes

Sensitive Multiplexed MicroRNA Spatial Profiling and Data Classification Framework Applied to Murine Breast Tumors

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