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Rolling Circle Amplification (RCA) Reporters – a new generic tool for the detection of DNA, RNA and proteins
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (molecular tools)ORCID iD: 0000-0002-5226-1427
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Many methods to detect biomolecules with rolling circle amplification (RCA), for example padlock probes and proximity ligation assay (PLA), are tedious and includes several reaction steps combined with long incubation times. The present investigation evaluates a new tool to be included in the toolbox of RCA based detection methods that we refer to as RCA Reporter. The main idea with the RCA Reporter is to use pre-ligated circles protected from RCA initiation by almost entire hybridization to a protector molecule that unlocks through a well characterized and highly specific strand displacement process. The RCA Reporters are a generic tool that can be used to directly detect ssDNA or RNA in a sample, to detect proteins via coupling to proximity recognition using a pair of antibodies or to further amplify ongoing RCA reactions. The latter can be used to allow for efficient digital readout of single molecules via flow cytometry with the potential to develop simple and highly accurate molecular counting assays.

National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-331743OAI: oai:DiVA.org:uu-331743DiVA: diva2:1150009
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
In thesis
1. Molecular Tools for Biomarker Detection
Open this publication in new window or tab >>Molecular Tools for Biomarker Detection
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The advance of biological research promotes the emerging of new methods and solutions to answer the biological questions. This thesis describes several new molecular tools and their applications for the detection of genomic and proteomic information with extremely high sensitivity and specificity or simplify such detection procedures without compromising the performance.

In paper I, we described a general method namely super RCA, for highly specific counting of single DNA molecules. Individual products of a range of molecular detection reactions are magnified to Giga-Dalton levels that are easily detected for counting one by one, using methods such as low-magnification microscopy, flow cytometry, or using a mobile phone camera. The sRCA-flow cytometry readout presents extremely high counting precision and the assay’s coefficient of variation can be as low as 0.5%. sRCA-flow cytometry readout can be applied to detect the tumor mutations down to 1/100,000 in the circulating tumor cell-free DNA.

In paper II, we applied the super RCA method into the in situ sequencing protocol to enhance the amplified mRNA detection tags for better signal-to-noise ratios. The sRCA products co-localize with primary RCA products generated from the gene specific padlock probes and remain as a single individual object in during the sequencing step. The enhanced sRCA products is 100% brighter than regular RCA products and the detection efficiency at least doubled with preserved specificity using sRCA compared to standard RCA.

In paper III, we described a highly specific and efficient molecular switch mechanism namely RCA reporter. The switch will initiate the rolling circle amplification only in the presence of correct target sequences. The RCA reporter mechanism can be applied to recognize single stranded DNA sequences, mRNA sequences and sequences embedded in the RCA products.

In paper IV, we established the solid phase Proximity Ligation Assay against the SOX10 protein using poly clonal antibodies. Using this assay, we found elevated SOX10 in serum at high frequency among vitiligo and melanoma patients. While the healthy donors below the threshold.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1387
Keyword
Rolling circle amplification, padlock probe
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331745 (URN)978-91-513-0114-3 (ISBN)
Public defence
2017-12-08, BMC/A1:111a, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-11-14 Created: 2017-10-17 Last updated: 2017-11-14

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