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Rolling circle amplification reporters – a general tool to simplify molecular detections
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]

Methods to detect biomolecules via rolling circle amplification (RCA), for example padlock probes and proximity ligation assay (PLA) tend to be complex and include several reaction steps. Herein, we evaluated a new tool for the toolbox of RCA-based detection methods - RCA Reporters. Briefly, RCA Reporters represent inert DNA structures that, upon contact with a specific nucleic acid sequence, unravel via a highly specific strand displacement process to initiate local enzyme-assisted RCA reactions. The RCA Reporters can be used to directly detect ssDNA or RNA in a sample, or proteins via oligonucleotide-conjugated antibodies. The reagents can also enable faster RCA reactions or extremely selective genotyping of RCA products with repeated copies of a target sequence through a majority-vote mechanism. Further amplification of ongoing RCA reactions via RCA Reporters can allow efficient digital enumeration of single molecules via flow cytometry, with potential for simple and highly accurate molecular counting assays. The intrinsic simplicity of RCA Reporter also renders them attractive for applications at the point of care.

National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-331743OAI: oai:DiVA.org:uu-331743DiVA, id: diva2:1150009
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-11-03
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. p. 48
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1387
Keywords
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: 2018-03-07
2. Dried blood sampling and digital readout to advance molecular diagnostics
Open this publication in new window or tab >>Dried blood sampling and digital readout to advance molecular diagnostics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A drastically increased capacity to measure large sets of molecular features in numerous patient samples in great detail will be required to fulfill the vision of precision medicine and wellness, which may characterize molecular diagnostics in the 21st century. Also sampling procedures need a renaissance to permit continuous sampling at population levels at reasonable cost.

Blood sampling is typically performed via venipuncture to draw several milliliters of blood for plasma isolation. This is inconvenient, time-consuming and costly, as well as hard to standardize. The effect on plasma protein profiles by pre-centrifugation delay was investigated in Paper II, demonstrating time- and temperature-dependent release of proteins from blood cells upon delayed plasma isolation, but almost no protein degradation as analyzed by two 92-plex protein panels (Olink® Proteomics). An alternative sampling method, where blood drops from a finger stick are collected dried on paper, is relatively non-invasive, potentially home-based and cheap. Dried blood spots can also be shipped via regular mail and compactly stored. The effect of drying and long term storage stability of a large set of proteins from dried blood spots was investigated in Paper I using Olink® technology. The main findings were that drying slightly but consistently influenced the recorded levels of blood proteins, and that long-term storage decreased the detected levels of some of the proteins with half-lives of decades.

Some molecular diagnostic investigations require great accuracy to be useful, arguing for digital enumeration of individual molecules. Digital PCR is the gold standard but Paper III presents an alternative approach based on rolling circle amplification of single molecules. Another instance where extreme assay performance is required is for rare mutation detection from liquid biopsies. Paper V presents a new method offering essentially error-free genotyping of individual molecules by majority-vote decisions for counting rare mutant DNA in blood. Yet other diagnostic investigations require very simple assays. Paper IV presents a novel one-step method to detect nucleic acid sequences by combining the power of rolling circle amplification and the specificity of DNA strand displacement in a format simple enough to be used at the point of care.   

Altogether, the thesis spans technologies for advanced molecular diagnostics, from sample collection over assay techniques to an improved readout.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1615
Keywords
molecular diagnostics, dried blood sample, DBS, digital readout, digital enumeration, DNA detection methods, proximity extension assay, protein stability, genotyping, rare mutations, cell free DNA, multiplex protein measurement.
National Category
Medical Biotechnology Biochemistry and Molecular Biology Medical Laboratory and Measurements Technologies
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-396325 (URN)978-91-513-0810-4 (ISBN)
Public defence
2019-12-20, A1:111a, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-11-28 Created: 2019-11-03 Last updated: 2019-11-28

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